Laboratory Trainer Theory

The Training Program for Health Institute Graduates has been developed for the health institute graduates of the Kingdom of Saudi Arabia. The Ministry of Health General Directorate of Training and Scholarship, in collaboration with subject matter experts from other Ministry of Health bodies that include, but are not limited to: The General Directorate of Pharmaceutical Affairs; The General Directorate of Nursing Affairs; The General Directorate of Radiology and Applied Services; The General Directorate of Lab and Blood Banks; Medical Records Administration; and The Field Epidemiology Training Program have produced the enclosed Training Program.

A special thank you to Teesside University and others for their contribution to the development of the materials for the First Edition of the Training Program for Health Institute Graduates

Kingdom of Saudi Arabia - Ministry of Health General Directorate of Training and Scholarship Training Program for Health Institute Graduates

Table of Contents Program Outline ............................................................................................................................................................. 1 General Orientation (day 1).................................................................................................................................. 10

General Orientation (day 2).................................................................................................................................. 21

Collaboration and Teamwork in Health Care...................................................................................... 21

Notes for trainer .............................................................................................................................. 21

Lecture................................................................................................................................................. 29

Infection Prevention and Control............................................................................................................. 40


Lecture................................................................................................................................................. 45

Workplace Safety and Injury Prevention .............................................................................................. 58


Lecture................................................................................................................................................. 61

Bacteriology ................................................................................................................................................................... 68

Notes for trainer .............................................................................................................................................. 69 An Introduction to the Bacterial Cell, its Organization and Members ...................................... 71

Elements of Microbial Nutrition ............................................................................................................... 82

Sterilization and Disinfection .................................................................................................................... 90

Tools of the Laboratory ............................................................................................................................. 102

Antimicrobial Chemotherapy and Sensitivity Testing ................................................................. 114

The Cocci of Medical Importance .......................................................................................................... 122 Gram Negative Bacilli of Medical Importance.................................................................................. 134 Gram Positive Bacilli of Medical Importance ................................................................................... 145

Cerebrospinal Fluid (CSF) Culture........................................................................................................ 156

Blood Culture................................................................................................................................................. 161

Urine Culture ................................................................................................................................................. 166 Respiratory Tract Infections ................................................................................................................... 175

Parasitology ................................................................................................................................................................ 184

Notes for trainer ........................................................................................................................................... 185

Medical Parasitology Lab.......................................................................................................................... 186

The Parasites of Medical Importance - Protozoa............................................................................ 212

The Parasites of Medical Importance - Helminth ........................................................................... 220

Clinical Chemistry.................................................................................................................................................... 226

Technology in Clinical Chemistry (Part I) ......................................................................................... 227

Kingdom of Saudi Arabia - Ministry of Health General Directorate of Training and Scholarship Training Program for Health Institute Graduates Technology in Clinical Chemistry (Part II) ........................................................................................ 231

Units and Calculation in Clinical Chemistry ...................................................................................... 236

Blood Glucose Tests .................................................................................................................................... 238

Kidney Function Tests ............................................................................................................................... 242

Electrolytes and Minerals......................................................................................................................... 246

Clinical Enzymology.................................................................................................................................... 253

Liver Function Tests ................................................................................................................................... 259 Cardiac Biomarkers .................................................................................................................................... 264

Lipid Profile.................................................................................................................................................... 275 Special Tests in Clinical Chemistry ....................................................................................................... 281

Body Fluid Chemistry (Urine – CSF- Other Body Fluids) ............................................................ 287

Blood Bank .................................................................................................................................................................. 295

Notes for trainer ........................................................................................................................................... 296 Donor Selection ............................................................................................................................................ 299

Blood Collection ........................................................................................................................................... 305 Blood Donor Adverse Reactions ............................................................................................................ 307 Donor Recruitment and Retention ....................................................................................................... 313

Blood Component Preparation and Storage ..................................................................................... 328

Blood Component Quality Control........................................................................................................ 333 Leukoreduced and Irradiated Blood Components......................................................................... 336

Basic Immunology ....................................................................................................................................... 343

Blood Grouping Discrepancies ............................................................................................................... 348 Pretransfusion Guidelines and Neonatal Transfusion Policy .................................................... 356

Antibody Identification ............................................................................................................................. 360

Transfusion Transmitted Diseases ....................................................................................................... 372

Bacterial Contamination ........................................................................................................................... 382 Screening and Confirmatory ................................................................................................................... 393 NAT Types....................................................................................................................................................... 400

Hematology ................................................................................................................................................................. 406


Introduction to Hematology .................................................................................................................... 408

Red Cells .......................................................................................................................................................... 412

Anemia ............................................................................................................................................................. 415 Abnormal Hemaglobins ............................................................................................................................ 418

Kingdom of Saudi Arabia - Ministry of Health General Directorate of Training and Scholarship Training Program for Health Institute Graduates White Cells ...................................................................................................................................................... 420

Lymphocytes and their Disorders ........................................................................................................ 422 Acute Leukemia ............................................................................................................................................ 424 Chronic Leukemia ........................................................................................................................................ 426

Investigation of Bleeding Disorders..................................................................................................... 428

Blood Coagulation ....................................................................................................................................... 430

Thrombin Time (TT) Test ........................................................................................................................ 431

Automation .................................................................................................................................................... 432

Specimen Collection ............................................................................................................................................... 434


Lecture ............................................................................................................................................................. 436

Quality Control and Assurance......................................................................................................................... 445

Lecture ............................................................................................................................................................. 446

Kingdom of Saudi Arabia – Ministry of Health General Directorate of Training and Scholarship Training Program for Health Institute Graduates

Competency Based Training Flow Chart for Laboratory Technician Trainee Laboratory Technician

Repeat 2months

English Course 6 Months

Fail Pass

Orientation to program (2 days) 23 days teaching

MCQ on completion of theory block

16 weeks practical period

Fail

Pass

Assigned to availability of vacant positions Dept. needs

Extend the training for 2 months and repeat the Technical Exam or MCQ

Pass

Fail End the training Period

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Course Title Hospital-based Training for the Laboratory Technician

Introduction The Ministry of Health believes in the value of their human resources. Every effort is made to provide Saudi and non-Saudi health professionals with the tools and resources that are necessary for success. An important first step in the process is an inclusive training program for new graduate trainees who have not worked in their profession for more than one year. This is a 6-month program which will provide them with the classroom and clinical experiences needed to embark on a successful career in the laboratory field. The program has been designed as a transition program to link the gap between the academic and the service settings and to prepare the target group to utilize decision skills in the care of patients. The program's theoretical foundations are based on Kolb’s model of experiential learning (1984), Knowles' adult-learning principles (1970), and Kramer's classic research on reality shock (1974).

Kolb’s (1984) model for experiential learning This 6-month training period supports the competency and professional development of novice laboratory technicians. The program components include both theoretical knowledge and practical learning experience.

Course Description This Unit will provide students with a background of the analytical methods, skills and instrumentation used in healthcare sciences. Core classes in this program study human diseases and laboratory tests that identify them. Students learn to operate equipment in medical laboratories and perform a wide range of procedures. Didactic and clinical instruction emphasize proper specimen collection and handling, understanding test procedures, safety, quality control, acquisition of technical skills, and troubleshooting techniques. Lectures and laboratory practical will cover several key areas including clinical chemistry, haematology, blood bank, specimen collection, parasitology and microbiology / bacteriology.

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Program Goals

The goals of the program are the following: • To provide students with a body of knowledge and clinical training to develop entry-level competencies in all routine areas of the clinical laboratory; • To produce graduates who demonstrate ethical behavior and professional attitudes; • To provide a quality program, which is assessed, evaluated, and revised as needed; • To provide graduates who will enrich the laboratories in which they are employed; • To provide a stimulating educational experience that encourages continuing education in both students and participating laboratory staff.

Program Objectives Theoretical Part Upon successful completion of the program and initial employment, graduates should be able to demonstrate entrance-level competencies in the following major areas of professional practice:  1. 2. 3. 4.

Knowledge: Demonstrate a basic knowledge of bacteria, parasites and viruses Demonstrate a basic knowledge of haematology and how this relates to abnormal results Demonstrate a basic knowledge of Biochemistry Demonstration of knowledge of the relationship of laboratory findings with common diseases processes

Clinical part Upon successful completion of the program and initial employment, graduates should be able to demonstrate entrance-level competencies in the following major areas of professional practice:  Skills: 1. Collection, handling, preparation, and storage of biological specimens for laboratory analysis; 2. Performance of technical analyses on body fluids, cells, products, and organisms; 3. Recognition of factors that affect procedures and results and take appropriate action within predetermined limits; 4. Ability to operate basic laboratory instrumentation; 5. Performance of quality control measures on instrumentation and technical analyses; 6. Recognition of and adherence to clinical laboratory safety policies; 7. Ability to troubleshoot instrumentation and technical analyses; 8. Ability to perform preventative and corrective maintenance on basic laboratory equipment and instrumentation; 9. Ability to recognize when to refer instrumentation problems to the appropriate sources; 10. Demonstration of professional conduct with patients and health care workers both within and outside the laboratory; 11. Demonstration of effective interpersonal communication skills

Practice Experience Following theoretical training, trainees will go into clinical practice to enable them to practice and consolidate skills under the supervision of a clinical preceptor. Trainees will rotate between different areas of the laboratory. These areas include: • Clinical Chemistry

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Kingdom of Saudi Arabia – Ministry of Health General Directorate of Training and Scholarship Training Program for Health Institute Graduates • • • • •

Hematology Microbiology / Bacteriology Blood Bank Specimen Collection Parasitology

Placements will be on a rotational basis and during each placement the clinical supervisor will offer formative and summative feedback on the trainee’s progress and tick the relevant box when a competency has been met. At the end of each placement a summative assessment will take place of all competencies.

Methods of Teaching The unit will be taught through a mixture of lecture and practical sessions. This will take the form of 6 hours of lectures and practicals per week. Competencies will be acquired in the following coursework through didactic presentation and laboratory experience. The program coursework is designed to show student progression of knowledge and skill.

Methods of Assessment • •

Theory Assessment: One MCQ assessing learning outcomes for knowledge areas 1 – 4 Practical Assessment: – Continuous evaluation of competencies at the end of each practical rotation according to the form of evaluation available assessing outcomes S1-11.

Summative assessment Summative assessment will take place at various points: 1. During the theoretical component trainees will be given a 2 hour MCQ examination of 60 questions at the end of the theory block. 2. By the end of trainees’ rotation in the relevant area, trainees will be assessed against the relevant competencies. By the end of the practical component trainees should have been assessed against all competencies. Competencies A-D are to be assessed at every point of assessment in the practical component of the program. The overall breakdown of marks awarded is as follows: • 20% theory • 70% practice • 10% attendance (trainee will receive a 0% if he is absent for more than 10% of the total duration of the course) Trainees are to achieve a weighted average of 60% in order to pass the course Should the trainee fail either part of the assessment then they will have 2 months to resit the component. If at this point they are still unable to pass then the training program may be discontinued.

Evaluation As this program is new and developed for a specific purpose it is essential to gain a full evaluation to inform future developments. Evaluation will be undertaken in several ways.

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Kingdom of Saudi Arabia – Ministry of Health General Directorate of Training and Scholarship Training Program for Health Institute Graduates Evaluation from Trainee Laboratory Technicians Ongoing feedback will be obtained from trainees through collection of qualitative data and a final course evaluation questionnaire will be given on completion. Evaluation from Laboratory Educators in clinical practice A focus group will be facilitated in each of the delivery sites to bring together educators from clinical practice. This feedback will identify if trainees are ‘fit for practice and purpose’ on completion. Qualitative data will be collected and experiences shared which will be fed back into the program design. Evaluation from speakers Speakers will be given a short questionnaire to evaluate the delivery of their session. This will include time to deliver content, student engagement, teaching methods and any future changes.

References Monica Cheesbrough, (2006). District Laboratory Practice in Tropical Countries 2nd Edition. Cambridge university press

Marshall, WJ and Bangert, SK. (2008). Clinical Biochemistry: Metabolic and Clinical Aspects 2nd Edition. Churchill Livingstone

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Laboratory Trainee Technician Program Outline

Theoretical Component Duration of session

Topic

2 days

General Orientation

4 days

Clinical Chemistry

Details of Session • • • • • • •

Orientation to the program Communication, Collaboration & Teamwork Delegation in Health Care Setting Infection Control in Health Care Setting Workplace Safety & Injury Prevention Technology in Clinical Chemistry (Part I) Technology in Clinical Chemistry (Part II) Units and Calculations in Clinical Chemistry

Objective is to identify: • Different methods (with their principles) applied in clinical chemistry field. • Units Used in Clinical Chemistry & how calculate some concentrations. • Blood Sugar Tests • Kidney Function Tests • Electrolytes and Minerals Objective is to know: • Investigations done for blood glucose. • Parameters for evaluating the kidney function & how calculate creatinine clearance. • Types of electrolytes, minerals of bone & iron profile • Clinical Enzymology • Liver Function Tests • Cardiac Biomarkers Objective is to be aware of: • Enzymes of clinical significance and their role in diagnosis & prognosis of some diseases. • Parameters for evaluating the liver function & their role in diagnosis & prognosis of liver diseases. • Investigations for diagnosis of myocardial infarction, heart failure & pulmonary embolism • Lipid Profile • Special Tests in Clinical Chemistry • Body Fluid Chemistry (Urine – CSF – Other Body Fluids) To clarify: • Parameters of lipogram and how calculate LDL-cholesterol from other parameters. • Types of non-routine investigations done in Clinical Chemistry.

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Kingdom of Saudi Arabia – Ministry of Health General Directorate of Training and Scholarship Training Program for Health Institute Graduates 4 days

4 days

Haematology Placement

Blood Bank Placement

•

Chemical tests done on urine, CSF & other body fluids

• • •

Function of blood, haematopoisis Collection of blood , Anticoagulants ,stains used in Hematology

• • •

Red cell function , morphology, PCV, and indices Reticulocytic count Haemoglobin function, structure, Introduction to Hb abnormalities

• • •

Anaemia Classifications: According to RBCs Indices According to cause of defect: o Membrane defect (Spherocytosis)

• •

Quantitative Hemoglobinopathies (Thalathaimias) Enzymopathies (G6PD)

•

White Blood Cells: Granulocytes, Monocytes and their Benign Disorders

• • •

Lymphocytes and their disorders Spleen Acute Leukaemias

•

Chronic Leukaemias

• • • • • • •

Investigations of Bleeding Disorders: Vascular endothelium damage Platelet Number, Structure, Function B.T Test Blood Coagulation External and Internal Pathways PT, PTT tests introduction

• •

Screening for a Circulating Anticoagulants PT, APTT, TT test method

• •

Fibrinolytic System FDP test Information Provided by Platelet count , PT, APTT, TT Automation role & principle Criteria of donor selection and deferral Blood donor sampling and collection Management of donor adverse reaction Donor recruitment and retained strategies

• • • •

Objective is for trainees to be able to:

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Kingdom of Saudi Arabia – Ministry of Health General Directorate of Training and Scholarship Training Program for Health Institute Graduates • • • • • • •

Prepare a donation program & deal with the factors affecting motivation of volunteers to donate blood. Determine if the donor is eligible to donate, temporally deferred, or permanently deferred. Counsel donors about the benefits and risk of donating blood products. Recognize adverse events related to blood donation and manage them appropriately. Blood component preparation and storage Blood component quality control Special blood component (irradiated – leucoreduced – frozen)

Objective is for trainees to be able to: • Prepare, process, store, release and quality control for blood components. • Manage and participate actively in all processes needed for safe effective manufacturing and storage of blood components. • Recognize plasma derivatives that are prepared commercially. • Can explain the metabolic changes that occur during storage • Lymphocytes and their disorders • Principles of immunology • Blood grouping systems • Compatibility testing policy and procedure • Neonatal transfusion policy • Antibody screening and identification Objective is for trainees to be able to: • Perform the identification and pre-transfusion testing of patient/ unit, including ABO/Rh testing, RBC antibody screen, and antibody identification. • Perform crossmatch: minor, major, Direct antiglobulin test rapid spin and type & screen • BTTDs • Bacterial contamination of blood • Screening and confirmatory testing • Principles and different methods of NAT Objective is for trainees to be able to: • Be able to perform various methodologies such as ELISA, chemiluminescence and rapid assays used in screening of TTDs. • Can perform the various methodologies such as western blot, neutralization and reba testing used in confirmatory of transfusion transmitted infection and interpret the results. • Can perform the NAT testing and interpret the results.

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Kingdom of Saudi Arabia – Ministry of Health General Directorate of Training and Scholarship Training Program for Health Institute Graduates 4 days

Microbiology / Bacteriology Placement

2 days

Parasitology Placement

1 day

Specimen Collection Placement

1 day

Quality Control and Assurance Placement

• • • •

• • • • • • • • • • • • • • • • • • • • • • • • • • • •

Introduction to the bacterial cell, its Organization, and Members Elements of Microbial Nutrition, Ecology, and Growth Sterilization & Disinfection Tools of the Laboratory: The Methods for Studying Microorganisms Antimicrobial chemotherapy and sensitivity testing The Cocci of Medical Importance The Gram-Negative Bacilli of Medical Importance The Gram-Positive Bacilli of Medical Importance Cerebrospinal Fluid (CSF) Culture Blood culture Urine sample process Respiratory sample culture Medical Parasitology Lab. Part1 Medical Parasitology Lab. Part2 The Parasites of Medical Importance “Protozoa” Part 1 The Parasites of Medical Importance “Protozoa” Part 2 The Parasites of Medical Importance “Helminth” Part 1 The Parasites of Medical Importance “Helminth” Part 2 Receiving samples Patient registration/data Preparing patients Handling of specimens Blood specimen tubes Blood withdrawal Types of blood samples Preparation of sample Transport of samples Sample retention Sample rejection The policies and procedures associated with Quality Control and Assurance Good Laboratory Practice (GLP) The principles of Good Clinical Practice (GCP)

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Ministry of Health Kingdom Of Saudi Arabia

Lecture Outline • About the Training Program for Health Institute Graduates • Laboratory Domain – Course Description • Laboratory Program Outline • Roles and Responsibilities – Trainer • Roles and Responsibilities – Trainee • Policies and Procedures • Important Program Dates

Training Program for Health Institute Graduates

Day 1 General Orientation 2

Introduction • • •

Welcome to the Training Program for Health Institute Graduates

The Training Program for Health Institute Graduates has been undertaken by the Ministry of Health. The aim is to assist and promote the transition of technicians within the Kingdom of Saudi Arabia (KSA) across various health care delivery settings The Training Program for Health Institute Graduates design reflects a model of delivery encompassing ten registered technician role domains. The ten specialty area domains developed for training under the program are:          

Pharmacy Nursing Radiology Laboratory Medical Records Epidemiology, Public Health, and Environmental Health Biomedical Anesthesia and Recovery Health and Hospital Administration Occupational Health and Safety

The successful completion of the Training Program by trainees of the domains will foster a stronger human resources development strategy and enhance the delivery of competency based practices to Kingdom of Saudi Arabia citizens. 3

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Historical Context

Program Values

• His Royal Majesty, King Abdullah bin Abdulaziz Al Saud has proactively identified the need to build capacity amongst its youth population.

Under the Kingdom of Saudi Arabia, Ministry of Health, Training Program for Health Institute Graduates ‐ a key goal of the program is to experience and build human resources capacity reflecting the following values:

• Thousands of Saudi youth have attended health institutes across the Kingdom to advance their knowledge in their areas of interest. However, the educational experience has created a theory‐practice gap for learners, thus posing difficulties for graduates searching for employment in their field.

        

• In response to Royal Decree A/29, dated 20/3/1432H, this unique program provides health institute graduates with a unique opportunity to participate in a Training Program for Health Institute Graduates. 5

Patient first Justice Professionalism Quality Honesty and transparency Teamwork Academic Integrity Initiative and productivity Societal involvement 6

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Program Description

Program Description Phase One Trainee’s participation in a 6 month English language training course and its successful completion is a prerequisite to entering the clinical learning phase of the Training Program for Health Institute Graduates.

The Training Program for Health Institute Graduates was envisioned for delivery in two important training / learning phases.

Phase Two Requires the trainee’s participation in a 6 month domain‐specific “on the job” training. This involves a theoretical component, as well as a practical placement component

Training Program for Health Institute Graduates ‐ Phase One Kingdom of Saudi Arabia, Training Program for Health Institute Graduates ‐ Phase Ministry of Health ‐ Trainee Two selection process completed Trainees participate in Phase Two of the Training Program. All Trainees will participate in a) 6000 + Trainees participate course based learning evaluation in a 6‐month Language This entails completion of a 6 month and b) domain‐specific trainee Study Course to Learn Hospital / Health Care Agency based placement practice evaluation. English for application in a course & placement site practice health care setting. based Training Program. This will include pretest / post test MCQ's, trainee self Trainees must successfully The goal of this phase of the assessment with multi‐source complete the language competency based learning program feedback, trainer assessment training to progress to as delivered in each one of the and feedback evaluation of both Phase Two. domain specific areas ‐ is a novice global and domain specific to beginner level trained Registered competencies at various points Technician who can understand and of the Training Program. demonstrate safe and competent scope of practice roles and responsibilities.

The theoretical component will allow the trainee to gain a fundamental understanding and knowledge of the skills they will be acquiring. The practical placements will allow the trainee to develop their professional practice role(s) and the delivery of practice based competencies for use in the health care / hospital setting.

Outcome Evaluation

Evaluation of each trainee’s learning outcomes will be based on both global core competencies and domain specific scope of practice competencies – as a novice level technician.

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Program Design: Kolb’s Model for Experiential Learning

Program Design • The Training Program has been developed from KSA Ministry of Health mission, vision and values • The Program was designed with local and international professional input from subject matter experts • The competencies developed for the program are designed in compliance with the Saudi Commission’s standards for health knowledge and skills and/or best practices for the respective domains, while simultaneously aligned with the MoH’s commitment to high quality and safe healthcare delivery across all of its health services. • The Program utilizes a participatory learning and competency guided approach, from the novice to more advanced beginner level practice

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Introduction to Laboratory domain • The Ministry of Health believes in the value of their human resources. Every effort is made to provide their human resources with the tools and resources that are necessary for success. • An important first step in the process is an inclusive training program for technicians who have not worked in their domain for more than one year.

Laboratory Technician

12

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Program Synopsis This Unit will provide students with a background of the analytical methods, skills and instrumentation used in healthcare sciences. Core classes in this program study human diseases and laboratory tests that identify them. Students learn to operate equipment in medical laboratories and perform a wide range of procedures. Didactic and clinical instruction emphasize proper specimen collection and handling, understanding test procedures, safety, quality control, acquisition of technical skills, and troubleshooting techniques. Lectures and laboratory practical will cover several key areas including clinical chemistry, haematology, blood bank, sample collection, parasitology and microbiology.

Course Description

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Course Description: Program Objectives (theory)

Course Description

Upon successful completion of the program and initial employment, graduates should be able to demonstrate entrance‐level competencies in the following major areas of professional practice:

• To include the following: – – – – – – –

Program objectives Chosen program rollout Program outline Materials needed for the program Teaching‐learning process Learning methods Methods of assessment (in theory and practical training)

Knowledge: • Demonstrate a basic knowledge of bacteria, parasites and viruses • Demonstrate a basic knowledge of haematology and how this relates to abnormal results • Demonstrate a basic knowledge of Biochemistry • Demonstration of knowledge of the relationship of laboratory findings with common diseases processes

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Course Description: Program Objectives (practical)

Course Description: program rollout

Upon successful completion of the program and initial employment, graduates should be able to demonstrate entrance‐level competencies in the following major areas of professional practice:

• The six month Training Program has a theoretical component and a practical component • Program rollout will be as follows:

Skills: • Collection, handling, preparation, and storage of biological specimens for laboratory analysis; • Performance of technical analyses on body fluids, cells, products, and organisms; • Recognition of factors that affect procedures and results and take appropriate action within predetermined limits; • Ability to operate basic laboratory instrumentation; • Performance of quality control measures on instrumentation and technical analyses; • Recognition of and adherence to clinical laboratory safety policies; • Ability to troubleshoot instrumentation and technical analyses; • Ability to perform preventative and corrective maintenance on basic laboratory equipment and instrumentation; • Ability to recognize when to refer instrumentation problems to the appropriate sources; • Demonstration of professional conduct with patients and health care workers both within and outside the laboratory; • Demonstration of effective interpersonal communication skills

– Two days of General Orientation to the program – One month of theory – Five months of practical

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18

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Course Description: Materials needed for the Program

Course Description: Program Outline (theory) The following areas will be covered in theory: • Bacteriology • Parasitology • Clinical chemistry • Haematology • Blood bank • Specimen collection • Quality control and assurance

• Trainees will receive – Booklet for theory • To include lecture slides • To include materials for classroom activities – Booklet for practice • To include competencies to be met • To include weekly self‐evaluation to be completed at the end of every week

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Course Description: Teaching‐Learning Process

Course Description: Learning Methods

Supporting the Trainee in the teaching – learning placement experience(s) are:  Preceptor  Clinical Supervisor  Course Lecturer  Local Staff Teams

• The unit will be taught through a mixture of lecture and practical sessions. Competencies will be acquired in the following coursework through didactic presentation and laboratory experience. The program is designed to show student progression of knowledge and skill.

Preceptor

Course Lecturer & Classes

Trainee

Clinical Supervisor

Local Staff Teams

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Course Description ‐ Methods of Assessment

Trainee Evaluation in Practice

Trainees will be assessed based on the following breakdown: • 20% theory

The assessment of trainees in the Program against global and domain specific standardized practice competencies will occur in the following ways:

– Assessment tools to include: MCQs

• 70% practical

 Trainee – Self assessment of competency based practice  Trainee – Trainer assessment & feedback of competency based practice  Trainee ‐ Individual Weekly Self‐Evaluation

– Based on the global and domain‐specific competencies – Trainees to complete weekly self‐evaluation (but will not receive a final grade towards weekly self‐evaluation)

• 10% attendance

• Trainees to achieve a weighted average score of 60% 23

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Trainee Assessment & Evaluation – Scope of Practice Competencies & Performance

Competency Grading Scale Self‐Assessment • 0 = No Knowledge and /or experiences (No competency) • 1 = Limited knowledge and /or experiences (Some competency) • 2 = Knowledgeable and feels confident (Complete competency)

Self‐ Assessment

0

1

Competencies (The trainee will be able to )

2

Placement

0

1

Placement

2

0

1

2

Final Summative Assessment • 0 = Not able to perform (No competency) • 1 = Limited ability to perform (Some competency) • 2 = Is able to perform (Complete competency) 25

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Weekly Self‐Evaluation

Trainee Evaluation – Global Practice Performance

Competencies (the trainee will be able to ): A. Professionalism, Ethical Practice & Teamwork Demonstrates a professional, ethical approach to practice & teamwork B. Maintaining Practice & Professional Development Takes responsibility for keeping own knowledge and skills up to date through continuing professional development C. Communication / Therapeutic Communication in Practice Demonstrates effective communication and inter‐personal skills to develop and maintain effective relationships with patients and colleagues D. Workplace Environment & Safety Practises safely and demonstrate understanding of the correct use, limitations and hazards of the environment 28

Trainers in Training Program • For theory training: – Course Lecturer

Roles and Responsibilities ‐ Trainer

• For practical training: – Clinical Supervisor – Preceptor

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Role of the Course Lecturer

Responsibilities of Course Lecturer

• The Course Lecturer member oversees the trainee and is responsible for the delivery of classroom sessions supporting the Training Program for Health Institute Graduates. • The content presented and discussed in the classroom sessions is then integrated by the trainee into practice within the practical placement experience environment.

• • • • • • • •

Provide sufficient information during general orientation to facilitate trainee preparation for the program; Support a constructive teaching / learning process in the classroom setting; Set meetings with the trainee to discuss the progress or learning challenges as needed; Encourage trainees to build on their own experience, personal knowledge and wisdom; Promote professional growth of trainees Support trainees in understanding the provision of safe care in the placement setting; Monitor and mediate interactions and concerns between other staff and trainees if warranted; Assign a classroom session final grade based on the trainee’s performance on class room tests, exams and assigned classroom work for evaluation in the Training Program

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Responsibilities of the Course Lecturer (continued) •

Coordinate with the responsible administrative domain‐specific representative for batch of trainees regarding the following: – Register Trainee in the First Week – Report Attendance ‐ Per Month – Tests – retrieve tests from representative and make necessary copies – Report Test Result per trainee

•

Submit incident report – upon the occurrence of an incident, download incident report and complete. Form to be sent to Ministry of Health General Directorate of Training and Scholarship. The course lecturer is to indicate the level or urgency of the incident

Responsibilities of the Clinical Supervisor •

• • • •

• Extremely urgent – for incidents that are detrimental to the patient and/or the trainee • Urgent – for incidents that cause potential harm to the patient and/or the trainee • Moderate ‐ for incidents that are important and require the immediate attention of the General Directorate of Training and Scholarship, but are not detrimental to the patient and/or trainee • Not urgent – for incidents that are to be brought to the attention of the General Directorate of Training and Scholarship, but do not require immediate attention

•

Overall responsibility for the management and administration of trainees within the clinical environment. Key roles include organizing the rotas to ensure that the learning outcomes are achieved and that adequate supervision is provided by the preceptors Make regular contact with the preceptor Provide support and advice to preceptors Speak to the preceptor and the trainee and review progress, trainee forms and workbooks Signs off on trainee weekly self‐evaluation and provides support to trainee where there are areas for improvement Formative signing off of relevant placement competencies once competency has been achieved

**Trainer reserves the right to suspend trainee, but the ultimate decision to terminate a trainee rests with the General Directorate of Training and Scholarship 33

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Responsibilities of the Clinical Supervisor (continued)

The role of the Preceptor • Orientate the trainee to the department or unit, procedures, reference manuals, and members of the health care team • Collaborate with the trainee’s clinical supervisor about progress throughout the semester; • Assist the trainee to access resources and relevant experiences; • Support the trainee to help increase their level of competence and confidence; • Share verbal and written feedback with both the trainee and clinical supervisor • Contribute (in writing where possible) information for the trainee’s final appraisal; • Immediately report concerns about unsafe practice to both the trainee and clinical supervisor • Review trainee learning goals and selecting appropriate learning experiences from personal assignments to meet the trainee’s learning objectives

• Coordinate with the responsible administrative domain‐specific representative for batch of trainees regarding the following: – Report Attendance ‐ Per Month – Submit trainee competency result

• Submit incident report – upon the occurrence of an incident, download incident report and complete. Form to Indicate the level or urgency of the incident • Extremely urgent – for incidents that are detrimental to the patient and/or the trainee • Urgent – for incidents that cause potential harm to the patient and/or the trainee • Moderate ‐ for incidents that are important and require the immediate attention of the General Directorate of Training and Scholarship, but are not detrimental to the patient and/or trainee • Not urgent – for incidents that are to be brought to the attention of the General Directorate of Training and Scholarship, but do not require immediate attention

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The role of the Preceptor (continued) • Take note of any trainee absences and informing immediate superiors (and clinical supervisor) and Program leads as required • Provide supervision and learning facilitation to the trainee in the practical area • Collaborate with the trainee to ensure learning needs are identified, and that supervision is appropriate to trainee’s knowledge and skill for each situation • Regularly consult with other staff members based on individual needs and the needs of the trainee to obtain feedback on the trainee’s performance • Provide feedback to the trainee through regularly established meeting times to identify new learning needs, and to plan the next day’s activities

Roles and Responsibilities ‐ Trainee

**The role does not involve marking the written assignments that the trainee completes as part of their practice experience or assigning a final grade. 37

Trainee roles and responsibilities

Trainee roles and responsibilities •

• • • • • • • • • • •

Complete all preparatory Training Program – Trainee Placement Prerequisites Review and complete with signature the Training Program agreements Complete all preparatory classroom and activities in a self –directed learning style to enhance successful learning outcomes and safe practice delivery Complete all required evaluation tools, as per domain‐specific requirements Understand that attendance is mandatory Act professional and be responsible at all times Orientate self to the organizational, departmental and practice environment Orientate self to roles and responsibilities of the various health care staff / team members; Orientate self to available human and physical resources Orientate self to care and service delivery and documentation protocols Build on one's own experience, personal knowledge and wisdom

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Know (and operate at all times) under the policies and procedures of the agency and the Training Program for Health Institute Graduates Constantly check the Program website for any announcements Work collaboratively and interdependently with others Be prepared to submit required evidence of your trainee practice & learning Maintain confidentiality Know /acknowledge that all trainee and evaluation data including trainee learning outcomes data belongs to the Training Program and will be used in reports and publications by the Ministry of Health Understand /know the Training Program conditions of trainee grading are based on the trainee successfully completing all Training Program tests, assignments and activities in accordance with prescribed deadlines after which a final grade will be determined based on the weighted distribution Understand / know the final grade weighted distribution Understand /know that the Training Program requires all trainees to achieve a trainee final grade (60%) from the weighted average distribution in order to achieve a grade status of PASS

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Trainee roles and responsibilities (continued) • • • • • • • •

Trainee Prerequisites for Entry to Placement Each trainee will meet the following prerequisites prior to entering the placement site – clinical environment:

Arrive on the unit or department and report to the preceptor for each assigned shift of the practical experience Work under the guidance and supervision of the preceptor and other qualified personnel as necessary Inform the preceptor past experience in relation to learning goals, scope of practice limitations and reliably conveys the amount of guidance and supervision required Seek feedback daily on personal performance and adjusts learning goals and creating remedial plans as necessary Participate as a collaborative team member, documents care, demonstrate knowledge of all unit/facility policies and measures for fire and other emergencies Notify clinical unit or department and preceptor of all absences in advance of a planned clinical shift. Report and document all critical incidents to the trainer in practice immediately (medication errors, procedure or treatment errors, a patient or personal fall or injury, needle stick injuries etc.) Complete the weekly self‐evaluation

Note: All prerequisites will be completed prior to entry to the placement setting and will be completed / signed off to the Ministry of Health by 26/7/1435H:  Trainee to complete the Placement Practice Agreement (PPA) by 12/7/1435H  Trainee Health Examination by Physician – current, completed and deemed “fit and able” to fully participate in writing  Trainee to present confirmation of TB Skin Testing  Identification of Allergies & Workplace Hazard Risks – medication, environmental sensitivities, latex allergy etc. 41

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Program & Professional ‐ Code of Conduct Each Trainee acknowledges and will practice the following elements supporting the values, attitudes and behaviors of the program at all times:

Policies & Procedures

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Respect Trainee Responsibilities for Professional Service Dress Code Integrity & Accountability Confidentiality & Protection of Privacy Teamwork & Collaboration Safety & Competency in Practice Attendance & Punctuality “Academic Integrity” Incident & Injury Reporting 44

Safe & Ethical Care Provision

Trainee Practices & Code of Ethics

• As professionals, trainees are responsible to protect the rights and interests of the patient / client.

• Trainees will reflect moral & ethical standards of practice. • Trainees, as members of a domain specific profession are required to uphold the standards of practice and scope of competencies of practice.

• There is a great deal of trust placed in health professionals, as they are expected to: – – – –

• Trainees – will seek guidance for decision‐making in concerns related to patient and service delivery ethical matters.

Be qualified Provide safe & competent care Respect basic human rights of all individuals Be truthful and act with integrity in their conduct and interactions with others

• The trainees code of ethics supports the primary values of ensuring patient / client health & well being, choice, dignity, confidentiality, fairness, accountability, and the environment of practice and its delivery are conducive to safe, competent and ethical care. 45

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Trainee Domain – Scope of Practice Boundaries

Trainee Role & Practice Liability

 It is the responsibility of each trainee to read, understand and practice within their trainee role and scope of practice competencies

• There may be liability if actions cause harm – the assignment of liability is dependent upon the individual situation

 It is the responsibility of the trainee to read, understand and adhere to all policies and procedures

• Within a preceptor– trainee supervised relationship the liability is usually shared by the trainer in practice, trainee & hospital

 Practice under the supervision and direction of their preceptor

• Trainees are expected to perform as a registered professional – and provide safe patient / client care

 Understand the limitations and boundaries of their role, scope and related practices and seek guidance and clarification if they are unsure ‐ prior to undertaking any task or accepting responsibility for care and services.

• Trainees do not practice outside of role / domain job description and practice scope AND require ongoing supervision. 47

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Dress Code in Placement Setting

Trainee ‐ Personal Hygiene & Infection Control

Trainees are required to comply with the dress code policies and procedures AND all mandatory dress code health and safety requirements (example: PPE – personal protective equipment) to entre and practice in the placement setting.

Trainee Personal Hygiene & Infection Prevention • Restrain hair – keeps hair from falling forward in field of vision, hair may be a vehicle to transport, carry or drop microorganisms especially if aseptic / sterile technique is required • Keep fingernails short • Cover any open wounds with an occlusive dressing

 Domain‐specific uniform may be required and if a trainee is no appropriately attired or is unkempt, the trainee may be refused admission to the agency  Footwear – designated by site & domain specific work, and occupational safety requirements. *Note: all footwear must be closed toed  Photo – ID name badges must be worn at all times  Adhere to scent‐free environment request  Adhere to all health & safety and infection control dress code requirements

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Trainee – Hand Hygiene Practices

Placement Site & Practice Delivery – Safety

When should hands be washed? • When visibly soiled • Before and after client contact • After contact with source of microorganisms (blood, body fluids, mucus membranes, non intact skin or inanimate objects that might be contaminated • Prior to performance of invasive procedures (IV catheters, indwelling catheters) • Before and after removing gloves (wearing gloves does not remove the need to wash hands) • At the beginning and end of every shift

All Trainees must read, review and are responsible for knowing the following practice & workplace safety requirements:  Confidentiality & privacy of personal patient and placement organization information, practices, policies & procedures;  Documentation standards, policies & procedures;  Workplace Occupational Health & Safety requirements, policies & procedures program and placement site specific policies;  Environmental and Workplace Hazards – practices, policies & procedures, and guidelines. 51

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Placement Site & Practice Delivery – Safety

Handbook Review & Preparing for Placement Trainees will complete the following:

All Trainees will read, review and are responsible for knowing the following practice & workplace safety requirements:

 Review of Training Program ‐ Handbook  Complete all prerequisites for entry into the placement setting  Complete and sign off of all required forms / acknowledgement agreements  Read, understand and comply with all policies and procedures  Read, understand and comply with the code of conduct and your scope of practice Trainee role  Practice with safety in mind, know your boundaries or limitations in knowledge and scope  Comply with dress code and all safety regulations

 Infection Prevention and Control practices, policies & procedures and guidelines;  Emergency Situations, Hazardous Threats, Fire Safety and Security related practices, policies & procedures  Personal Protective Equipment & Hand Hygiene practices, policies & procedures  Any specific domain related safety and quality assurance elements that trainees need to be aware of / or needs to practice in the domain specific department or in the delivery of their specific practice role  Others specific to the Placement Site but not noted here. 53

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Trainee Evaluation in Program

Trainee Evaluation Requirements & Data

The Training Program for Health Institute Graduates – Trainee evaluation will allow for the assignment of a final grade.

As a condition of understanding for entry into the Training Program for Health Institute Graduates:

The trainee’s final grade at completion of the program will be determined on the following basis:

• Every trainee is required to complete all Training Program ~ Trainee Evaluation and Program Evaluation requirements and Pre / Post Program Feedback Surveys as requested by the Training Program and Ministry of Health

Every Training Program trainee is aware and understands that all Training Program conditions for determining a trainee final grade are based on and subject to the trainee successfully completing all Training Program tests, assignments and activities in accordance with prescribed deadlines and reflective of academic integrity requirements of the program.

AND • Every trainee is aware and acknowledges his understanding that all Trainee and Program Evaluation data including trainee’s learning outcome data is the property of the Training Program and may be used in reports and publications as determined by the Ministry of Health. 55

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Tests and Exams: absences • • • •

Policy Guidelines for Trainee’s Scholarly Work •

Attendance is compulsory for all scheduled tests and examinations. Written documentation IS REQUIRED to support claims of extenuating circumstances, i.e., illness (Medical Certificate must be provided) or death of a family member. A trainee missing any type of evaluative method must contact their trainer prior to the test/exam/assignment or evaluation session due date and time and clarify the reason for absence. Failure to notify the trainer in advance will result in a grade of zero for that evaluative method.

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**Alternate make‐up activities could be negotiated and contracted with the instructor. If the reasons for missed test/exam/ assignment/evaluation are in keeping with expectations of the Training Program policy AND all appropriate documentation is provided as stated above, an instructor may choose to offer a trainee an alternative due date and time for the test/ exam / assignment or evaluation if the trainee meets the criteria warranting an alternative time & date to complete the evaluative method.

• It is the trainee’s responsibility to follow up with the trainer regarding missed, late or absent assignments, tests or exams. The trainer in consultation with the Ministry of Health have the right and obligation to make the final decision about course evaluation activities, noting that certain policies may need to be upheld in support of fairness to all trainees in the program.

Trainees are responsible for promoting academic integrity within their training program and the development of their scholarly work. If trainees feel the faculty have made a mistake in computing the assigned grade, trainees have a responsibility to come to faculty as soon as possible prepared to provide a rational re why they feel a mistake has been made. Academic dishonesty includes any misrepresentation of any part or whole of the process or product in preparation of academic scholarly work Plagiarism specifically can be understood as, the act of copying, reproducing, or paraphrasing significant portions of someone else’s published or unpublished material and representing someone else’s thinking as one’s own thinking by not acknowledging the appropriate source or by the failure to use appropriate quotation marks. Plagiarism is one of many forms of academic misconduct (such as cheating, misrepresentation, and submission of false information) that are described in the Code of Academic Conduct. The minimum penalty is a mark of “0” for the work. Trainees may fail the course or be expelled from the program (as per the discretion of the General Directorate of Training and Scholarship)

Please be advised: Records of absence from tests/examinations may be kept in trainee files 57

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Statement of Agreement / Acknowledgement Each trainee will read, understand and agrees to comply with:  Training Program for Health Institute Graduates – Trainee Placement Practice Agreement (PPA) (Form 1)  Training Program for Health Institute Graduates ‐ Trainee Confidentiality Statement of Understanding & Agreement (Form 2)  Code of Conduct Policy  Placement Site specific Trainee Confidentiality Acknowledgement Form (if applicable) * Note: This is mandatory to enter the placement site

Important Program Dates

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Program – General Timetable & Key Dates Training Program for Health Institute Graduates – General Timetable & Key Dates Program Begins

2/7/1435H English Language Study Course ends 12/7/1435H Day 1 – Training Program for Health Institute Graduates – General Orientation 13/7/1435H Day 2 – Training Program for Health Institute Graduates – General Orientation

Eid Al Fitr

25/9/1435H – 4/10/1435H Eid Al Fitr Holiday No classes

National Day

28/11/1435H National Day Holiday No classes

Eid Al Adha 2/12/1435H – 17/12/1435H Eid Al Fitr Holiday No classes Program Ends

6/1/1436H Training Program for Health Institute Graduates ends

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General Orientation Trainer

Communication Collaboration & Teamwork Delegation in Health Care Setting Slide 5 - Communication & Therapeutic Interaction What does communication in the health care setting mean to you ? [ Lecturer to explore this by asking trainees in the classroom setting the above question and inviting a brief dialogue ] Following the short dialogue with trainees … continue the presentation by sharing the following: Health care workers utilize appropriate communication skills and personal interactions, in order to effectively build and maintain relationships, and to facilitate optimal health care. Relationships may be with patient / clients, families, or other health care professionals, for example. Each relationship can contribute something important to health and health decision making, therefore learning about communication and therapeutic interaction is an important topic to review today. - Slide 6 - Communication and Human Interaction Communication in some form allows expression and supports all human interaction  Verbal and non verbal (Verbal communication refers to spoken language. Non verbal communication may include facial expressions, posture, eye contact, body language, tone of voice, displayed emotion, etc., and often provides valuable information, for both conscious and unconscious individuals)  Written and unwritten (Written information records and communicates important information, that may be vital to care, for example. Unwritten information can include utilizing picture tools, or using computer technology, for example.) - Slide 7 - What other Factors Influence Communication ? Several factors may influence the process of communication. The Transactional Model of Communication is depicted here. This model shows: 1) The communicators – There is the person who is sending outgoing information, and the person who is receiving incoming information. 2) The message or messages – This is the information being transmitted. Remember, this information may be verbal or non verbal information, written and unwritten. 3) Noise – This can be anything that interferes with effective communication. This could include other conversations, phones ringing, or being called away to attend to something else. “Transactional” refers to the fact that communication is a continuous and ongoing process. Additionally, there are a variety of people with whom we are communicating with. It is important to remember that our personal beliefs, experiences, attitudes, (and other factors) impact the way we send information and also impact the way we interpret incoming information. This can be helpful at times, but it may also impair communication or lead to misinterpretation of information, for example. -

Communication is considered developmental in the experience of human interaction. As such recognizing or having a common language is an important aspect of our communication. When we speak or have a different language from one another, it can make it difficult to establish communication with one another. If you think back about your own development or the development of a baby to a child to a young adult … you may recall some developmental changes in the way the individual communicated with their environment or with people around them.

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Thus, how a baby communicates is different than that of a toddler or young child. Each is progressing in their development of communication and language. This development will be influenced by their own brain and body’s functioning ability but also by the influences of their environment. Slide 9 - Communication Process – Influencing Factors When we think about the communication process … There are many influencing factors that can impact what we send and receive as messages in the communication process. These may include: (As above - read slide) Slide 10 - Communication in Health Care Setting In the health care setting communication is essential in every aspect of the care we provide and the meaning it has to our patients and families Therefore, being able to support effective communication is of vital importance to our work and care delivery in the health care setting. As effective communication may be essential to: (As above - read slide) Slide 11 - The Communication Process This slide depicts the communication process as a 2-way dialogue between the sender and receiver. Understanding how the communication process works is necessary to building effective communication skills and abilities as a health care provider. This includes your ability to identify and focus on understanding the other factors that may influence the communication process or interactions as we engage with others. This includes understanding the way an incoming message is interpreted/or decoded. The Sender transmits information, in verbal and non verbal ways, each having the ability to influence the way the message will be received and interpreted. The Receiver (or listener) decodes the verbal and non verbal information together, and in doing so, interprets the message. Once a message is decoded, feedback occurs (a response), which is once again communicated in verbal and non verbal ways. This can be an ongoing process, providing both/all people are open to the communication. Many other factors can influence the communication process. Our interpersonal variables, such as personal experiences and attitudes, affect what information is sent and how it is received. Additionally, environmental factors, such as a noisy environment, a lack of privacy, etc., may also have an impact on the effectiveness of sending and receiving the communication. Slide 12 - Communication & Gender There can be communication differences related to ones gender. (As above - read slide) The differences may be due to growth and development variations by gender OR they may be differences due to one’s environment while growing up (such as differences in social, cultural diversity or even educational / learning opportunity differences). Therefore, how one communicates can be influenced by the norms, values and behaviors which they have learned from their life experiences. As a result, improving one’s communication and its effectiveness can also be learned or improved at any time in the life span. Slide 13 - The Meaning in Communication Notice the differences between left and right sides of the brain. The left side - we see logical thinking and analysis, a fondness for knowing facts, strength in mathematics, and areas involved in understanding language and speaking.

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We may keep this in mind, because it helps us understand differences in ways of thinking and ways of communicating. It also helps us to understand the variation in communication and the interpretation of meaning from communication we may notice in others like our patients. It is also worthy of noting: The left side of the brain “controls” the right side of the body and the right side of the body “controls” the left side of the body. For example: damage to the left side of the brain may affect the patient’s ability to speak, or understand language. Or it may cause weakness or paralysis of the right side of the body. Slide 14 - Right Brain – Left Brain & Communication The important message here is the following: It is important to appreciate how differences in thinking, perception and therefore the influence of the brain create meaning in the communication process and how this may vary from person to person ~ It can even offer some explanation of the differences in how people receive and interpret information in the communication process differently. As a result of these differences – it may impact your relationships with other people including those you work with. This is being presented to you to help you understand one possible explanation for the “diversity in the workplace” we sometime experience as a result of communication. Slide 15 - Influences - Values, Perceptions & Behaviors Our values, perceptions, and behaviors also influence the communication processes, by way of how information is transmitted, and by how information is interpreted/perceived. (As above - read slide) It is important to be mindful of each of these influences, and how they may impact things like communication, health, decision making, and delivery of health care. These differences may also impact your relationships with other people including those you work with. This is being presented to you to help you understand one possible explanation for the “diversity” in the workplace that you may experience as a result of communication. Slide 16 - Types of Communication in Health Care There are different types or ways that communication can occur in the health care setting. Verbal communication is one type. (As above - read slide) It is important in verbal communication to validate that the messages are being received and interpreted accurately in the 2- way dialogue as a shared interaction. Slide 17 - Types of Communication in Health Care Another type or way that communication can occur in the health care setting is written communication. (As above - read slide) As health care providers you will also be engaged in the written communication process through your documentation of the patient’s shared information and messages that were being shared with you. Slide 18 - Types of Communication in Health Care Another type or way that communication can occur in the health care setting is non verbal communication. (As above - read slide) This can occur when the patient can not or is unable to speak verbally. Keep in mind a patient may be able to communicate in other ways (eye contact, facial expressions, gestures, writing, use of pictures etc.)

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Kingdom of Saudi Arabia – Ministry of Health General Directorate of Training and Scholarship Training Program for Health Institute Graduates -

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In non verbal communication it can be important to try and confirm or validate your interpretation of messaging when ever possible … as not every thing may have the same cultural significance or is universally understood. Slide 19 - Types of Communication in Health Care Another type or way that communication can occur in the health care setting is through electronic and digital communication. (As above - read slide) The use of electronic and digital communication is often guided or controlled by strict policies and procedures to protect the privacy and confidentiality of information, who gets to access it and where and who can share it. Please refer to your placement organization’s policies regarding your use of all electronic and digital communication. Slide 20 - Personal Space & Communication In understanding the communication process, it is important to recognize the influence of one’s personal space as measured between the sender and receiver during the communication episode. Personal space can actually change the perception of how the message is received and interpreted by the receiver. (As above - read slide) You will want to keep this in mind when dealing with patients and other health care providers in the health care setting … as the measure of personal space can influence how others perceive your messages and the “appropriateness” of the message and any corresponding actions that may follow. The greater the congruency between what we say and how say it – the clearer and more accurate the message by the receiver. When congruency does not exists then misinterpretation and misunderstanding results in the communication process. The strength of the communication process for use in the workplace is essential in health care settings. Misinterpretation and misunderstandings can have very negative effects on collaboration, teamwork, productivity and patient care outcomes. Slide 21 - Therapeutic Communication Therapeutic communication simply refers to the way a health care provider communicates and interacts with their patient and the patient’s family. Therapeutic communication occurs in such a way that a trusting and respectful professional relationship can be established. Communicating and interacting in a compassionate, kind, confidential, and authentically caring way, can help health care workers develop this relationship, which is essential if patients are going to communicate their concerns and needs with us. It is essential if we are also going to be able to develop a health care plan with the patient, and establish how best to meet his/her health needs. Establishing a therapeutic relationship may also help to create an optimal healing environment for the patient. Slide 22 - Patient – First (Person Centred Care) Patient First = is also known as person centred care In health care delivery settings, the patient is our priority. They are at the center of their own care and within an ethical practice care delivery setting we are at all times advocating this for the patient and their care. This means that we need to get to know the patient. This sometimes also means we will get to know the patient’s family or others who are important in their lives. As health care providers, we need to gather information about what is important to the patient, what their goals and hopes are for their health situation. Where do they live? Do they

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live alone? Do they have friends and family nearby, who can help them with their physical, emotional, or other health related needs? We need to be compassionate, ask the right questions, show concern and respect, and really listen to the information that the patient wishes to share with us. This information will help ensure that an appropriate health care plan can be developed with the patient and help ensure that the patient’s health needs can be best met. Slide 23 - Characteristics of Therapeutic Relationship There are several important characteristics you will need to consider in establishing or building the therapeutic relationship with a patient. Slide 24 - Communication & Patient Documentation Another important component of communication and the patient care process is reflected in documentation … as in the patient’s chart or health record. Slide 25 - Types of Documentation There can be several different types of documentation that occur specific to the patient’s care process and the health care provider’s roles and responsibilities. (As above - read slide) You are responsible for and will need to become familiar with the policies and procedures for documentation in your trainee role while in the placement setting. Slide 26 - Do’s & Don’ts of Documentation Please keep in mind the following general aspects of documentation in the health care setting: (As above - read slide)

Slide 27 - Communication & Collaborative Practice Communication is important in building a therapeutic rapport with a patient and the documentation of our communication is important in the ongoing sharing the needs, wishes, goals and progress of the patient’s care as a process. Communication and documentation are also critically important in our work delivery (as care or services) both as and with other health care providers in the hospital or health care setting. (As above - read slide) Thus, our ability to communicate with and work with others on the health care team toward the patient’s goals – which we often refer to as collaborative care or collaborative practice can be an important aspect of success as a registered technician in the health care setting. In concluding this section of the presentation: Hopefully, you have gained greater understanding of the concepts of communication and therapeutic communication for use within the health care setting. As you continue your experience in the Entry to Practice Training Program – we invite you to continue building your abilities to demonstrate and exhibit a strong therapeutic communication style of behavior --- as this will be a very important component to your ongoing success during the placement experience and as you continue your career path in health care. Slide 30 - Collaboration & Teamwork During the next part of this lecture session on collaboration and teamwork, our learning objective and outcomes will include building an: Understanding of who is on your “team” Increased understanding of the benefits/barriers to collaboration and teamwork And Understanding of delegation and how this may occur in the delivery of work in the health care setting. Slide 31 - Communication & Collaborative Practice In our previous presentation we talked about the importance of communication and collaborative practice in the health care setting. 25


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To begin our next session, let us begin by reviewing some of those definitions of the key terminology … (As above - read slide) Slide 32 - What does Teamwork Mean to You ? (As above - read slide) Now lets take a moment and consider … what teamwork means to you ? So … in reflecting on this photograph … what does it say to you about teamwork ? How does it fit with your own beliefs about teamwork ? [With gentle encouragement … invite the trainees to share their thoughts] To summarize … Sometimes things can be easier to achieve when we have help. Thinking of your own life … we all may have certain responsibilities. Our parents, our children, our spouse … but when we distribute these responsibilities, with each person having their own duties to perform, we are acting like a team, and as a result the entire family and household may function properly because of this sharing. This is like teamwork. Teamwork … is necessary if we are to make health care function properly for the sake of the patient. Slide 33 - The Patient’s Healthcare Team (As above - read slide) [Invite the trainees to share their ideas] Slide 34 - Patient’s Interprofessional Healthcare Team (As above - read slide) The patient’s interprofessional health care team can include the following … There may also be others not identified here who will or can also be invited to assist in the patient’s care or its planning. Slide 35 - Why is teamwork - as the health care team important? (As above - read slide) So … why is teamwork as a health care team important ? [Invite the trainees to share their ideas] Can anyone think of an example to share with the group? [Invite the trainees to share their ideas] Perhaps drawing from personal experience? [Invite the trainees to share their ideas from the perspective of the registered technician] Slide 36 - Importance of Teamwork There are several important aspects to consider when thinking about teamwork in the health care setting. (As above - read slide) Slide 37 - Benefits of Teamwork Approach There are also some important benefits in using a “teamwork” approach which you should keep in mind as you develop your role in the health care setting. (As above - read slide) Slide 38 - Barriers of Working on a Team There Role boundaries and limits can create barriers – therefore team members must have clearly defined roles and responsibilities, which are clearly communicated to all team / staff members. Some overlap may occur, but major responsibilities are designated to appropriately qualified professionals. Expressing your needs and views - Every team member needs to feel comfortable in expressing valid concerns, needs, information, and views, and each team member needs to have the opportunity to do so.

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Teams can be too flexible and avoid decisions – Often teams can try to be flexible or agreeable, instead of contributing important information, and this can hinder team effectiveness and efficiency. Power and decision making imbalance – when an unequal or unfair distribution of power and decision making abilities, this can affect team sharing/contributions and team decision making. Workload imbalances - can lead to anger, frustration, conflict, missed deadlines, as well as physical and mental fatigue and even illness can occur which can all negatively impact team functioning. Disagreements and conflicts may occur, therefore utilizing appropriate conflict resolution techniques are essential to handle conflict and support ongoing healthy team functioning. Slide 39 - What do you Share with the Team? As a team member … it is important to understand what it means to be a fellow professional and what may or may not be appropriate and ethical to share within teams. Generally, the following three areas of focus are a good guide to ensure your communication of information on teams is appropriate. (As above - read slide) Slide 40 - Benefits of Teamwork in Health Care In summary, this has been a brief overview of collaboration and teamwork … to assist you in the health care setting. The benefits of teamwork are significant in this type of workplace environment. Teamwork is also a critical aspect of the workplace values and workplace culture of the hospital or health care setting. The benefits of teamwork can … (As above - read slide) Your developing and being open to a strong collaborative - teamwork style of behavior will be an important aspect of your success during this placement and learning experience. In concluding this section of the presentation: Hopefully, you have gained greater understanding of the concepts of collaboration and teamwork for use within the health care setting. As you continue your experience in the Entry to Practice Training Program – we invite you to continue building your abilities to demonstrate and exhibit a strong collaboration and teamwork style of behaviors --- as they will be very important components in your ongoing success during the placement experience and as you continue your career path in health care. Slide 42 - Delegation & Technician Practice In this next lecture session, we hope to learn about and understand the following objective: To review the process of delegation and the benefits and barriers to its use for teamwork and practice delivery in the health care setting. Slide 43 - Delegation of Work Tasks The importance of delegation in work tasks is vital to the success of work in health care settings. More specifically, (As above - read slide) Slide 45 - Understanding the Terminology It is important to have a common understanding of some key terms as it relates to delegation in the workplace. (As above - read slide) Slide 54 - Supervision & Reporting on Teams It is important that as a trainee in the role of a registered technician that you recognize that supervision and reporting is an expectation of all staff who work on or with teams in the health care setting.

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Within your trainee role you will need to identify and clarify these expectations with your preceptor / supervisor and clinical instructor especially as it relates specifically to your unique placement setting Thus … (As above - read slide) Slide 59 - Teamwork Choices & Decisions In team work and delegation, there are reasons why an individual may decline the delegation of a task. As a trainee, it is important that understand the limits of your role and practice and under what conditions you should decline the delegation of a task or responsibility. Slide 61 - Quality of Care Impact Participating in teamwork and more specifically the delegation process which supports patient care and services provision in the health care setting - is an important concept that you as trainees in the placement setting need to understand. Hopefully, you have gained a greater understanding of the concept of delegation and appreciate its impact on care and service delivery, collaborative practice and teamwork as used in the health care setting. You will need as a trainee, to understand more specifically how this is done in your placement and departmental setting. As you continue your experience in the Entry to Practice Training Program – we invite you to continue building your abilities to demonstrate and exhibit a strong response to participate in roles of delegation as a work style behavior --This is important --- as it will be a very important component to your ongoing success during the placement experience and as you continue your career path in health care as a registered technician.

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Ministry of Health Kingdom of Saudi Arabia


Training Program for Health Institute Graduates Collaboration, Communication & Teamwork in Health Care

Day 2 – Communication, Collaboration and Teamwork in Health Care

Lecture Outline 1) Introduce the concepts of communication and therapeutic communication in the health care setting 2) To reflect and review the importance of teamwork and collaboration in care and service delivery to patients 3) To review the process of delegation and the benefits and barriers to its use for teamwork and practice delivery

Training Program for Health Institute Graduates Communication & Therapeutic Interactions

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Communication & Therapeutic Interaction

Communication and Human Interaction What is communication & why is it important ?  Allows the exchange of information  Permits thoughts and ideas to be shared  Conveys feelings and desires  Encourages questions and exploration of new things  Promotes diversity  Communication in some form allows expression and supports all human interaction Verbal and non verbal Written and unwritten

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Language & Communication Development

What other Factors Influence Communication ?

• Language and communication skills develop through stages across a lifespan • How a person communicates as Newborn – Infant – Toddler – Young Child – Adolescent – Adult – Older Adult changes • Language and communication can be expressed through movement, play, drawing , reading, writing, story telling , facial expressions / body language and sound / vocalization / speech 7

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Communication in Health Care Setting

Communication Process – Influencing Factors • • • • • • • •

Effective communication is essential to:

Attitudes and biases Values and perceptions Roles and relationships Environment (level of noise, personal space proximity) Gender Life stages – growth & development, emotional state Social / Cultural characteristics Proximity – personal space / territoriality

 Understand and identify and the patient’s health status understand and identify illness /disease based needs  Provide all aspects of safe, patient – centered care  Collaborate with others in the delivery of care and services  Creating meaning and learning through shared experiences 9

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The Communication Process

Communication & Gender

Sender – as source encoder

Communication differences are noted between males and females from an early age:

Message – what is actually said / written, body language, how words are transmitted – or channelled

• Boys – learn to establish independence and negotiate status • Girls – seek confirmation, belonging and acceptance

Receiver – is listener – decoder – perception of intention

• Challenges to successful communication, can arise from cultural, personality, age, and gender differences. If you can recognize them in the interaction, you may be better able to deal with them ~ in the process of communication.

Response – Feedback 11

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The Meaning in Communication • •

Right Brain – Left Brain & Communication • Research suggests, left‐brained subjects focus on logical thinking, analysis, and accuracy.

Good communication is the key to success in just about everything in life. Understanding how the two hemispheres of the brain are responsible for different ways of thinking can help. People use both hemispheres, but some have a distinct preference for one over the other.

• Right‐brained subjects focus on aesthetics, feeling, and creativity. Right‐brained people can remember details better if they have a story quality. • Thus, appreciating how differences in thinking, perception and therefore the influence of the brain creating meaning in the communication process ~ could impact your relationships with other people including those you work with. 13

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Influences ‐ Values, Perceptions & Behaviors

Types of Communication in Health Care

 Values – standards that influence behaviors  Perceptions – personal views of an event  Unique personality traits and experiences  Validation of previous experiences  Role modeling the behaviors of others

• Verbal Communication:

 Sociocultural characteristics such as culture, education and socio economic level can impact development of language and the communication process 15

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Use of common language, requires comprehension and receivers perception / value judgement of information shared for understanding of its meaning in the specific situational context

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Can range from sharing of simple to complex thought and ideas

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A person’s level of literacy (knowledge and ability to understand the spoken and written word in that language) and the ability to actually understand the language being used will influence their ability to process the message and understand the information or data shared

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Pace, intonation of voice, clarity of speech , noise level in the environment, “body language”, perceived congruence of the information (verbal and nonverbal messages match), perceived credibility of the speaker etc. – Can all impact what and how information is heard and interpreted by the receiver

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Types of Communication in Health Care Effective Written Communication: (legible) ‐ Can be similar to verbal communication, but it does not convey nonverbal cues ‐ It typically depicts appropriate use of language and terminology (impacts credibility / “believability” of information) ‐ It typically depicts logical organization of thoughts / ideas ‐ It can reflect a more permanent recording of events or interactions 17

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Types of Communication in Health Care

Personal Space & Communication

Electronic and Digital Communication:

Personal space is defined as the distance people prefer in interactions with others

‐ Electronic communication offers advantages due to speed of transmission, efficiency and legibility however it has disadvantages – as it compromises patient / client confidentiality , may not comply with policy guidelines and may not be accessible to everyone due to variation in socioeconomic status

• Intimate distance – often see in direct care • Personal distance – less overwhelming • Social distance – increased eye contact & decreased touch • Public distance – farther away

‐ Trainee need to recognize agency specific guidelines and will need consent to use electronic communication related to sharing of patient information and care. 19

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Therapeutic Communication

Patient – First (Person Centred Care)

• At the core of health care and services are therapeutic relationships based on caring, mutual respect and dignity. • Communication is the way that these helping – healing relationships are established and reflects an interactive process between the health care provider and patient / client • Therapeutic communication between health care providers and patients ‐ empowers and enables the patient to know themselves and make choices for themselves especially in times of stress

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Characteristics of Therapeutic Relationship • • • • • • • • •

Communication & Patient Documentation

Focus on the patient / client, build rapport Respect the patient as an individual Respect patient confidentiality Focus on patient / client well‐being Based on mutual trust, respect and acceptance Is supported by empathy, listening and uses silence Is non judgemental and avoids personal information Educate and empower the patient / client Maintain truth telling

• Communication related to documentation in the patient record or chart is vital to the care delivery process • Communication as documentation can reflect discussion (teams) recording (charting of care and related health / illness information) and formally documenting (as legal documents) • Only those formally engaged in the care of the patient / client have access to the patient’s record or chart 24

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Do’s & Don’ts of Documentation

Types of Documentation • Source oriented record (examples – admission sheet, vital signs graphic record, MAR, diagnostic reports etc)

Do’s

Don’ts

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      

Chart changes in condition of patient Be timely, read prior notes Be objective & factual Properly correct errors Write legibly and neatly Date & time all entries Adhere to professional standards Follow agency policy and procedures, ethical guidelines  Treat as legal document

• Problem‐oriented record where the documentation is arranged according to the patients’ problems which can encourage collaboration and alert caregivers to patient’s needs • Charting by exception and reflecting standards of care with the aid of bedside flow sheets and forms • Trainees will need to review documentation policies and procedures in their placement setting for adoption and their compliance in care and service delivery requirements

Do not leave blank spaces Do not chart in advance Do not use vague terms Do not chart for others Never alter the record Do not record assumptions Do not remove pages or destroy any part of the patient record / chart  If chart is electronic – do not leave computer terminal unattended  Do not share personal password or security codes

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Communication & Collaborative Practice

Questions / Discussion

• Collaboration is a way of working together that recognizes collective responsibility and the need for interdependency of relationships to achieve outcomes • Collaborative practice is defined as working toward mutually identified goals while valuing different perspectives and accountabilities of team members • Communication skills are used to gather, analyze and transmit the information and to accomplish the “goal work” at each step in the process • Good communication and collaborative practices can impact a healthy teamwork environment and delivery of patient care quality (Potter & Perry, 2009)

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Collaboration & Teamwork Learning Outcomes:


• Understand who is on your “team” • Increase understanding of the benefits/barriers • Understand delegation

Collaboration & Teamwork

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Communication & Collaborative Practice

What does Teamwork Mean to You ?

• Collaboration is a way of working together that recognizes collective responsibility and the need for interdependency of relationships to achieve outcomes • Collaborative practice is defined as working toward mutually identified goals while valuing different perspectives and accountabilities of team members • Communication skills are used to gather, analyze and transmit the information and to accomplish the “goal work” at each step in the process • Good communication and collaborative practices can impact a healthy teamwork environment and delivery of patient care quality (Perry & Potter, 2009) 31

The Patient’s Healthcare Team

Patient’s Interprofessional Healthcare Team • Patient/client • Family/significant others

Who makes up the patient’s Healthcare Team?

• Physician • Specialists Registered Staff • Registered Nurses • Physiotherapists • Occupational therapist • Registered Technician – Domain Specific • Others

Why is teamwork ‐ as the health care team important?

Importance of Teamwork • Provides “patient first” holistic approach to care • Provides a consistent approach in supporting the patient’s health goals / outcomes • Promotes all aspects of patients life – – – – –

Physical Emotional Social Intellectual Spiritual

• We depend on each other to perform roles and communicate effectively with each other

Registered Technician

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Benefits of Teamwork Approach

Barriers of Working on a Team • • • • • •

Role boundaries and limits Expressing your needs and views Being too flexible and avoiding decisions Power and decision making imbalance Workload imbalances Handling conflict

What do you Share with the Team?

Benefits of Teamwork in Health Care

• What was done for the patient • What needs to be done for the patient • How the patient is responding to care & treatment

• • • • • • •

Supports collaboration Supports communication Assist with learning & knowledge building Can improve decision making Can improve problem solving Distribution of work can be cooperatively shared Assists with access to expertise as needed in care and services delivery


Training Program for Health Institute Graduates Delegation & Technician Practice

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Delegation of Work Tasks

Delegation – Defined

• Some professional disciplines that are registered often exhibit a scope of practice which provides them the authority to delegate a task (physicians, registered nurses, radiologists, pharmacists, medical laboratory directors, etc)

• The process by which responsibility and authority are transferred to another individual • Transferring the authority to perform a controlled act procedure to a person • Delegation is a mutual transfer of responsibility & authority that occurs on the basis of competence & trust

• When a task is delegated do not be offended or angry if you are not allowed to perform a task that you usually do

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Understanding the Terminology

Principles of Delegation in Practice

Responsibility • An obligation to accomplish a task

1. You can only delegate those tasks for which you are responsible 2. You must transfer responsibility and authority for the performance of an activity 3. You must know the task to be delegated 4. You must keep in mind the goal is to accomplish the care or work safely 5. Registered staff may remain “accountable” for the outcome

Accountability • The act of accepting ownership for the results or lack thereof • Delegation is a learnable skill and involves good communication techniques, the spirit of teamwork and understanding of the principles of delegation 45

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Benefits of Delegation

The Delegation Process

Benefits to the Delegator

1. Define the task • What can and can’t be delegated? • You can only delegate what you have responsibility and authority over • You can only delegate effectively what you know best • Need to define the aspects of the task

       

Time & efficiency Development of new skills and abilities Increase self‐esteem & confidence Job satisfaction Increase teamwork Increase goals and objective outcomes Increase the quality of care Decrease absenteeism and overtime 47

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2. Determine who to delegate to • What skill and / or qualifications is required • Scope of practice • Who is available • Who is willing to accept the delegation

3. Clearly communicate and describe expectations • Clear and complete communication of expectations • Describe the task • Provide rationale for the task • Describe the outcome expected • Validate understanding of the person being delegated the task • Be clear and reasonable about time frames

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4. Mutual agreement • Seek agreement that he is willing to accept responsibility and authority

5. Monitor performance and give feedback • Assessment and follow up evaluation • Be accessible • Give support • Praise and give recognition • Be public about accomplishments

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Trainee ‐ Accepting Delegation

Supervision & Reporting on Teams

• Have a clear understanding of what is being asked of you • Be realistic and examine whether you have the skills and abilities for the task • Do you have the time? If you do not be honest! • Ask questions and seek clarification if uncertain

Registered Technicians are responsible to the patient / client and co‐workers in their service provision Trainees will be accountable to their trainer in practice in the placement setting however they will work collaboratively with other Team members in the practice setting As an employee the Registered Technician is accountable to their “supervisor” and also within the “chain of reporting / decision making command “ to:

Five Rights (5) of Delegation: Right task, right circumstances, right person, right direction & communication, right supervision & evaluation .

 Department Head / Department Manager who may report to a more senior executive leader 53

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Common Environmental Obstacles to Delegation • • • • • • •

Obstacles to Delegation – Delegator  Lack of trust & confidence  Believe others incapable  Believe self indispensable  Fear of competition  Fear of criticism  Fear of liability  Fear of blame

Compliance with Standards Job Descriptions which limit roles & responsibilities Organizational policies which limit roles Organizational structures which limit roles Management styles Challenging norms of practice Resource limitations

 Fear of loss of control  Fear of overburdening  Fear of decreased job satisfaction  Insecurity  Inexperience in delegation  Inadequate organizational skills

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Obstacles to Delegation by Delegatee

Why Delegation can be Ineffective

• • • • •

Under Delegation  Major reason for under delegation is the need for control and perfection  It occurs when full authority is not given  Delegator fails to provide proper equipment or instruction

Inexperience Fear of failure and reprisal Lack of confidence Overdependence on others Avoidance of responsibility

Reverse Delegation  Delegator takes back responsibility Over Delegation  Too much authority and/or responsibility is given 57

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Teamwork Choices & Decisions

Trainee – In Delegation what do I “report”

Trainees have right to say no to a delegated task when:

• Completion of the task / responsibility, any issues or concerns in delivering the task, anything that is “different” from what was expected to happen

Beyond the practice scope or limits of your role Not prepared to perform the task safely Could harm the person Condition of patient has changed You do not know how to use the supplies or equipment in the task • Do not ignore a delegated act • Must communicate concerns to delegator • Must have sound reason for your refusal • • • • •

• Report any changes in the patient’s presentation, their abilities or presenting signs / symptoms • Need to develop trust & rapport with the staff member delegator which emerges from you responsibility for practice task and reliability of the information reported

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Quality of Care Impact


• By participating as a collaborative team member, who values and embraces the spirit of teamwork as well as, communicates clearly his scope of practice roles and responsibilities ‐ a Trainee can have a significant impact on quality patient care and services delivery.

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References Potter, P.A., & Perry, A. G. (2009). Canadian fundamentals of Nursing (4th ed.). Toronto, Canada: Elsevier Canada.

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Infection Control in Health Care Setting -

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Slide 3 - Trainee Orientation to Infection Control Every health care worker has the responsibility to provide safe health care. This responsibility includes knowledge of how to keep themselves safe from infection, and knowledge of how to prevent the spread of infection to patients. Health care workers need to: Recognize patient and health care staff safety as an important responsibility in practice delivery and build awareness of the role(s) of infection prevention and infection control protocol systems. Apply required knowledge in preventing and/or minimizing infection in daily practice. Perform appropriate behaviors required to prevent health care associated infections. Demonstrate required competence to provide patients with safe care and reduce / protect others from the risk of hospital acquired infections. Slide 5 - Infection Control There is an important need to control the spread of infection. There may be many ways to help control the spread, and each strategy plays an important part in infection control. Each strategy can also protect both healthcare workers and patients, as well as everyone else that each of us are near to or may come in contact with. Thus, infection prevention and infection control is an important practice in the hospital – health care setting given the high risk of infectious – illness and disease type agents that may be found in the setting. Infection control typically, Includes all of the practices used to prevent the spread of microorganisms that could cause disease in a person, and Infection control practices help to protect clients and healthcare providers and families and visitors from disease by reducing and/or eliminating sources of infection. Slide 6 - What are Nosocomial Infections ? What are nosocomial infections? Nosocomial infections are also known as hospital acquired infections. They result from delivery of health services in a healthcare setting, and clients are at an increased risk of “catching” these infections. Unfortunately, HAIs (Hospital Acquired Infections) may lead to extended hospital length of stay, healthcare costs and potential mortality. This is another reason why we need to learn how to prevent the spread of infection. Slide 7 - Health Care - Hospital Associated Infections Here are interesting facts about hospital associated infections or HAI’s. (As above - read slide) It is easy to see how costly HAI’s can be. Costly to the health of loved ones and costly to treat. This is another reason why it is important for health care workers to learn how they can help prevent the spread of infection. Slide 11 - What is the Chain of Infection ? (As above - read slide) These factors involved in the transmission of infection and are considered part of the Chain of Infection (as seen in the diagram). From the diagram we can see that the Chain of Infection involves: the Causative agent (the micro-organism that has the ability to cause infection or disease) the Reservoir (the place where this organism can live, reproduce, thrive. It may be a person, or a table top)

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the Portal of Exit (the place where this organism may “exit” or “escape” from, such as a nose or mouth during a cough, or the bowel as feces, are examples), the Mode of Transmission (the method by which the organism is transferred from one place to the next. For example, from the hands of a healthcare worker to another person, through the air, or through sharing another contaminated object) the Portal of Entry (the place where the organism enters a host, such as through broken skin or mucous membranes.) the Susceptible Host (the person who cannot resist the micro-organism from invading their body and causing infection or disease. Someone who has a chronic illness, or who is elderly, are more susceptible than healthier individuals, for example.) Healthcare workers need to be aware of the chain of infection, as well as the opportunities to break the chain. In doing so, workers protect themselves and their patients from the spread of infectious disease. The chain of infection, and ways of breaking this chain, will be discussed a little later in this presentation. Slide 15 - Breaking the Chain of Infection – Transmission In this diagram we can see the Chain of Infection depicted, We also see ways of breaking that chain of infection identified. This list outlines several ways of reducing transmission of infectious organisms, and therefore they are strategies which we can utilize to prevent spread of infection. (As above - read slide) The chain of infection, and the strategies listed above, will be discussed further, in subsequent slides. Slide 16 - Chain of Infection Understanding some of the key terminology used in the Chain of Infection, that we are discussing will be helpful in facilitating a better understanding of infection prevention and infection control practices that will now begin to explore in this presentation. (As above - read slide) Slide 17 - Chain of Infection Airborne – refers to infectious agents/organisms which are carried long distances by air currents. They are carried through very small airborne particles, and are not seen with the eye.

Example of infectious disease, which can be transmitted through the air, include Measles and Tuberculosis. Contact – refers to infectious agents which are spread through touch. For example, touch/contact of an infected person of the infectious organism coming in contact with things like furniture, or equipment may allow for transmission of the infectious agent / organism from one person to another Droplet – refers to infectious agents which are transmitted by large respiratory droplets, that may travel up to 1-2 meters from the patient. Slide 18 - Chain of Infection & Mode of Transmission Understanding the mode of transmission can be helpful in understanding how to break or interrupt the chain of infection and therefore reduce the transmission or spread of infection. Slide 21 - Early Recognition & Planned Precautions An important key in the prevention of infection begins with the health care worker recognizing and identifying that there may be a potential source of infection and therefore reason for concern. Slide 22 - Epidemiology and Surveillance Outbreak This slide contains common terminology to be aware of, regarding the study of population based infection, illness and disease patterns and trends.

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Each healthcare worker has the responsibility of helping to break the chain of infection. Additionally, health care providers should remember that this may involve educating the patient and the patient’s family / visitors about ways to also prevent the spread of infectious organisms or agents. This may include ways of isolating the pathogen and eliminating its mode of transmission thereby breaking the chain of infection. Breaking any potential area in the chain (as in the diagram) can reduce the risk of further transmission. Slide 29 - Infection Prevention & Control Practices In your hospital – health care placement setting there are resources to help educate and guide your practice, regarding infection prevention and infection control strategies. This includes the organizations key policies and procedures related to safe practices. As trainees, you will need to identify and understand the key policies, procedures and practices related to infection prevention and infection control that will be required to safely fulfill your registered technician role and responsibilities. Slide 31 - Infection Control – Know the Precautions Infection Control – Know the Precautions: Standard Precautions Standard precautions should be applied for ALL patients. Standard precautions include the need to wash your hands between any contact with or before and after any direct contact with a patient. This includes for example, contact with blood, other body fluids or excretions, non-intact skin, and wound dressings. You should also wash your hands after touching anything in the patient’s area and after removing gloves. You should also wear personal protective equipment (like gloves or gowns) if you anticipate contact with bodily fluids, You should discard sharp instruments and needles in a puncture resistant container, and clean shared equipment between patient use, You should teach patients to cover mouth when coughing, and teach patients to wash their hands. Standard precautions are used for all patients regardless of their diagnoses to ensure protection of the patient, the health care worker and any other patients being cared for in the same location. For certain highly transmissible or epidemiologically important pathogens, other transmission-based precautions are used in addition to standard precautions. Transmission-Based Precautions to Isolate Infectious Agents Contact Droplet Airborne Contact, droplet, and airborne precautions are meant to block the different routes or modes of transmission. Slide 35 - Contact Precautions Contact precautions offer a simple prevention strategy: In hospital – health care setting it is helpful to post signs called practice precautions (sometimes also called isolation precautions) at the entrance/doors of the rooms containing patients with infectious illness or disease. The signs/precautions are an important way of informing staff and visitors that infection has been identified and that certain protective measures may be require before entering the room. This is an important step in preventing the further spread of infection.

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Essentially – the signs alert healthcare staff, family and visitors to the presence of infection, and advise them of how to protect themselves before entering the room. Notice the sign on top – for contact precautions in the diagram. It informs people the patient is isolated for contact precautions. It also informs people to clean their hands, wear gloves and a gown before entering, and to disinfect equipment before using with other patients. Your will need to check the precautions required for use in your own placement setting. Slide 38 - Droplet / Contact Precautions This is an example of a Droplet/Contact Precautionary sign (as a prevention strategy). It contains instructions for visitors and healthcare staff, including the personal protective equipment that is required before entering the patient’s room. Notice – it advises people to wear a mask (example: an N95 mask, which filters out up to 95% of airborne agents, if fitted properly). It also advises gowns, gloves, hand washing, and eye protection. Family and visitors need to be educated about these precautionary requirements. Slide 41 - Airborne Precautions This is an example of an Airborne Precaution sign (a prevention strategy). It contains instructions for family, visitors and healthcare staff, including the personal protective equipment that is required before entering the room. Notice – this sign informs us that respiratory protection, such as a special N95 mask is required, hand washing and the need to keep the patient’s room door closed. Slide 43 - Prevent Infectious Disease Transmission by: In summary, this diagram illustrates some of the ways that infection is spread or transmitted in the hospital – health care setting environment. For example, this may include transmission through the use of shared contaminated equipment and furniture, being transported on the hands of healthcare workers, family members and visitors. The infectious organism / agent is then carried to the next susceptible patient - as a host. Also important in the diagram is the depiction of ways to help “break” or prevent the spread of infection. The precautionary measures for health care workers to follow – regarding droplet transmission of infection are: (As above - read slide) Slide 47 - PPE for Standard Precautions PPE as Standard Precautions include: (As above - read slide) This is true, unless additional precautions are necessary for airborne, droplet, or contact concerns. In that case, we have additional precautions (as discussed in earlier slides, such as N95 mask etc), that are utilized in addition to those of the above standard precautions. You will notice here – that standard precautions are used with all patients. Slide 48 - Bloodborne Pathogens In considering the necessary precautions for health care workers to be protected from bloodborne pathogens, the following approach should be utilized. It is important to understand, that bloodborne pathogens are the infectious organisms found and transmitted within the blood. (As above - read slide) Slide 50 - Bloodborne – Universal Precautions This is an example of a precaution sign that depicts the universal precautions taken to protect the health care worker and other patients from the risk of transmission of bloodborne infections. Slide 52 - Personal Protective Equipment (PPE)

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PPE is an important strategy in the prevention of the spread of infection. We have been discussing the use of personal protective equipment as a mandatory requirement of all health care workers in order to support the prevention and control of infection as being spread in the health care setting from person to person. In summary , there are some important things to remember. (As above - read slide) Slide 54 - Personal Protective Equipment – PPE Personal protective equipment is shown here. It includes gown, gloves, shoe covers, mask, eye shield, and hair net/cover. It may also be worn by a member of a surgical team wishing to support surgical asepsis. Slide 60 - Effective Hand Washing Technique Ensuring an effective hand washing technique also includes the following: (As above - read slide) Tip to remember: Keep in mind – dirty hands turned the tap/water on. Therefore, the handles/tap are dirty. You may use the paper towel to turn tap/water off. The paper towel can also be used to open the door and then throw the paper towel away. Slide 64 - Aseptic Technique – Types The word “aseptic” refers to being free from germs that cause disease or infection or being free from contamination of infection-causing organisms. Aseptic techniques are ways by which we prevent the spread of these germs, or ways we try to minimize contamination. The two types mentioned here are: (As above - read slide) Slide 74 - Summary of Infection Control Practices Participating in infection prevention and supporting infection control in patient care and services provision in the health care setting - are important concepts that you as trainees in the placement setting need to understand. Hopefully, you have gained a greater understanding of the concept of infection prevention and infection control and appreciate its impact on care and service delivery and practice precautions as used in the health care setting. You will need as a trainee, to understand more specifically how this is done in your placement and departmental setting. As you continue your experience in the Entry to Practice Training Program – we invite you to continue building your abilities to demonstrate and exhibit a strong response to participate in roles of infection prevention and control as your work behavior --This is important --- as it will be a very important component to your ongoing success during the placement experience and as you continue your career path in health care as a registered technician.

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Lecture Outline 1) Describe Healthcare Associated Infection (HAI) 2) Describe the chain of infection & standard / universal precautions 3) Be aware and understand the role(s) of isolation precautions in preventing the spread of transmission for certain infections 4) Describe each type of isolation precaution used to reduce the spread of contact, droplet , airborne infectious organisms in the placement setting 5) Review hand hygiene and its required use


Day 2 - Infection Prevention & Control

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Trainee Orientation to Infection Control

Infection

 Recognize patient and health care staff safety as an important responsibility in practice delivery and build awareness of the role(s) of infection prevention and infection control protocol systems.

• An invasion of pathogens or microorganisms into the body that are capable of producing disease.

 Apply required knowledge in preventing and/or minimizing infection in daily practice.

• The invasion and reproduction of microorganisms in a body tissue that can result in a local or systemic clinical response such as cellulitis, fever etc.

 Perform appropriate behaviors required to prevent health care associated infections.  Demonstrate required competence to provide patients with safe care and reduce / protect others from the risk of hospital acquired infections. 3

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Infection Control

What are Nosocomial Infections ?

• Includes all of the practices used to prevent the spread of microorganisms that could cause disease in a person.

• Result from delivery of health services in a healthcare setting, clients are at increased risk. • Unfortunately, HAIs may lead to extended hospital length of stay, healthcare costs and potential mortality.

• Infection control practices help to protect clients and healthcare providers and families and visitors from disease by reducing and/or eliminating sources of infection.

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Health Care – Hospital Associated Infection

Health Care ‐ Hospital Associated Infections According to the World Health Organization(2005):

 WHO defines a HAI as:  An infection acquired in hospital by a patient who was admitted for a reason other than that infection.  An infection occurring in a patient in a hospital or other health‐care facility in whom the infection was not present or incubating at the time of admission. This includes infections acquired in the hospital but appearing after discharge, and also occupational infections among staff of the facility.

 On average, 8.7% of hospital patients suffer health care‐ hospital associated infections (HAI)  Developed countries HAI is approximately 5‐10%  While in developing countries the risk of HAI is 2‐20 times higher and may affect more than 25% of patients  At any one time, over 1.4 million people worldwide suffer from infections acquired while in hospital. 7

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Main Sources of Infection

Impact of HealthCare Associated Infection

Healthcare associated infections:

HAI can: • Increase patients’ suffering. • Lead to permanent disability. • Lead to death. • Prolong hospital stay. • Increase need for a higher level of care. • Increase the costs to patients and hospitals.

Are caused by bacteria, fungi or viruses entering the body through one or more of the following routes:  Person‐person via hands of health‐care providers, patients and visitors;  Personal equipment (e.g. stethoscopes, computers) and clothing;  Environmental contamination;  Airborne transmission;  Carriers on the hospital staff;  Common‐source outbreaks

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What is the Chain of Infection ?

Transmission of Nosocomial Infections

• There are several factors which influence the opportunity and risk of acquiring an infection where transmission occurs from one person to another in a health care setting.

• Most nosocomial infections are transmitted by health care workers and clients as a result of direct contact. • We, must pay particular attention to washing hands after contact with clients or equipment.

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Infection Prevention & Control Practices

Prevention of Infections Requires health care providers who have:

Guidelines for Infection Prevention & Control Practices  Saudi Ministry of health reporting policies  GCC infection control manual  WHO guidelines  Centre for Disease Control Guidelines  Local Health Care Setting / Hospital Policies & Procedures

 Knowledge of common infections and modes of transmission  Knowledge of the extent of the problem;  Knowledge of the steps and strategies to protect self and others

Departmental Procedures  Infection Control  Management of Prescribed Contagious Conditions

‐ An attitude of cooperation and commitment ‐ Being an effective team player ‐ Commitment to preventing HAIs

 Knowledge of appropriate reporting procedure  Knowing when and who to ask for help

Fact Sheets / Safety Alerts  For patients, visitors and staff 13

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Chain of Infection

Breaking the Chain of Infection – Transmission

• Infectious Agent – micro organism (bacteria, viruses)

• Infection Control Practices

• Resident – normally reside on the skin in stable numbers • Transient – attach loosely to the skin by contact with another – easily removed by hand washing

• Cleaning and Disinfecting

• Reservoir ‐ or source of pathogen.

• Infectious Waste

Pathogen survives here but may or may not multiply.

• Contagious Conditions

• Portal of Exit –

• Immunization

From the reservoir, exit through the skin, respiratory tract, or blood. Site where micro organism leaves. 16

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Chain of Infection

Chain of Infection & Mode of Transmission

• Mode of Transmission or means of spread:

For transmission of infection, there must be sufficient quantities of the pathogen, AND the pathogen must be virulent enough to cause disease. The pathogen moves through a route of transmission, and reaches a “portal of entry,” such as eyes, nose, mouth, or puncture wound, to enter the susceptible host.

– Airborne – Contact – Droplet

Disease transmission can be prevented by breaking one or more of the links in this chain of transmission Most pathogens have a preferred portal of entry, one that gives ready access to an immediate environment suitable for the establishment of growth, or one that allows the pathogens to reach their target tissues or organs.

• Portal of Entry (to the host) Enters the same way they exit the host (example: open wound, breathe in)

Common portals of entry include the respiratory tract, Gastro intestinal tract, mucosa (e.g., conjunctiva, nose, mouth), genitourinary tract, breach of skin integrity, or mosquito bite Next, need to review the different routes of transmission from an infected patient to a susceptible host. 17

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Susceptibility of Host

Patient Risks for Developing Infections

• Host must be susceptible to the strength and numbers of the microorganisms.

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Lower resistance to infectious microorganisms (due to illness or disease).

• To reduce susceptibility – provide adequate nutrition & rest, promote body defenses against infection & provide immunization

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Exposure to an increased number of and more types of disease‐causing organisms. (Hospital harbors a high population of virulent strains of microorganisms that are resistant to antibiotics) MRSA, VRE – super bugs.

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The performance of invasive procedures. (IV cathetars etc.. Anything that crosses protective barriers)

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Early Recognition & Planned Precautions

Epidemiology and Surveillance Outbreak

Early Recognition • Health care facility staff must quickly identify and separate potential sources of infection from susceptible hosts

• Outbreak is a term used in epidemiology to describe an occurrence of disease greater than would otherwise be expected in a particular time and place. It may be small and localized group or impact upon thousands of people across an entire continent.

• Since the infecting agent often is not known at the time of admission to a health care facility, transmission‐based precautions are used empirically, according to the clinical syndrome and the likely etiologic agent at time, and then modified when the pathogen is identified or a transmissible infectious disease etiology is ruled out.

• Two linked cases of a rare infectious disease may be sufficient to constitute an outbreak. Outbreaks may also refer to epidemics, which affect a region in a country or a group of countries, or pandemics, which describe global disease outbreaks.

• These infection control principles are also used for laboratory and procedure‐specific safety.

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Required Skills to Prevent Infections

Infection Control Practices

• • • • •

Apply standard / universal precautions* Use personal protection equipment methods Know what to do if you become exposed Encourage others to use universal precautions Report breaks in technique that increase patient risks • Observe patients for signs and symptoms of infection and report for isolation and outbreak risk management

Trainee’s Role in Prevention and Infection Control

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Principles of Infection Control


1. Microorganisms move through space on air currents – avoid shaking or tossing linen.

3. Microorganisms are transferred by gravity when one item is held above another, avoid passing dirty items over clean items, eg. Clean items on upper shelves – dirty items on lower shelves (bedpan).

2. Microorganisms are transferred from one surface to another whenever objects touch, a clean item touching a less clean item becomes “dirty” – keep hands away from face, keep linens away from uniforms, an item dropped on the floor is considered dirty.

4. Microorganisms are released into the air on droplet nuclei whenever a person breathes or speaks – avoid breathing directly in someone’s face; when someone coughs/sneezes, cover mouth with tissue, discard, wash hands. 25

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Breaking the Chain of Infection

5. Microorganisms move slowly on dry surfaces, but very quickly through moisture – use paper towel to turn facets off, dry bath basin before returning to bedside table.

• Various types of infection prevention & control strategies by isolation of the pathogen and transmission mode can reduce the risk(s) of person to person spread of hospital acquired infections

6. Proper handwashing removes many of the microorganisms that would be transferred by the hands from one item to another – always wash hands between patients. 27

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Infection Prevention & Control Practices

Practice Precautions for Route of Transmission

Guidelines for Infection Prevention & Control Practices  Saudi Ministry of health reporting policies  GCC infection control manual  WHO guidelines  Centre for Disease Control Guidelines  Local Health Care Setting / Hospital Policies & Procedures

• Contact: Infections spread by direct or indirect contact with patients or the patient‐care environment • Droplet: Infections spread by large droplets generated by coughs, sneezes, etc.

Departmental Procedures  Infection Control  Management of Prescribed Contagious Conditions

• Airborne (droplet nuclei): Infections spread by particles that remain infectious while suspended in the air

Fact Sheets / Safety Alerts  For patients, visitors and staff 29

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Infection Control – Know the Precautions

Transmission by Direct & Indirect Contact

Standard Precautions

• Some pathogens are spread through direct contact, while others are spread through indirect contact.

 Should be applied for ALL patients Standard precautions are used for all patients regardless of their diagnoses to ensure protection of the health care worker and the patient.

• Examples of direct contact include person‐to‐person mechanisms such as kissing, skin‐to‐skin contact and sexual contact.

For certain highly transmissible or epidemiologically important pathogens, transmission‐based precautions are used in addition to standard precautions.

Transmission‐Based Precautions to Isolate Infectious Agents

• As well there can be direct contact with animals, soil or vegetation.

Contact, droplet, and airborne precautions are meant to block the different routes of transmission that we discussed earlier.

• Indirect contact occurs when an agent is carried from a reservoir (the source of infection) to a susceptible host without direct contact with the source. For example, by touching a contaminated surface a person can become infected.

 Contact  Droplet  Airborne

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Contact Transmission – Precautions

Contact Transmission ‐ Precautions

Trainees should avoid contact with their face, eyes or mouth when their non gloved or gloved hands may be contaminated

Used for protection against contact (i.e., hand contamination or self‐ contact) with large droplets

Limit patient movement outside of their designated room

The key elements of Contact Precautions for patients suspected or confirmed of having a disease spread by droplets or some common respiratory pathogens are:

Limiting patient contact with other well patients Contact Transmission Persons infected with some common respiratory pathogens can spread their disease by either contaminating their own hands, the hands of a healthcare worker or an environmental surface. Hands can transmit these diseases when they have contact with contaminated surfaces followed by contact with either another body surface such as the conjunctiva or nasal mucosa or an intermediate object.

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 Using clean, non sterile gloves for all episodes of direct patient contact  Changing the gloves after each patient contact  Use a gown (disposable or re‐washable) for each patient contact and disposing of it as either waste or laundry depending on its type, after each episode  Use dedicated specific equipment (preferable single‐use) for a single patient and clean and disinfect shared equipment between patient uses. 33

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Contact Precautions

Droplet Transmission

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Droplet transmission involves contact of the conjunctivae (the mucous membrane that lines the inner surface of the eyelid and the exposed surface of the eyeball) or the mucous membranes of the nose or mouth of a susceptible person with large‐particle droplets containing microorganisms generated from a person who is infected with the microorganism.

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Droplets are generated primarily during coughing, sneezing, or talking and during the performance of certain procedures such as suctioning and bronchoscopy.

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Transmission via large‐particle droplets is associated with close contact between people, probably because large droplets typically do not travel beyond about 1 meter.

•

Some examples of diseases that spread via large droplets are pharyngeal diphtheria, pertussis, and meningococcal disease. 36

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Droplet Transmission – Precautions

Droplet / Contact Precautions

• Droplet Precautions are used in addition to Standard Precautions to provide protection to clinicians and others protection from infections spread by large droplets generated by coughs and sneezes Additional protection measures critical under Droplet Precautions are: • Use of a medical/ procedural mask (by Trainees) when within a meter of a patient • Physically maintaining distance between the infected patient and other persons by a distance of at least one meter from all other persons, • Limit patient movement. If a patient has to leave the area, the patient should wear a medical mask, if tolerated, for the duration of their time away. 37

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Airborne Transmission

Airborne Transmission – Precautions

• Airborne transmission occurs by dissemination of inhalable infectious material.

Airborne precautions are used for protection against inhalation of tiny infectious droplet nuclei In addition to Standard Precautions: • Use a particulate respirator (example: N95 Mask) when entering the patient isolation room. Perform a mask – seal check before each use.

• Microorganisms carried in this manner can be dispersed widely by air currents and those that remain infectious while in the air may cause infection when inhaled or deposited on a susceptible host’s respiratory tract, potentially over a long distances from the source patient, depending on the environmental factors.

• Place the patient in adequately ventilated room (≥ 12 air changes per hour)

• Certain therapeutic procedures (e.g., endotracheal intubation, bronchoscopy) are associated with the generation of aerosols.

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Limit patient movement and ensure that the patient wears a medical mask if outside their room.

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Airborne precautions should also be performed during the performance of any aerosol‐generating procedures associated with pathogen transmission (e.g., endotracheal intubation, bronchoscopy)

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Airborne Precautions

Drug Resistant Organisms & Carriers  Epidemiological evidence suggests that multidrug‐ resistant organisms are carried from person‐to‐person by health‐care professionals.  Carriers are individuals who harbor disease organisms in their body without visible symptoms and may pass the infection to another person.  It is possible to carry an organism without being aware of it for example, Typhoid Mary a woman who carried the typhoid bacillus and unknowingly started an epidemic in the US in the 1880s. 41

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Prevent Infectious Disease Transmission by:

Required Performance of Trainee

Source Control Measures Cough etiquette, cleaning and disinfection of environment

Trainees need to: • • • • • •

Modes of Transmission Contact: hand hygiene Droplet: distance from source less than1 ‐2meters Airborne: isolation rooms and ventilation Portal of Entry into the Host Adding barriers, e.g., PPE Host Strengthen host defences, e.g., vaccination, immunization

Apply standard / universal precautions Consider immunized against Hepatitis B Use personal protection equipment (PPE) methods Know what to do if exposed to blood / bodily fluids Encourage others to use universal precautions Trainees need to make every effort to minimize the spread of infection and to encourage patients and other health‐care workers to actively engage in practices that minimize the spread of infection in the hospital.

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Centre for Disease Control Guidelines

Standard Practice Precautions

• Based on the concept that bodily fluids from ANY person can be infectious • Therefore, standard precautions should be used on every patient

• Trainees should approach every situation as having the potential to infect a patient or a health‐care worker or themselves. • Infections are preventable when health care workers use the right techniques and remain on the look out for unclean and unsafe situations.

All necessary PPE for protection use by Trainees is mandatory and should comply with the Centre for Disease Control Guidelines which requires that the use of category specific isolation for infection in addition to the use of all standard precautions be used when a patient is known or suspected to have an infection.

• Guidelines for preventing expose to blood, body fluids, secretions, excretions, broken skin or mucous membranes should be followed. 45

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PPE for Standard Precautions

Bloodborne Pathogens • Do not touch or try to clean up any bodily fluids such as blood, urine, stool or vomit unless you have been trained in the use of proper PPE

• IF direct contact with blood & body fluids, secretions, excretions, mucous membranes, non‐ intact skin – Gloves – Gown • IF there is the risk of spills onto the body and/or face – Gloves – Gown – Face protection (mask plus eye protection goggle or visor; face shield)

• Trained staff with latex gloves (and safety glasses) are authorized to handle this type of hazardous waste material and its proper disposal • If you are exposed to any bodily fluids notify your supervisor immediately (example: needle stick injury, contact through an open area of the body or mucous membrane etc.) 47

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If exposed to blood / body fluids

Bloodborne – Universal Precautions

• Guidelines if exposed to blood or body fluids • If puncture type wound (needle stick injury) occurs it should be washed immediately and should be caused to bleed. • If skin contamination should occur, wash the area immediately • If splashes to the nose or mouth occur it should be flushed with water • Eye slashed require irrigation with clean water or saline • Report incident to supervisor / preceptor for follow up protocol instructions, check with occupational health or physician as the situation warrants • Trainers must complete an Incident Report Form 49

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Universal Precautions

Personal Protective Equipment (PPE)

• Universal precautions: "Universal precautions," as defined by CDC, are a set of precautions designed to prevent transmission of human immunodeficiency virus (HIV), hepatitis B virus (HBV), and other bloodborne pathogens when providing first aid or health care. Under universal precautions, blood and certain body fluids of all patients are considered potentially infectious for HIV, HBV and other bloodborne pathogens. Retrieved from CDC web site

 Personal protective equipment includes the use of gowns, gloves, aprons, eye protection and face masks.  The use of these equipment is usually based on assessment of the risk of micro‐organism transmission to the patient or to the carrier as well as the risk of contamination of the health‐care practitioner’s clothing and skin by the patient’s blood, bodily fluids, secretions or excretions.  Health care workers should wear a face mask, eye protection and a gown if there is the potential for blood or other bodily fluids to splash.

http://www.cdc.gov/ncidod/dhqp/bp_universal_precautions.html 51

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PPE & Contact with Infectious Agent

Personal Protective Equipment – PPE

• All of the PPE listed here prevent contact with the infectious agent, or body fluid that may contain the infectious agent, by creating a barrier between the worker and the infectious material. • Gloves, protect the hands, • Gowns or aprons protect the skin and/or clothing, • Masks and respirators protect the mouth and nose, goggles protect the eyes, and face shields protect the entire face. The respirator, has been designed to also protect the respiratory tract from airborne transmission of infectious agents. We’ll discuss this in more detail later. • Goggles – protect eyes • Face shields – protect face, mouth, nose, and eyes

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Infection Prevention through Hand Washing

Hand Washing – Hand Hygiene

• Hand washing is the single most important infection control intervention strategy for use in the health care – hospital setting

• Hand washing is the single most important intervention to be performed ‐ it is done before and after patient contact. • Also preform hand hygiene immediately before donning and after removing PPE

• Trainees require knowledge & skill: – – – – –

• Every health‐care worker is required to act responsibly and without fail to apply the techniques for hand washing at every patient encounter. They also should advise patients and families of the importance of hand washing and give them permission to remind the staff.

How to clean hands Rationale for choice of clean hand practice Techniques for hand hygiene Protect hands from contaminants Promote adherence to placement site hand hygiene guidelines

• Decontamination refers to the process for physical removal of blood, bodily fluids and the removal or destruction of micro‐ organisms from the hands. 55

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Five Moments of Hand Hygiene

Five Moments of Hand Hygiene

• Before patient contact • Before an aseptic task • After body fluid exposure even if wearing gloves! • After patient contact • After contact with patient surroundings or equipment

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When Should Hands be Washed

Effective Hand Washing Technique

• When visibly soiled. • Before and after client contact. • After contact with a source of microorganisms (blood, body fluids, mucus membranes, non intact skin or inanimate objects that might be contaminated. • Prior to performance of invasive procedures (IV catheters, indwelling catheters). • Before and after removing gloves (wearing gloves does not remove the need to wash hands). • At the beginning and end of every shift.

• • • • •

Remove all wrist and hand jewelry. Cover cuts and abrasions with waterproof dressings. Keep fingernails short, clean, and free from nail polish. Wet hands under tepid running water Apply soap or antimicrobial preparation – solution must have contact with whole surface area of hands – vigorous rubbing of hands for 20–30 seconds – especially tips of fingers, thumbs and areas between fingers

• Rinse completely • Dry hands with good quality paper towel. 59

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Use of alcohol based Hand Sanitizer

Personal Protection & Skin Integrity

 Apply a 1‐2 squirts of product in your cupped hand  Rub hands palm to palm  Right palm over left hand with interlaced fingers  Palm to palm with fingers interlaced  Backs of fingers to opposing palms with fingers interlocked  Rub between thumb and forefinger  Rotational rubbing, backwards and forwards with clasped fingers of right hand in left palm and vice versa  Once dry, your hands are cleaned

• Frequent hand washing dries skin. Skin can breakdown and crack, breaking our skin barrier protection. • Use hand moisturizer frequently. • Protection of the client is priority, however, we must also protect ourselves – we are at risk for contact with infectious materials or exposure to a communicable disease. 61

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Before Aseptic Contact with Each Patient

Aseptic Technique – Types

A trainee should clean hands before an aseptic task.

• Medical Asepsis – Clean technique; procedures used to reduce & prevent spread of microorganisms ** Hand washing**

 It is essential that trainees clean their hands immediately before any aseptic task. This is necessary to protect the patient against harmful micro‐organisms, including the patient’s own micro‐ organisms, entering his or her body. Trainees must protect against transmission through contact with mucous membrane: oral/dental care, giving eye drops, secretion aspiration.

• Surgical Asepsis – Sterile technique; procedures used to eliminate microorganisms **Sterilization**

 Often trainees will be treating patients who have open wounds and any contact with non‐intact skin: skin lesion care, wound dressing, any type of injection is an opportunity for transmission.  Medical devices are well known for harbouring potentially harmful micro‐organisms and contact with devices such as a catheter insertion, opening a vascular access system or a draining system must be done with careful preparation. Trainees should also be diligent in preparation of food, medications and dressing sets. 63

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Equipment Needs ‐ Masks & Gloves

Handling of Sharps & Injection Equipment

Masks should be worn:  If aseptic or sterile technique is required  To protect an immune compromised patient

• Trainees should be aware of the significant problem for health‐care workers caused by needle stick injuries, which are as prevalent as injuries from falls and handling and exposure to hazardous substances.

Masks or respirator mask (N 95) should be worn  If airborne infection is suspected or confirmed

• If puncture type wound (needle stick injury) occurs it should be washed immediately and should be caused to bleed.

Gloves must be worn for:  All invasive procedures  Contact with sterile sites  Contact with non‐intact skin or mucous membranes  All activities assessed as having a risk of exposure to blood, bodily fluids, secretions and excretions, and handling sharps or contaminated instruments.

• Report incident to supervisor / preceptor for follow up protocol instructions, check with occupational health or physician as the situation warrants

 Hands should be washed before and after gloving 65

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Safe Use and Disposal of Sharps

Environmental Decontamination

• Keep handling of sharps / needles to a minimum

• Cleaning MUST precede decontamination • Disinfectant ineffective if organic matter is present • Use mechanical force

• Do not recap needles, bend or break after use • Discard each needle into a sharps container at the point of use

– Scrubbing – Brushing – Flush with water

• Do not overload a bin if it is full • Do not leave a sharp bin in the reach of children 67

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Environmental Cleaning & Wastes

To Minimize the Spread of Infection

Environmental Cleaning: Use appropriate procedures for the routine cleaning and disinfection of environmental and other frequently touched surfaces

REMEMBER … Before contact with each and every patient:

Waste Disposal:

• • • • •

Treat waste contaminated with blood, bodily fluids, secretions and excretions as clinical waste, in accordance with local regulations, disposal protocols and storage in properly marked containers

clean hands before touching a patient clean hands before an aseptic task clean hands after touching a patient clean equipment used with a patient clean hands when leaving patient surroundings

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Trainee Practice Responsibilities

Trainee Role & Liability

• Review and understand the key guidelines, policies and procedures for infection control and safety within your health care placement site and clinical domain environments. • Demonstrate responsibility for minimizing infection transmission in your practice delivery. • Ask questions if you are unsure of a patient’s infection control – mode of transmission precautions • Let staff know if supplies are inadequate or depleted. • Educate others about clean hands and infection transmission protocols / etiquette.

• Liable if actions cause harm – usually shared by instructor, trainee & hospital • Expected to perform as registered professional – safe client care • As a trainee – you do not practice outside of role / domain job description and you require supervision

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Summary of Infection Control Practices

Summary of Infection Control Practices

 Maintain cleanliness of the hospital

● Early recognition and reporting ● Infection control precautions ● Hand hygiene: alcohol‐based hand rub, hand washing ● PPE: gloves, gowns, masks/respirators, eye protection ● Patient accommodation‐ isolation room ● Environmental cleaning and waste disposal ● Occupational health management

 Clean and disinfect hospital & patient care equipment  Personal attention to hand washing before and after every contact with a patient or object  Use personal protective equipment whenever indicated  Use and dispose of sharps safely 73

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References


• World Health Organization. (2010). WHO Patient Safety Curriculum Guide for Medical Schools. • World Health Organization. (2010). Topic 1: What is patient safety? • World Health Organization. (2010). Topic 9: Minimizing infection through improved infection control.

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Workplace Safety & Injury Prevention -

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Slide 3 - Trainee Safety in the Health Care Setting Work related injuries not only cause time away from the workplace activities but can also result in time away from activities with your family & friends … Therefore it is very important for those working in health care settings to have a good understanding of safety measures and work place policies and procedures promoting both patient, staff / trainee and visitor safety. In order to do this, as a trainee you will need to have a better understanding of the meaning of working safely in the hospital / health care setting. (As above - read slide) Slide 4 - Definition of Patient Safety It is also important to understand that patient safety is everyone’s responsibility. In the workplace, we may have different roles and responsibilities and even different titles related to our “work” but as health care professionals … We all have an equal responsibility to ensure the safety of each other and this is especially important in how we promote the prevention of accidents and injury for patients. Slide 5 - Adverse Events & Workplace Safety In the health care setting - an adverse event can cause harm to another person be it a patient or health care provider. When we refer to an adverse event for a patient --- it is typically the result of an error, lapse or breach in safety or system of safety measures or protocols. This means that as a health care worker … we can be a contributor to the cause of an adverse event and the event could have otherwise been prevented. As a basic example, consider hand washing and your contact with a patient. By not properly completing your hand washing or not hand washing before working directly with each patient - you become a risk to a patient’s safety as you may transmit or carry the potential for transmission of infection from one patient to another patient by not complying with the safe hand washing protocol. Slide 6 - Emergency Situations & Response Many unanticipated situations can arise in a hospital or other health care settings. This can include Emergency situations that require all personnel to respond in accordance with organizational planned safety responses. Slide 7 - Emergency Evacuation Anticipating what to do in the event of an emergency situation is critical to saving lives in a real emergency situation. Therefore, hospital – health care settings have established policies and procedures with a defined plan of action to guide staff on the proper way to respond in these situations. As trainees, it is important that you familiarize yourself with the emergency and safety related policies, protocols and procedures of the organization in which you are completing your placement. An example of an unanticipated emergency situation might be that of an emergency evacuation situation. It is not unusual for teams to practice their safety responses to build their preparation and sense of readiness in the event of an actual emergency. (As above - read slide) Slide 8 - Fire Safety Trainees understanding of fire safety in the hospital – health care setting is also critical, as time and knowing what to do can save lives in a real fire situation. (As above - read slide) Slide 9 - Fire Extinguishers & Training Knowledge and understanding of fire safety is important … as is how to utilize fire safety equipment. 58


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This may be very important as there can be higher risks when unsafe work practices are undertaken by people in the workplace for example - when flammable substances are not stored properly and safely which can then lead to a fire situation. In thinking about fire safety and your training keep in mind … (As above - read slide) Each trainee needs to review the fire safety manual, be familiar with the fire safety protocols of their department and the location of fire equipment in the event of an actual fire emergency. Slide 12 - Workplace Adverse Events & Safety Every job has some risk to the worker if they do not comply with work related training or they do not follow safety guidelines and protocols as established in the workplace setting. Therefore, as trainees you will need to become familiar with all the safety precautions related to your role and responsibilities while in the placement setting. Slide 15 - Trainees have a duty to … As a trainee it is especially important to recognize what you know … and to acknowledge what you do not know and find out Not being truthful about your knowledge, gaps in your knowledge or scope of practice & role limitations can hurt you and hurt others in the workplace. Professionalism and ethics requires you to share this information. (As above - read slide) Slide 17 - Your Use of Personal Protective Equipment (PPE) In the hospital – health care setting the need for your use of personal protective equipment or PPE as it is called … is prescribed as a mandatory requirement according to the policies and procedures of the placement organization. By properly using PPE – you help to ensure the safety of the patients, other health care workers, the organization and the general public who enters the organization. (As above - read slide) Slide 18 - Personal Protective Equipment – PPE By building a better understanding of transmission and spread of infection and its prevention / control through the proper use of PPE, you are promoting the safety of others and yourself as a health care provider. By complying with the guidelines and protocols for infection prevention and infection control – as we have briefly covered in today’s session, you will also be protecting yourself in the work environment. Slide 19 - Body Mechanics and Movement A basic understanding of proper body mechanics and movement is important in the prevention of accidents and injury and future health problems for the health care worker. (As above - read slide) Slide 27 - Lifts & Transfers When asked to assist in lifting or transferring a patient - keep in mind you may become trained in using other types of special equipment to assist in such lifts and transfers. For the safety of the patient and your fellow team members, if you are not familiar with or have not been trained in the use of such equipment, you will need to disclose this prior to engaging in any patient transfer or lift and allow for another staff member to assist in the procedure. Once you have been trained in the safe use of the equipment and lift / transfer procedures you are encouraged to assist others as needed. Slide 30 - Material Safety Data Sheets – MSDS To assist all staff in identifying safety information for the workplace setting, MSDS or material safety data sheets should be available to staff / employees and individuals working in the organization. Speak with your department preceptor / supervisor regarding where they can be found in your placement setting’s department. 59


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Slide 31 - Bloodborne Pathogens As a trainee in the hospital – health care setting it is very important for you to understand the precautions and risks associated with bloodborne pathogens and their contact precautions. This includes understanding the PPE needs in accordance with organizational and work task related guidelines and policies. (As above - read slide) Slide 33 - Safe Use and Disposal of Sharps To avoid accidents and injuries to yourself and others in the workplace related to blood and body fluids transmission of infection -- you will need to practice the safe use and you must dispose of all sharps in the appropriate disposal / biohazard marked containers. Your compliance with this practice is mandatory (As above - read slide) Slide 36 - Injuries & Accidents – Trainee Reporting It is essential that should you, as a trainee experience an accident or injury in the placement setting that this is reported in accordance with the Entry to Practice Training Program policies and guidelines. It is also essential that you report all injuries and accidents to your supervisor / preceptor in the placement setting immediately (As above - read slide) Slide 37 - Breaching Safety Practice & Disciplinary Action The consequence of disregarding or not practicing required occupational health and safety practice guidelines in the work place setting can result in your dismissal from the placement setting and the program. (As above - read slide) Slide 38 - Key Messages – Safety & Injury Prevention (As above - read slide) In concluding this lecture session you should have gained a better understanding of the following : You recognize patient safety as an important responsibility in your practice and as a healthcare delivery system You may apply the required knowledge in preventing and/or minimizing workplace injury and accidents You will support performance of appropriate behaviors required to prevent health care associated injuries, and You will demonstrate the required competence to provide patients with safe care and to avoid injuries. As a trainee, you will need to understand more specifically how this applies to you in your placement and departmental setting. Thus, as you continue your experience in the Entry to Practice Training Program – we invite you to continue building your abilities to demonstrate and exhibit a strong response to participate in roles of safe practice delivery and the prevention of accidents & injuries in the work place as a valued work attitude and behavior. This is also important --- as it will be a very important component to your ongoing success during the placement experience and as you continue your career path in health care as a registered technician.

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Lecture Outline 1. Recognize patient safety as an important responsibility in your practice and as a healthcare delivery system. 2. Apply required knowledge in preventing and/or minimizing workplace injury and accidents. 3. Perform appropriate behaviors required to prevent health care associated injuries. 4. Demonstrate required competence to provide patients with safe care and to avoid injuries.


Day 2 - Workplace Safety & Injury Prevention 2

Trainee Safety in the Health Care Setting

Definition of Patient Safety

Work related injuries not only cause time away from the workplace activities but can also result in time away from activities with your family & friends

• Patient safety is a discipline in the health care sector that applies safety science methods toward the goal of achieving a trustworthy system of health care delivery.

What is the meaning of working safely? • Completing every task the correct way and not taking hazardous shortcuts • Understanding organizational policies & safety procedures • Wearing required personal protective equipment (PPE) • Being rested, alert and paying attention to the task you are undertaking • Asking for instruction and guidance prior to completing unfamiliar tasks

• Patient safety is also an attribute of health care systems; it minimizes the incidence and impact of, and maximizes recovery from, adverse events. • According to Institute of Medicine, safety is defined as “freedom of accidental injury”. (Emanuel, et al, 2008)

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Adverse Events & Workplace Safety

Emergency Situations & Response

• Adverse events are widespread and preventable

In Emergency Situations – in the health care setting you must know what to do:

(Emanuel et al., 2008)

• Much unnecessary harm is caused by health-care errors, safety lapses and system failures. Example: Example:

Hospital acquired infections (HAI) from poor hand-washing.

Example:

Work related accidents and injuries.

• KNOW the Emergency Response Codes and Responses for your Placement setting by reviewing the policies and procedures and discussing what your trainee role will be with your supervisor / preceptor

Complications from errors in medication orders or administering the wrong medication.

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Emergency Evacuation

Fire Safety • If you find a small fire smaller then a small trash can you should call for help and then you may try to put it out.

• Evacuation of a building may be required if an emergency situation threatens the life and safety of patients, visitors and employees

• If the fire is anything larger, you should sound the fire alarm, calling the emergency response number with the location of the fire and then following the fire protocols of the health care placement setting

• Situations that may require evacuation may include: – – – – –

Fire or smoke Chemical spills Power failure Bomb threat Violence or terrorist attacks

• This may include removing any person in the immediate area of the fire to an area of safety and • Reporting / assembling in your designated area with coworkers to await other instructions

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Fire Extinguishers & Training

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Electrical Safety

• Fire extinguishers only have a minute of retardant in each extinguisher • So you will only be able to put out a fire the size of a small trash can with it

• Only trained maintenance employees are authorized to investigate and conduct electrical repairs • Do not attempt any maintenance activities you are not trained or authorized to conduct

To use a fire extinguisher REMEMBER … PASS • Pull the pin • Aim at the base of the fire • Squeeze the handle • Sweep the base of the fire

• Never use a damaged extension cord or any other piece of damaged equipment • Never use electrical equipment in a damp or wet area 9

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Workplace Safety in Health Care Setting

Workplace Adverse Events & Safety

• Patient safety is a discipline in the health care sector that applies safety science methods toward the goal of achieving a trustworthy system of health care delivery.

• Workplace safety can assist to decrease the risk of adverse events that can occur in the workplace environment • Unnecessary harm can be caused by healthcare worker fatigue, errors, taking shortcuts, lack of attention to environmental safety and poor planning for emergency or system failures.

• Patient safety is also an attribute of health care systems; it minimizes the incidence and impact of, and maximizes recovery from, adverse events

• Many accidents and injuries that occur in the workplace are preventable

(Emanuel et al., 2008)

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How Can Someone be Hurt at Work

Patient & Environmental Safety

You have a role to play in staying safe by:

• Prevent and/or minimize the adverse events and eliminate preventable harm in the health care setting

• Learning to recognize hazards and the potential for fall risks • Looking for hazards everywhere

• As all health care professionals, you are responsible for ensuring patient and environmental safety • Help ensure a safe workplace protects your health

• Thinking of all four hazard categories • • • •

Physical Chemical Biological Ergonomic

• Safety is everyone’s responsibility - if everyone does their part it can make a real difference • Practicing health & safety “on the job” lowers the rate of accidents and injuries in the workplace

Report hazards to your preceptor / trainer in practice for appropriate action 13

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Trainees have a duty to …

Health & Safety is Your Responsibility

• Do your “trainee role / job” safely and in the way you have been trained, using the right safety devices and personal protective equipment

• Your are encouraged to report any safety problems to your supervisor / preceptor

• Comply and meet the prerequisites for entry into the health care setting work environment

• Before taking on any task – consider the necessary safety measures and check policy / procedures

• Know and comply with the health care setting work place policies and procedures

• LISTEN, ask questions and talk to one another

• Ask questions and obtain clarification of the things you do not know or understand

• REPORT immediately any accident, injury or health & safety problem to your supervisor / preceptor 15

16

Personal Protective Equipment – PPE

Your Use of Personal Protective Equipment (PPE) • Safety glasses / face shields • Hearing protection • Gloves • Respirators / Masks • Gowns • Protective footwear (enclosed shoes) • X‐ray / radiation shields These are considered part of your required UNIFORM 17

18

63

Body Mechanics and Movement

Definition of Body Alignment • Body alignment refers to the relationship of one body part to another body part along a horizontal of vertical line. • It is the condition of joints, tendons, ligaments and muscles in various body positions where correct alignment reduces strain on musculoskeletal structures , maintains adequate muscle tone and contributes to balance. • Typically when at rest and where the correct alignment is supported it does not cause pain

• Body Mechanics are the coordinated efforts of the musculoskeletal and nervous systems as the person moves, lifts, bends, stands, sits and completes daily activities (Thibbeau & Patton, 2007)

(Thibbeau & Patton, 2007) 19

20

Definition of Body Balance

Lifting Technique & Proper Body Mechanics

• Body Balance is achieved when a relatively low centre of gravity is balanced over a wide, stable base of support is present, and t a line falls from the centre of gravity vertically through the base of support. The body loses balance when the line from the centre of gravity does not fall vertically through the base of support

Maintaining balance, proper body alignment and posture during work activity can be supported by doing the following: 1) Maintain a broad base of support by separating your feet to a comfortable distance while standing 2) To increase your balance – bring your centre of gravity closer to your base of support by bending your knees slightly, flexing your hips and keeping your trunk erect in proper back alignment

• Body Balance is also enhanced by proper posture and body positions that most favours the functions of movement and requires the least amount of muscular work / strain on muscles, ligaments and bones

3) Bring the load (item you are carrying / lifting ) close to body to help maintain your balance

(Thibbeau & Patton, 2007) 21

22

KNOW Your Back Safety Guidelines

Object Assessment Prior to Lifting

• More than 50% of all back pain in health care workers is associated with manual lifting tasks • The most common back injury is strain on the muscle group around the lumbar vertebrae • Injury in this area affects the ability to bend forward, backward, and from side to side • It can also decrease your ability to rotate the hips and lower back

• Always check the weight of the object prior to lifting it person and determine if assistance is needed and resources are available • If moving a patient, use safe patient‐handling equipment and engage other knowledgeable staff as your “lift / transfer team member” supports

• KNOW your health care facility’s safety information regarding transfer, positioning and lifting patients and use the recommended back safety guidelines to prevent injury

• Manual lifting of the patient should be the last resort and should only be used when it does not involve lifting most or all of the patient’s weight 23

24

64

Body Mechanics & Lifting Objects • Improper versus proper object lifting technique

Lifting Technique Instructions • If the object seems heavy, get help from another person or more

• Proper lifting & standing technique

• Plan the path of travel before the lift and ensure the path is clear, allows adequate space for movement and is safe to move the object freely • Determine who will lead / give instructions for lifting • Always lift with your legs / knees and keep your back straight • Never twist while carrying a load / object, move your feet in the direction you wish to move 26

25

Lifts & Transfers

Environmental Awareness & Safety for Others • It is important to maintain a high level of environmental cleanliness throughout the health care setting for the safety of others • Falls and accidents from tripping hazards • Water / Spills cause slip hazards • Clean up or immediately notify your supervisor of any environmentally unsafe conditions • Call bells within reach for all patients • Bedrails / stretcher railings up for patients 27

28

Hazardous Materials

Material Safety Data Sheets – MSDS

• All chemicals and substances in the health care setting should be labeled with the name of the chemical and manufacturer

• Health care settings often use MSDS or material safety data sheets to assist in identifying important safety information to staff / employees

• Bulk chemicals and chemicals with a hazard must be labeled with a Hazard Management Information System Label

• The MSDS provides in‐depth information on health hazards, reactivity, flammability, chemical properties, guidelines on usage and storage

• This label may use symbols or a number rating – the higher the number the more hazardous the chemical (dangerous vapor, ignites, flammable or reactivity with contact etc.) Note: hazardous materials are typically stored requiring special safety conditions.

• Typically, MSDS for all products used at a facility are in binders within department areas where the materials are being used • Identify with your Supervisor / Preceptor where the MSDS binder is located for your reference & review any special precautions for the department work delivery

• If your trainee work area houses hazardous materials you must KNOW & review the related policies & procedures

EXAMPLE: Eye wash stations do exist for washing of hazardous materials from eyes, then seek medical attention

29

65

30

Bloodborne Pathogens

If Exposed to Blood / Body Fluids

• Do not touch or try to clean up any bodily fluids such as blood, urine, stool or vomit unless you have been trained in the use of proper PPE

• Follow healthcare guidelines if exposed to blood or body fluids • If puncture type wound (needle stick injury) occurs it should be washed immediately and should be caused to bleed. • If skin contamination should occur, wash the area immediately • If splashes to the nose or mouth occur it should be flushed with water • Eye splashed require irrigation with clean water or saline • Report incident to trainer in practice immediately for follow up protocol instructions, and for the need of immediate visit to the occupational health physician for the assessment for the need of post exposure prophylaxis. • Trainer in practice will be requested to complete an Incident Report Form

• Trained staff with latex gloves (and safety glasses) are authorized to handle this type of hazardous waste material and its proper disposal • If you are exposed to any bodily fluids notify your supervisor immediately (example: needle stick injury, contact through an open area of the body or mucous membrane etc.) 31

32

Safe Use and Disposal of Sharps

Handling Patient Care Equipment

• Keep handling of sharps / needles to a minimum

• Handle patient care equipment (soiled with blood or other body fluid secretions or excretions) in a way (PPE) that prevents contact with skin and mucous membranes

• Do not recap needles, bend or break after use

• Handle patient care equipment in a way that prevents contamination of clothing and the spread of microorganisms to other patients

• Discard each needle into a sharps container at the point of use

• Appropriately dispose of single use equipment

• Do not overload a bin if it is full

• Clean and disinfect reusable equipment after each patient episode of use. Do not share equipment with other patient’s without properly cleaning it first

• Do not leave a sharp bin in the reach of children 33

34

Serious Accident or Medical Emergency

Injuries & Accidents – Trainee Reporting

If a trainee has a serious accident or injury or needs Emergency medical care follow the guidelines below:

• All injuries and accidents must be reported to your supervisor / preceptor immediately • This includes first aid injuries and “close calls”. • Accidents and injuries resulting in medical treatment must be documented on an Incident Report Form

• Summon a first responder to the injured person and call for help • Inform the Trainee’s supervisor / preceptor • Transport the trainee to receive medical attention (example: to Emergency Department) • Notify the Entry to Practice Training Program – Ministry of Health Staff and complete required documentation forms as required • Trainee requires a physicians note indicating he is “fit” and able to return to placement and perform duties

Disciplinary Action: • Disregarding occupational health & safety rules or established safety practice in the health care setting can result in dismissal from the placement 35

36

66

Breaching Safety Practice & Disciplinary Action

Key Messages – Safety & Injury Prevention

Disciplinary Action: • Disregarding occupational health & safety rules or established safety practice in the health care setting can result in dismissal from the placement

• Workplace safety and injury prevention is the responsibility of every professional health care provider • It is critical to become knowledgeable on the plan of action, policies / procedures , and key team member roles essential for response ~ in advance of an emergency situation • All Trainees must review the Emergency Preparedness Policies & Procedures for their assigned Placement Site

Example of Violations: • Not wearing required PPE • Not immediately reporting an injury or damage • Committing an unsafe act such as tampering with equipment, removing safety warnings etc. • Operating a piece of equipment you are not authorize to operate 37

References


• Emanuel, L., Berwick, D., Conway, J., Combes, J., Hatlie, M., Leape, L., Reason, J., Schyve, P., Vincent, C., & Walton, M. (2008). What exactly is patient safety? Advances in Patient Safety, Vol. 1: Assessment. Retrieved from http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=ap s2v1&part=advances‐emanuel‐berwick_110 • Thibbeau, G.A., & Patton, K.T., (2007). Anatomy and physiology (6th ed.)., St.Louis, MO: Mosby. http://www.slideshare.net/gtwaddell/general‐safety‐ presentation

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67


Bacteriology

68

Kingdom of Saudi Arabia – Ministry of Health General Directorate of Training and Scholarship Training Program for Health Institute Graduates Bacteriology

Sterilization and Disinfection - Slide 24 – Moist Heat Methods Most effective when air is removed initially and replaced by super heated steam. This vacuum allows steam to get to places not normally reached as pockets of air would normally insulate that area and kill temperature would not be reached. - Slide 52 – Alcohols 70% alcohol gives maximum kill for bacteria and viruses. - Slide 55 – Aldehydes Not used these days routinely for disinfection due to their toxicity. Tools of the Laboratory - Slide 33 Can also use Eosin as counterstain (brighter than Safranin)

The Cocci of Medical Importance - Slide 20 – Prevention of Staphylococcal Infections Universal now known as standard precautions - Slide 71 – Other Gram-negative Cocci and Coccobacilli Now known as Moraxella catarrhalis

The Gram-Negative Bacilli of Medical Importance - Slide 12 – Burkholderia Used to be known as Pseudomonas pseudomallei - Slide 13 - Acinetobacter and Stenotrophomonas Used to known as Pseudomonas maltophilia

The Gram-Positive Bacilli of Medical Importance - Slide 56 – Diagnosis Now available to differentiate between BCG vaccination and true TB is Quantiferon Gold

Cerebrospinal Fluid (CSF) Culture - Slide 13 – Microscopic Examination Care needs to be taken when staining as the dried sediment can easily come off the slide and you are only looking at stain deposit. Take extreme care in the staining process. Preferable to do manual staining.

Urine Culture - Slide 23 – Specimen Transport This is one option. Not necessarily preferred option. Usually when delay in transport is envisaged, but not routinely - Slide 27 – Laboratory Examination of Urine This is not normally classed as abnormal result if urates seen in urine. - Slide 29 – Examine the specimens microscopically -

Slide 44 – Materials MacConkey plate can be used instead of CLED plate as well.

Respiratory Tract Infections

69


-

-

Slide 6 – Flora of respiratory tract Host defense mechanisms normally remove any microbes which invade the bronchial tract or lung alveoli in healthy people. These mechanisms keep LRT sterile Slide 7 – Types of specimens received Blood cultures should not always be collected for a LRT. Maybe sometimes if warranted but not every time. Slide 18 – Endotracheal and Tracheostomy secretions A tracheostomy is a hole made in the front of the neck, opens through trachea. breathing tube can be placed into the windpipe. Slide 21 - *Important Delays in processing of sputums can give false results. Yeasts especially grow well in sputum Slide 29 – Rejection criteria for tracheal aspirates This could also be indicative of TB and should be checked to rule this out.

70


Characteristics of Cells and Life All living things (single and multicellular) are made of cells that share some


common characteristics: – Basic shape – spherical, cubical, cylindrical


– Internal content – cytoplasm, surrounded by a membrane – DNA chromosome(s), ribosomes, metabolic capabilities Two basic cell types: eukaryotic and prokaryotic

An Introduction to the bacterial Cell Its Organization, and Members

2

Characteristics of Cells

Characteristics of Life

Eukaryotic cells: animals, plants, fungi, and protists

• Reproduction and heredity – genome composed of DNA packed in chromosomes; produce offspring sexually or asexually

– Contain membrane‐bound organelles that compartmentalize the cytoplasm and perform specific functions

• Growth and development

– Contain double‐membrane bound nucleus with DNA chromosomes

• Metabolism – chemical and physical life processes

Prokaryotic cells: bacteria and archaea

• Movement and/or irritability – respond to internal/external stimuli; self‐ propulsion of many organisms

– No nucleus or other membrane‐bound organelles

• Cell support, protection, and storage mechanisms – cell walls, vacuoles, granules and inclusions • Transport of nutrients and waste 3

Prokaryotic Profiles

4

Prokaryotic Profiles • Structures that are essential to the functions of all prokaryotic cells are a cell membrane, cytoplasm, ribosomes, and one (or a few) chromosomes

5

6

Structure of a bacterial cell

External Structures • Appendages – Two major groups of appendages: • Motility – flagella and axial filaments (periplasmic flagella) • Attachment or channels – fimbriae and pili

• Glycocalyx – surface coating

7

8

Flagella

Flagella

• 3 parts: – Filament – long, thin, helical structure composed of protein Flagellin – Hook – curved sheath – Basal body – stack of rings firmly anchored in cell wall

• Rotates 360o • Number and arrangement of flagella varies: – Monotrichous, lophotrichous, amphitrichous, peritrichous

• Functions in motility of cell through environment 9

10

Flagellar Arrangements 1.

Monotrichous – single flagellum at one end

2.

Lophotrichous – small bunches emerging from the same site

3.

Amphitrichous – flagella at both ends of cell

4.

Peritrichous – flagella dispersed over surface of cell; slowest

Electron micrographs of flagellar arrangements

11

12

72

Flagellar Responses

The operation of flagella

Guide bacteria in a direction in response to external stimulus: Chemical stimuli – chemotaxis; positive and negative Light stimuli – phototaxis

Signal sets flagella into rotary motion clockwise or counterclockwise: Counterclockwise – results in smooth linear direction – run Clockwise – tumbles

13

14

Chemotaxis in bacteria

Periplasmic Flagella • Internal flagella, enclosed in the space between the outer sheath and the cell wall peptidoglycan • Produce cellular motility by contracting and imparting twisting or flexing motion

15

16

Periplasmic flagella

Fimbriae • Fine, proteinaceous, hair‐like bristles emerging from the cell surface

• Function in adhesion to other cells and surfaces

17

18

73

Pili

Glycocalyx

• Rigid tubular structure made of pilin protein

Coating of molecules external to the cell wall, made of sugars and/or

•

proteins

• Found only in gram‐negative cells

Two types:

•

• Function to join bacterial cells for partial DNA transfer called conjugation

1.

Slime layer ‐ loosely organized and attached

2.

Capsule ‐ highly organized, tightly attached

Functions:

• –

Protect cells from dehydration and nutrient loss

–

Inhibit killing by white blood cells by phagocytosis, contributing to pathogenicity

–

Attachment ‐ formation of biofilms

19

20

21

22

Biofilm on a catheter

The Cell Envelope • External covering outside the cytoplasm • Composed of two basic layers: – Cell wall and cell membrane • Maintains cell integrity • Two different groups of bacteria demonstrated by Gram stain: – Gram‐positive bacteria: thick cell wall composed primarily of peptidoglycan and cell membrane – Gram‐negative bacteria: outer cell membrane, thin peptidoglycan layer, and cell membrane 23

24

Structure of Cell Walls • Determines cell shape, prevents lysis (bursting) or collapsing due to changing osmotic pressures

• Peptidoglycan is primary component: – Unique macromolecule composed of a repeating framework of long glycan chains cross‐linked by short peptide fragments

25

26

Peptidoglycan

Gram‐Positive Cell Wall • Thick, homogeneous sheath of peptidoglycan – 20‐80 nm thick – Includes teichoic acid and lipoteichoic acid: function in cell wall maintenance and enlargement during cell division; move cations across the cell envelope; stimulate a specific immune response – Some cells have a periplasmic space, between the cell membrane and cell wall

27

28

Gram‐Negative Cell Wall • Composed of an outer membrane and a thin peptidoglycan layer • Outer membrane is similar to cell membrane bilayer structure – Outermost layer contains lipopolysaccharides and lipoproteins (LPS) • Lipid portion (endotoxin) may become toxic when released during infections • May function as receptors and blocking immune response • Contain porin proteins in upper layer – regulate molecules entering and leaving cell – Bottom layer is a thin sheet of peptidoglycan • Periplasmic space above and below peptidoglycan 29

30

75

Comparison of Gram‐Positive and Gram‐ Negative

31

32

33

34

The Gram Stain • Differential stain that distinguishes cells with a gram‐positive cell wall from those with a gram‐negative cell wall – Gram‐positive ‐ retain crystal violet and stain purple – Gram‐negative ‐ lose crystal violet and stain red from safranin counterstain

• Important basis of bacterial classification and identification • Practical aid in diagnosing infection and guiding drug treatment

Extreme variation in shape of Mycoplasma pneumoniae

Nontypical Cell Walls • Some bacterial groups lack typical cell wall structure, i.e., Mycobacterium and Nocardia – Gram‐positive cell wall structure with lipid mycolic acid (cord factor) • Pathogenicity and high degree of resistance to certain chemicals and dyes • Basis for acid‐fast stain used for diagnosis of infections caused by these microorganisms

• Some have no cell wall, i.e., Mycoplasma – Cell wall is stabilized by sterols – Pleomorphic 35

36

76

Cell Membrane Structure

Cell membrane structure

• Phospholipid bilayer with embedded proteins – fluid mosaic model • Functions in: – Providing site for energy reactions, nutrient processing, and synthesis – Passage of nutrients into the cell and the discharge of wastes • Cell membrane is selectively permeable

37

38

Bacterial Internal Structures


• Cell cytoplasm:

• Chromosome – Single, circular, double‐stranded DNA molecule that contains all the

– Dense gelatinous solution of sugars, amino acids, and salts

genetic information required by a cell

– 70‐80% water

– Aggregated in a dense area called the nucleoid • DNA is tightly coiled

• Serves as solvent for materials used in all cell functions

39

Chromosome structure

40

Bacterial Internal Structures • Plasmids – Small circular, double‐stranded DNA – Free or integrated into the chromosome – Duplicated and passed on to offspring – Not essential to bacterial growth and metabolism – May encode antibiotic resistance, tolerance to toxic metals, enzymes, and toxins – Used in genetic engineering ‐ readily manipulated and transferred from cell to cell

41

42


Prokaryotic ribosome

• Ribosomes – Made of 60% ribosomal RNA and 40% protein – Consist of two subunits: large and small – Prokaryotic differ from eukaryotic ribosomes in size and number of proteins – Site of protein synthesis – Present in all cells

43

44


Bacterial inclusion bodies

• Inclusions and granules – Intracellular storage bodies – Vary in size, number, and content – Bacterial cell can use them when environmental sources are depleted – Examples: glycogen, poly ‐hydroxybutyrate, gas vesicles for floating, sulfur and phosphate granules (metachromatic granules), particles of iron oxide

45

46



• Endospores – Inert, resting, cells produced by some G+ genera: Clostridium, Bacillus, and

• Cytoskeleton

Sporosarcina

– Many bacteria possess an internal

• Have a 2‐phase life cycle:

network of protein polymers that is

– Vegetative cell – metabolically active and growing

closely associated with the cell wall

– Endospore – when exposed to adverse environmental conditions; capable of high resistance and very long‐term survival – Sporulation ‐ formation of endospores • Hardiest of all life forms • Withstands extremes in heat, drying, freezing, radiation, and chemicals • Not a means of reproduction – Germination ‐ return to vegetative growth

47

78

48

Endospores

Sporulation cycle

• Resistance linked to high levels of calcium and dipicolinic acid • Dehydrated, metabolically inactive • Thick coat • Longevity verges on immortality, 250 million years • Resistant to ordinary cleaning methods and boiling • Pressurized steam at 120oC for 20‐30 minutes will destroy 49

50

Bacterial Shapes, Arrangements, and Sizes

Common bacterial shapes

• Vary in shape, size, and arrangement but typically described by one of three basic shapes: – Coccus – spherical – Bacillus – rod • Coccobacillus – very short and plump • Vibrio – gently curved – Spirillum – helical, comma, twisted rod, • Spirochete – spring‐like 51

52

Bacterial Shapes, Arrangements, and Sizes

Comparison of Spiral‐Shaped Bacteria

• Arrangement of cells is dependent on pattern of division and how cells remain attached after division: – Cocci: • Singles • Diplococci – in pairs • Tetrads – groups of four • Irregular clusters • Chains • Cubical packets (sarcina) – Bacilli: • Diplobacilli • Chains • Palisades

53

54

79

The dimensions of bacteria

Arrangement of cocci

55

56

Classification Systems in the Prokaryotae

Diagnostic Scheme for Medical Use

1.

Microscopic morphology

• Uses phenotypic qualities in identification

2.

Macroscopic morphology – colony appearance

3.

Bacterial physiology

4.

Serological analysis

5.

Genetic and molecular analysis

– Restricted to bacterial disease agents – Divides bacteria based on cell wall structure, shape, arrangement, and physiological traits

57

58

Species and Subspecies • Species – a collection of bacterial cells which share an overall similar pattern of traits in contrast to other bacteria whose pattern differs significantly • Strain or variety – a culture derived from a single parent that differs in structure or metabolism from other cultures of that species (biovars, morphovars) • Type – a subspecies that can show differences in antigenic makeup (serotype or serovar), susceptibility to bacterial viruses (phage type) and in pathogenicity (pathotype)

59

80

Unusual Forms of Medically Significant Bacteria

Unusual Forms of Medically Significant Bacteria

• Obligate intracellular parasites

– Chlamydias

– Rickettsias • Tiny

• Very tiny, gram‐negative bacteria

• Obligate intracellular parasites

• Most are pathogens that alternate between mammals and blood‐sucking arthropods

• Not transmitted by arthropods

• Obligate intracellular pathogens • Chlamydia trachomatis – severe eye infection and one of the most

• Cannot survive or multiply outside of a host cell

common sexually transmitted diseases

• Cannot carry out metabolism on their own • Rickettsia rickettisii – Rocky Mountain spotted fever

• Chlamydia pneumoniae – lung infections

• Rickettsia typhi – endemic typhus 61

62

Comparison of Three Cellular Domains

63

81


Microbial Nutrition Nutrition – process by which chemical substances (nutrients) are acquired


from the environment and used for cellular activities Essential nutrients ‐ must be provided to an organism Two categories of essential nutrients:


– Macronutrients – required in large quantities; play principal roles in cell structure and metabolism • proteins, carbohydrates – Micronutrients or trace elements – required in small amounts; involved in enzyme function and maintenance of protein structure • manganese, zinc, nickel

Elements of Microbial Nutrition, Ecology, and Growth

2

Nutrients

Chemical Analysis of Microbial Cytoplasm

• Inorganic nutrients– atom or molecule that contains a

• 70% water

combination of atoms other than carbon and hydrogen

• Proteins

– metals and their salts (magnesium sulfate, ferric nitrate, sodium

• 96% of cell is composed of 6 elements:

phosphate), gases (oxygen, carbon dioxide) and water

– Carbon

• Organic nutrients‐ contain carbon and hydrogen atoms and are

– Hydrogen

usually the products of living things

– Oxygen

– methane (CH4), carbohydrates, lipids, proteins, and nucleic acids

– Phosphorous – Sulfur – Nitrogen 3

4

Sources of Essential Nutrients

Sources of Essential Nutrients Nitrogen Sources:

• Carbon sources

• Main reservoir is nitrogen gas (N2); 79% of earth’s atmosphere is N2.

• Heterotroph – must obtain carbon in an organic form such

• Nitrogen is part of the structure of proteins, DNA, RNA & ATP – these

as proteins, carbohydrates, lipids and nucleic acids, made

are the primary source of N for heterotrophs.

by other living organisms

‐

‐

• Some bacteria & algae use inorganic N nutrients (NO3 , NO2 , or NH3).

• Autotroph ‐ an organism that uses CO2, an inorganic gas as

• Some bacteria can fix N2.

its carbon source

• Regardless of how N enters the cell, it must be converted to NH3, the only form that can be combined with carbon to synthesis amino acids,

– not nutritionally dependent on other living things

etc. 5

6

82



Oxygen Sources

Hydrogen Sources

• Major component of carbohydrates, lipids, nucleic acids, and

• Major element in all organic compounds and several inorganic

proteins

ones (water, salts and gases)

• Plays an important role in structural and enzymatic functions of

• Gases are produced and used by microbes.

cell

• Roles of hydrogen:

• Component of inorganic salts (sulfates, phosphates, nitrates) and

– maintaining pH

water

– forming H bonds between molecules

• O2 makes up 20% of atmosphere

– serving as the source of free energy in oxidation‐reduction reactions of respiration

• Essential to metabolism of many organisms 7



Phosphorous (Phosphate Sources) • Main inorganic source is phosphate (PO4

8

Sulfur Sources ‐3)

derived from

• Widely distributed in environment, rocks; sediments contain

phosphoric acid (H3PO4) found in rocks and oceanic mineral

sulfate, sulfides, hydrogen sulfide gas and sulfur

deposits

• Essential component of some vitamins and the amino acids:

• Key component of nucleic acids, essential to genetics

methionine and cysteine

• Serves in energy transfers (ATP)

• Contributes to stability of proteins by forming disulfide bonds

9

10

Other Nutrients Important in Microbial Metabolism

Growth Factors: Essential Organic Nutrients

• Potassium – essential to protein synthesis and membrane function

• Organic compounds that cannot be synthesized by an organism because they lack the genetic and metabolic

• Sodium – important to some types of cell transport

mechanisms to synthesize them

• Calcium – cell wall and endospore stabilizer

• Must be provided as a nutrient

• Magnesium – component of chlorophyll; membrane and ribosome

– essential amino acids, vitamins

stabilizer • Iron – component of proteins of cell respiration • Zinc, copper, nickel, manganese, etc. 11

12

Nutritional Types • Main determinants of nutritional type are: – Carbon source – heterotroph, autotroph – Energy source – • chemotroph – gain energy from chemical compounds • phototrophs – gain energy through photosynthesis

13

14

15

16

17

18

Transport: Movement of Chemicals Across the Cell Membrane • Passive transport –does not require energy; substances exist in a gradient and move from areas of higher concentration towards areas of lower concentration – diffusion – osmosis – diffusion of water – facilitated diffusion – requires a carrier

• Active transport – requires energy and carrier proteins; gradient independent – active transport – group translocation – transported molecule chemically altered – bulk transport – endocytosis, exocytosis, pinocytosis

19

20

Environmental Factors That Influence Microbes

3 Cardinal Temperatures

• Environmental factors fundamentally affect the function of metabolic

• Minimum temperature – lowest temperature that permits

enzymes.

a microbe’s growth and metabolism

• Factors include:

• Maximum temperature – highest temperature that permits

– temperature

a microbe’s growth and metabolism

– oxygen requirements

• Optimum temperature – promotes the fastest rate of

– pH

growth and metabolism

– electromagnetic radiation – barometric pressure

21

22

23

24

3 Temperature Adaptation Groups 1.

Psychrophiles – optimum temperature below 15oC; capable of growth at 0oC

2.

Mesophiles – optimum temperature 20o‐40oC; most human pathogens

3.

Thermophiles – optimum temperature greater than 45oC

85

Gas Requirements

Categories of Oxygen Requirement

Oxygen • Aerobe – utilizes oxygen and can detoxify it

• As oxygen is utilized it is transformed into several toxic products: ‐

– singlet oxygen (O2), superoxide ion (O2 ), peroxide (H2O2), and

• Obligate aerobe ‐ cannot grow without oxygen

‐

hydroxyl radicals (OH ) • Most cells have developed enzymes that neutralize these chemicals:

• Facultative anaerobe – utilizes oxygen but can also grow in its absence

– superoxide dismutase, catalase • If a microbe is not capable of dealing with toxic oxygen, it is forced to

• Microaerophilic – requires only a small amount of oxygen

live in oxygen free habitats.

25

26

Categories of Oxygen Requirement

Carbon Dioxide Requirement

• Anaerobe – does not utilize oxygen

All microbes require some carbon dioxide in their metabolism.

• Obligate anaerobe ‐ lacks the enzymes to detoxify oxygen so cannot

• Capnophile – grows best at higher CO2 tensions than normally present

survive in an oxygen environment

in the atmosphere

• Aerotolerant anaerobes – do no utilize oxygen but can survive and grow in its presence

27

28

Effects of pH • Majority of microorganisms grow at a pH between 6 and 8 • Obligate acidophiles – grow at extreme acid pH • Alkalinophiles – grow at extreme alkaline pH

29

30

86

Osmotic Pressure

Other Environmental Factors

• Most microbes exist under hypotonic or isotonic conditions

• Barophiles – can survive under extreme pressure and will rupture if exposed to normal atmospheric pressure

• Halophiles – require a high concentration of salt • Osmotolerant – do not require high concentration of solute but can tolerate it when it occurs

31

32

Ecological Associations Among Microorganisms

Ecological Associations Among Microorganisms

• Symbiotic – organisms live in close nutritional relationships; required by

• Non‐symbiotic – organisms are free‐living; relationships not

one or both members

required for survival

– mutualism – obligatory, dependent; both members benefit

– synergism – members cooperate and share nutrients – commensalism – commensal member benefits, other member not

– antagonism – some member are inhibited or destroyed

harmed

by others

– parasitism – parasite is dependent and benefits; host is harmed

33

34

Interrelationships Between Microbes and Humans

Microbial Biofilms • Biofilms result when organisms attach to a substrate by

• Human body is a rich habitat for symbiotic bacteria, fungi,

some form of extracellular matrix that binds them together

and a few protozoa ‐ normal microbial flora

in complex organized layers

• Commensal, parasitic, and synergistic

• Dominate the structure of most natural environments on earth • Communicate and cooperate in the formation and function of biofilms – quorum sensing

35

36

87

The Study of Microbial Growth • Microbial growth occurs at two levels: growth at a cellular level with increase in size, and increase in population • Division of bacterial cells occurs mainly through binary fission (transverse) – parent cell enlarges, duplicates its chromosome, and forms a central transverse septum dividing the cell into two daughter cells

37

38

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40

Rate of Population Growth • Time required for a complete fission cycle is called the generation, or doubling time • Each new fission cycle increases the population by a factor of 2 – exponential or logarithmic growth. • Generation times vary from minutes to days.

Rate of Population Growth

The Population Growth Curve

• Equation for calculating population size over time:

•

In laboratory studies, populations typically display a predictable pattern over time – growth curve.

n

Nƒ = (Ni)2

•

Nƒ is total number of cells in the population.

Stages in the normal growth curve:

1.

Lag phase – “flat” period of adjustment, enlargement; little growth

2.

Exponential growth phase – a period of maximum growth will continue as long as cells have adequate nutrients and a favorable environment

Ni is starting number of cells. 3.

Exponent n denotes generation time.

Stationary phase – rate of cell growth equals rate of cell death caused by depleted nutrients and O2, excretion of organic acids and pollutants

n

4.

2 number of cells in that generation

Death phase – as limiting factors intensify, cells die exponentially in their own wastes

41

42

Methods of Analyzing Population Growth • Turbidometry – most simple • Degree of cloudiness, turbidity, reflects the relative population size • Enumeration of bacteria: – viable colony count – direct cell count – count all cells present; automated or manual 43

44

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89


Controlling Microorganisms • Physical, chemical, and mechanical methods to destroy or reduce


undesirable microbes in a given area (decontamination) • Primary targets are microorganisms capable of causing infection or spoilage:


– Vegetative bacterial cells and endospores – Fungal hyphae and spores, yeast – Protozoan trophozoites and cysts – Worms – Viruses

Sterilization and Disinfection

– Prions 2

Relative Resistance of Microbes •

Highest resistance

•

Moderate resistance

– Prions, bacterial endospores

– Pseudomonas sp. – Mycobacterium tuberculosis – Staphylococcus aureus – Protozoan cysts

•

Least resistance – Most bacterial vegetative cells – Fungal spores and hyphae, yeast – Enveloped viruses – Protozoan trophozoites

3

4

Terminology and Methods of Control • Sterilization – a process that destroys all viable microbes, including viruses, prions and endospores • Disinfection – a process to destroy vegetative pathogens, not endospores; inanimate objects • Antiseptic – disinfectants applied directly to exposed body surfaces • Sanitization – any cleansing technique that mechanically removes microbes • Degermation – reduces the number of microbes through mechanical means • ‐cide= to kill, bactericide, viricide, etc 5

• Statis/ static‐ stand still‐ bacteriostatic

6

Microbial Death • Permanent loss of reproductive capability, even under optimum growth conditions • Hard to detect, microbes often reveal no conspicuous vital signs to begin with

7

Factors That Affect Death Rate

8

Number of microbes

The effectiveness of a particular agent is governed by several factors:  Number of microbes  Nature of microbes in the population  Temperature and pH of environment  Concentration or dosage of agent  Mode of action of the agent  Presence of solvents, organic matter, or inhibitors  Length of exposure to the agent 9

Nature of microbes

10

Mode of action of the agent

11

12

Length of exposure to the agent

Practical Concerns in Microbial Control Selection of method of control depends on circumstances:  Does the application require sterilization?  Is the item to be reused?  Can the item withstand heat, pressure, radiation, or chemicals?  Is the method suitable?  Will the agent penetrate to the necessary extent?  Is the method cost‐ and labor‐efficient and is it safe?

13

14

15

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Antimicrobial Agents’ Modes of Action Cellular targets of physical and chemical agents: 1. The cell wall – cell wall becomes fragile and cell lyses; some antimicrobial drugs, detergents, and alcohol 2. The cell membrane – loses integrity; detergent surfactants 3. Protein and nucleic acid synthesis – prevention of replication, transcription, translation, peptide bond formation, protein synthesis; chloramphenicol, ultraviolet radiation, formaldehyde 4. Proteins – disrupt or denature proteins; alcohols, phenols, acids, heat

Methods of Physical Control 1. Heat – moist and dry 2. Cold temperatures 3. Desiccation 4. Radiation 5. Filtration

17

18

Mode of Action and Relative Effectiveness of Heat •

Moist heat – lower temperatures and shorter exposure time; coagulation and denaturation of proteins

•

Dry heat – moderate to high temperatures; dehydration, alters protein structure; incineration

19

20

Heat Resistance and Thermal Death

Thermal Death Measurements

Bacterial endospores most resistant – usually require temperatures above boiling

• Thermal death time (TDT) – shortest length of time required to kill all test microbes at a specified temperature • Thermal death point (TDP) – lowest temperature required to kill all microbes in a sample in 10 minutes

21

22

Moist Heat Methods • Steam under pressure – sterilization • Autoclave 15 psi/121oC/10‐40min • Steam must reach surface of item being sterilized • Item must not be heat or moisture sensitive • Mode of action – denaturation of proteins, destruction of membranes and DNA

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93

Nonpressurized Steam • Tyndallization – intermittent sterilization for substances that cannot withstand autoclaving • Items exposed to free‐flowing steam for 30–60 minutes, incubated for 23–24 hours and then subjected to steam again • Repeat cycle for 3 days • Used for some canned foods and laboratory media • Disinfectant 25

26

Boiling Water

Pasteurization

• Boiling at 100°C for 30 minutes to destroy non‐spore‐forming

• Pasteurization – heat is applied to kill potential agents of infection and

pathogens

spoilage without destroying the food flavor or value

• Disinfection

• 63°C–66°C for 30 minutes (batch method) • 71.6°C for 15 seconds (flash method) • Not sterilization – kills non‐spore‐forming pathogens and lowers overall microbe count; does not kill endospores or many nonpathogenic microbes 27

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Dry Heat Dry heat using higher temperatures than moist heat • Incineration – flame or electric heating coil –Ignites and reduces microbes and other substances • Dry ovens – 150–180oC – coagulate proteins

Desiccation

Cold

• Gradual removal of water from cells, leads to metabolic Microbiostatic – slows the growth of microbes

inhibition • Not effective microbial control – many cells retain ability to

• Refrigeration 0–15oC and freezing <0oC

grow when water is reintroduced • Lyophilization – freeze drying; preservation

• Used to preserve food, media, and cultures

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34

Radiation • Ionizing Radiation – deep penetrating power that has sufficient energy to cause electrons to leave their orbit, breaks DNA – Gamma rays, X‐rays, cathode rays – Cold (low temperature) sterilization – Used to sterilize medical supplies and food products

Radiation • Nonionizing radiation – little penetrating power – must be directly exposed • UV light creates pyrimidine dimers, which interfere with replication

35

36

Filtration • Physical removal of microbes by passing a gas or liquid through filter • Used to sterilize heat sensitive liquids and air in hospital isolation units and industrial clean rooms

37

38

Summary: Methods of Physical Control • Heat‐ moist vs. dry – Moist‐ steam (pressure or none), boiling water, pasteurization – Dry‐ incineration, hot air • Thermal death time (TDT) • Thermal death point (TDP) • Cold and dessication – lyophilization • Radiation • Filtration 39

40

Chemical Agents in Microbial Control

Levels of Chemical Decontamination

• Disinfectants, antiseptics, sterilants, degermers, and preservatives

• High‐level germicides – kill endospores; may be sterilants – Devices that are not heat sterilizable and intended to be used in sterile

• Some desirable qualities of chemicals:

environments (body tissue)

– Rapid action in low concentration

• Intermediate‐level – kill fungal spores (not endospores), tubercle bacillus, and viruses

– Solubility in water or alcohol, stable

– Used to disinfect devices that will come in contact with mucous

– Broad spectrum, low toxicity

membranes but are not invasive – Penetrating

• Low‐level – eliminate only vegetative bacteria, vegetative fungal cells, and some viruses

– Noncorrosive and nonstaining

– Clean surfaces that touch skin but not mucous membranes

– Affordable and readily available 41

42

Factors that Affect Germicidal Activity of Chemicals • Nature of the material being treated • Degree of contamination • Time of exposure • Strength and chemical action of the germicide

43

Germicidal Categories 1.

Halogens

2.

Phenolics

3.

Chlorhexidine

4.

Alcohols

5.

Hydrogen peroxide

6.

Detergents & soaps

7.

Heavy metals

8.

Aldehydes

9.

44

Halogens • Chlorine – Cl2, hypochlorites (chlorine bleach), chloramines –Denaturate proteins by disrupting disulfide bonds –Intermediate level –Unstable in sunlight, inactivated by organic matter –Water, sewage, wastewater, inanimate objects • Iodine ‐ I2, iodophors (betadine) –Interferes with disulfide bonds of proteins –Intermediate level

Gases

–Milder medical and dental degerming agents, disinfectants, ointments

10. Dyes 45

46

Phenolics • Disrupt cell walls and membranes and precipitate proteins • Low to intermediate level – bactericidal, fungicidal, virucidal, not sporicidal – Lysol – Triclosan – antibacterial additive to soaps

47

48

Chlorhexidine • A surfactant and protein denaturant with broad microbicidal properties • Low to intermediate level • Hibiclens, Hibitane • Used as skin degerming agents for preoperative scrubs, skin cleaning, and burns

49

50

Alcohols • Ethyl, isopropyl in solutions of 50‐95% • Act as surfactants dissolving membrane lipids and coagulating proteins of vegetative bacterial cells and fungi • Intermediate level

51

52

53

54

Hydrogen Peroxide • Produce highly reactive hydroxyl‐free radicals that damage protein and DNA while also decomposing to O2 gas – toxic to anaerobes • Antiseptic at low concentrations; strong solutions are sporicidal

98

Aldehydes • Glutaraldehyde and formaldehyde kill by alkylating protein and DNA • Glutaraldehyde in 2% solution (Cidex) used as sterilant for heat sensitive instruments • High level • Formaldehyde – disinfectant, preservative, toxicity limits use –Formalin – 37% aqueous solution • Intermediate to high level 55

56

57

58

Gases and Aerosols • Ethylene oxide, propylene oxide • Strong alkylating agents • High level • Sterilize and disinfect plastics and prepackaged devices, foods

Detergents and Soaps • Quaternary ammonia compounds (quats) act as surfactants that alter membrane permeability of some bacteria and fungi • Very low level • Soaps – mechanically remove soil and grease containing microbes

59

60

99

61

62

Heavy Metals • Solutions of silver and mercury kill vegetative cells in low concentrations by inactivating proteins • Oligodynamic action • Low level • Merthiolate, silver nitrate, silver

63

64

65

66

100

Dyes as Antimicrobial Agents

Acids and Alkalis

• Aniline dyes are very active against gram‐positive species of bacteria and

• Low level of activity

various fungi

– Organic acids prevent spore germination and bacterial and fungal growth

• Sometimes used for antisepsis and wound treatment

– Acetic acid inhibits bacterial growth

• Low level, narrow spectrum of activity

– Propionic acid retards molds – Lactic acid prevents anaerobic bacterial growth – Benzoic and sorbic acid inhibit yeast

67

68

69

101


Training Program for Health Institute Graduates Laboratories

Tools of the Laboratory: The Methods for Studying Microorganisms

2

Discovery of Microorganisms • magnification – ability to enlarge objects • resolving power – ability to show detail

• Antony van Leeuwenhoek (1632‐ 1723) – first person to observe and describe micro‐ organisms accurately

3

4

Lenses and the Bending of Light

Lenses

• light is refracted (bent) when passing from one medium to another • refractive index

• focus light rays at a specific place called the focal point • distance between center of lens and focal point is the focal length • strength of lens related to focal length

– a measure of how greatly a substance slows the velocity of light

– short focal length more magnification

• direction and magnitude of bending is determined by the refractive indexes of the two media forming the interface

5

6

102

7

8

• Working Distance — distance between the front surface of lens and surface of cover glass or specimen

9

10

Microscope Resolution • Ability of a lens to separate or distinguish small objects that are close together • Wavelength of light used is major factor in resolution Shorter wavelength  greater resolution

11

12

103

The Light Microscope • Many types – – – –

Bright‐field microscope Dark‐field microscope Phase‐contrast microscope Fluorescence microscopes

• Are compound microscopes – Image formed by action of 2 lenses

13

14

The Bright-Field Microscope

The Dark-Field Microscope

• Produces a dark image against a brighter background • Has several objective lenses

• Produces a bright image of the object against a dark background

– Parfocal microscopes remain in focus when objectives are changed

• Used to observe living, unstained preparations

• Total magnification – Product of the magnifications of the ocular lens and the objective lens

15

16

The Phase-Contrast Microscope • Enhances the contrast between intracellular structures having slight differences in refractive index • Excellent way to observe living cells

17

18

104

The Differential Interference Contrast Microscope

The Fluorescence Microscope

• Creates image by detecting differences in refractive indices and thickness of different parts of specimen

• Exposes specimen to ultraviolet, violet, or blue light • Specimens usually stained with fluorochromes

• Excellent way to observe living cells • Shows a bright image of the object resulting from the fluorescent light emitted by the specimen

19

20

21

22

Specimens Preparation

Preparation and Staining of Specimens

• Wet mounts & hanging drop mounts – allow examination of characteristics of live cells: motility, shape, & arrangement

• Increases visibility of specimen • Accentuates specific morphological features • Preserves specimens

• Fixed mounts are made by drying & heating a film of specimen. This smear is stained using dyes to permit visualization of cells or cell parts.

23

24

105

Fixation

Dyes and Simple Staining

• Process by which internal and external structures are preserved and fixed in position • Process by which organism is killed and firmly attached to microscope slide

• Dyes – Make internal and external structures of cell more visible by increasing contrast with background – Have two common features • Chromophore groups

– Heat fixing • Preserves overall morphology but not internal structures – Chemical fixing • Protects fine cellular substructure and morphology of larger, more delicate organisms

– Chemical groups with conjugated double bonds – Give dye its color

• Ability to bind cells

25

26

27

28

Dyes and Simple Staining • Dyes… – cationic dyes ‐ basic, with positive charges on the chromophore – anionic dyes ‐ acidic, with negative charges on the chromophore – surfaces of microbes are negatively charged and attract basic dyes – positive staining. – negative staining – microbe repels dye & it stains the background

Dyes and Simple Staining

Differential Staining

• Simple staining

• Divides microorganisms into groups based on their staining properties

– A single staining agent is used – Basic dyes are frequently used • Dyes with positive charges • E.G., Crystal violet

– E.G., Gram stain – E.G., Acid‐fast stain

29

30

106

Gram staining • Most widely used differential staining procedure • Divides bacteria into two groups based on differences in cell wall structure

primary stain

mordant

decolorization counterstain Positive negative 31

32

Acid-fast staining • Particularly useful for staining members of the genus mycobacterium E.G., Mycobacterium tuberculosis – causes tuberculosis E.G., Mycobacterium leprae – causes leprosy – High lipid content in cell walls is responsible for their staining characteristics

Escherichia coli – a gram‐negative rod

33

34

Staining Specific Structures

Staining Specific Structures

• Negative staining

• Spore staining – Double staining technique – Bacterial endospore is one color and vegetative cell is a different color

– Often used to visualize capsules surrounding bacteria – Capsules are colorless against a stained background

• Flagella staining – Mordant applied to increase thickness of flagella

35

36

107

Types of stains

Electron Microscopy • Beams of electrons are used to produce images • Wavelength of electron beam is much shorter than light, resulting in much higher resolution

37

38

39

40

The Transmission Electron Microscope • Electrons scatter when they pass through thin sections of a specimen • Transmitted electrons (those that do not scatter) are used to produce image • Denser regions in specimen, scatter more electrons and appear darker

Specimen Preparation

Other preparation methods

• Analogous to procedures used for light microscopy • For transmission electron microscopy, specimens must be cut very thin • Specimens are chemically fixed and stained with electron dense material

• Shadowing – Coating specimen with a thin film of a heavy metal

• Freeze‐etching – Freeze specimen then fracture along lines of greatest weakness (e.G., Membranes)

41

42

108

Ebola

43

44

The Scanning Electron Microscope • Uses electrons reflected from the surface of a specimen to create image • Produces a 3‐dimensional image of specimen’s surface features

Fly head

45

46

Newer Techniques in Microscopy • Confocal microscopy and scanning probe microscopy • Have extremely high resolution • Can be used to observe individual atoms

47

48

109

The 5 I’s of culturing microbes

Isolation

1. Inoculation – introduction of a sample into a container of media

• If an individual bacterial cell is separated from other cells & has space on a nutrient surface, it will grow into a mound of cells‐ a colony

2. Incubation – under conditions that allow growth

• A colony consists of one species

3. Isolation –separating one species from another 4. Inspection 5. Identification 49

50

51

52

Isolation technique

Media – providing nutrients in the laboratory

Types of media

• Most commonly used:

• Synthetic – contains pure organic & inorganic compounds in an exact chemical formula • Complex or nonsynthetic – contains at least one ingredient that is not chemically definable • General purpose media‐ grows a broad range of microbes, usually nonsynthetic • Enriched media‐ contains complex organic substances such as blood, serum, hemoglobin or special growth factors required by fastidious microbes

– Nutrient broth – liquid medium containing beef extract & peptone – Nutrient agar – solid media containing beef extract, peptone & agar

• Agar is a complex polysaccharide isolated from red algae – Solid at room temp, liquefies at boiling (100oc), does not resolidify until it cools to 42oc – Provides framework to hold moisture & nutrients – Not digestible for most microbes 53

54

110

Enriched media • Selective media‐ contains one or more agents that inhibit growth of some microbes and encourage growth of the desired microbes • Differential media – allows growth of several types of microbes and displays visible differences among desired and undesired microbes

55

56

Selective & Differential Media

Selective media

57

58

Differential media

Types of Agar – Blood Agar • Defining Feature: – Contains 5% blood cells from an animal, usually sheep.

• What Will Grow: – Most bacteria will grow on this.

59

60

111

Types of Agar – Chocolate Agar

Types of Agar – Chocolate Agar

• Defining Features:

• What Will Grow: Most bacteria will grow on this.

– Made of lysed red blood cells, typically sheep. – Nutrient medium used to culture fastidious organisms. – DOES NOT give haemolysis data

– Haemophilus grows due to X & V factors from the sheep blood – cannot differentiate between the species though! – Neisseria grows due to nutrient‐rich environment. 61

62

Types of Agar – MacConkey Agar

Types of Agar – Thayer‐Martin Agar

• Defining Feature:


– E. coli differentiates by growing into red colonies due to sugar fermentation. – Can be mixed with or without sugar lactose.

– Designed to isolate Neisseria gonorrhoeae.

• What Will Grow: – Specifically designed for Neisseria gonorrhoeae.

• What Will Grow: – Will only grow Gram Negative bacteria.

63

64

Types of Agar – Xylose Lysine Deoxychocolate Agar

Types of Agar – Sabouraud Agar



– Used for culturing stool samples. – Contains two different indicators. – Colonies of organism that ferment lactose will appear yellow.

– Used to grow fungi. – Has a low pH (kills most bacteria). – Contains gentamicin antibiotic (particularly useful for Gram Negative bacteria).

• What Will Grow: – Formulated to inhibit Gram‐ positive bacteria. – Formulated to encourage Gram‐ negative bacilli.

• What Will Grow: – Grows fungi. 65

66

112

Miscellaneous media

Carbohydrate fermentation media

• reducing medium – contains a substance that absorbs oxygen or slows penetration of oxygen into medium; used for growing anaerobic bacteria • carbohydrate fermentation medium‐ contains sugars that can be fermented, converted to acids, and a pH indicator to show the reaction; basis for identifying bacteria and fungi

67

68

113


Principles of Antimicrobial Therapy • Administer to an infected person a drug that destroys the


infective agent without harming the host’s cells.


• Antimicrobial drugs are produced naturally or synthetically.

Antimicrobial chemotherapy and sensitivity testing 2

3

4

Origins of Antibiotic Drugs

Interactions Between Drug and Microbe

• Antibiotics are common metabolic products of aerobic spore‐

• Antimicrobial drugs should be selectively toxic ‐ drugs should

forming bacteria and fungi.

kill or inhibit microbial cells without simultaneously damaging host tissues.

– bacteria in genera Streptomyces and Bacillus

• As the characteristics of the infectious agent become more

– molds in genera Penicillium and Cephalosporium

similar to the vertebrate host cell, complete selective toxicity

• By inhibiting the other microbes in the same habitat, antibiotic producers have less competition for nutrients and

becomes more difficult to achieve and more side effects are

space.

seen

5

6

114

Mechanisms of Drug Action 1.

Inhibition of cell wall synthesis

2.

Disruption of cell membrane structure or function

3.

Inhibition of nucleic acid synthesis, structure or function

4.

Inhibition of protein synthesis

5.

Blocks on key metabolic pathways

7

8

The Spectrum of an Antimicrobic Drug

Antimicrobial Drugs That Affect the Bacterial Cell Wall

• Spectrum – range of activity of a drug

• Most bacterial cell walls contain peptidoglycan.

– Narrow‐spectrum – effective on a small range of microbes

• Penicillins and cephalosporins block synthesis of peptidoglycan, causing the cell wall to lyse.

• Target a specific cell component that is found only in

• Active on young, growing cells

certain microbes – Broad‐spectrum – greatest range of activity

• Penicillins do not penetrate the outer membrane and are less effective against Gram‐negative bacteria.

• Target cell components common to most pathogens

• Broad spectrum penicillins and cephalosporins can cross the cell walls of Gram‐negative bacteria. 9

10

Antimicrobial Drugs That Disrupt Cell Membrane Function • A cell with a damaged membrane dies from disruption in metabolism or lysis. • These drugs have specificity for a particular microbial group, based on differences in types of lipids in their cell membranes. • Polymyxins interact with phospholipids and cause leakage, particularly in gram‐negative bacteria. • Amphotericin b and nystatin form complexes with sterols on fungal membranes which causes leakage.

11

12

115

Drugs That Inhibit Nucleic Acid Synthesis • May block synthesis of nucleotides, inhibit replication, or stop transcription • Chloroquine binds and cross‐links the double helix; quinolones inhibit DNA helicases. • Antiviral drugs that are analogs of purines and pyrimidines insert in viral nucleic acid, preventing replication.

13

14

15

16

17

18

Drugs That Block Protein Synthesis • Ribosomes of eucaryotes differ in size and structure from procaryotes; antimicrobics usually have a selective action against procaryotes; can also damage the eucaryotic mitochondria • Aminoglycosides (streptomycin, gentamycin) insert on sites on the 30S subunit and cause misreading of mRNA. • Tetracyclines block attachment of tRNA on the A acceptor site and stop further synthesis.

Drugs that Affect Metabolic Pathways • Sulfonamides and trimethoprim block enzymes required for tetrahydrofolate synthesis needed for DNA and RNA synthesis. • Competitive inhibition – drug competes with normal substrate for enzyme’s active site • Synergistic effect – an additive effect, achieved by multiple drugs working together, requiring a lower dose of each

Survey of Major Antimicrobial Drug Groups

Penicillin and Its Relatives • Large diverse group of compounds

• Antibacterial drugs

• Could be synthesized in the laboratory

– antibiotics

• More economical to obtain natural penicillin through

– synthetic drugs

microbial fermentation and modify it to semi‐synthetic forms

• Antifungal drugs

• Penicillium chrysogenum – major source

• Antiprotozoan drugs

• All consist of 3 parts:

• Antiviral drugs

– Thiazolidine ring

About 260 different antimicrobial drugs are classified in 20 drug

– Beta‐lactam ring – Variable side chain dictating microbial activity

families. 19

20

• Beta‐lactam antimicrobials ‐ all contain a highly reactive 3 carbon, 1 nitrogen ring • Primary mode of action is to interfere with cell wall synthesis. • Greater than ½ of all antimicrobic drugs are beta‐lactams. • Penicillins and cephalosporins most prominent beta‐lactams

21

22

Subgroup and Uses of Penicillins • Penicillins G and V most important natural forms • Penicillin is the drug of choice for Gram‐positive cocci (streptococci) and some Gram‐negative bacteria (meningococci and syphilis spirochete). • Semisynthetic penicillins – ampicillin, carbenicillin and amoxicillin have broader spectra – Gram‐negative enteric rods • Penicillinase‐resistant – methicillin, nafcillin, cloxacillin • Primary problems – allergies and resistant strains of bacteria

23

24

117

Cephalosporins

Cephalosporins

• Account for majority of all antibiotics administered

• 4 Generations exist: each group more effective against Gram‐negatives than the one before with improved dosing schedule and fewer side

• Isolated from Cephalosporium acremonium mold

effects

• Synthetically altered beta‐lactam structure

– First generation – cephalothin, cefazolin – most effective against gram‐ positive cocci and few gram‐negative

• Relatively broad‐spectrum, resistant to most penicillinases, &

– Second generation – cefaclor, cefonacid – more effective against gram‐

cause fewer allergic reactions

negative bacteria

• Some are given orally; many must be administered

– Third generation – cephalexin, ceftriaxone – broad‐spectrum activity against enteric bacteria with beta‐lactamases

parenterally.

– Fourth generation – cefepime – widest range; both gram‐ negative and gram‐

• Generic names have root – cef, ceph, or kef.

positive 25

26

The Acquisition of Drug Resistance • Adaptive response in which microorganisms begin to tolerate an amount of drug that would ordinarily be inhibitory; due to genetic versatility or variation; intrinsic and acquired • Acquired resistance: – Spontaneous mutations in critical chromosomal genes – Acquisition of new genes or sets of genes via transfer from another species • Originates from resistance factors (plasmids) encoded with drug resistance, transposons 27

28

Mechanisms of Drug Resistance • Drug inactivation by acquired enzymatic activity ‐ penicillinases • Decreased permeability to drug or increased elimination of drug from cell – acquired or mutation • Change in drug receptors – mutation or acquisition • Change in metabolic patterns – mutation of original enzyme

29

30

118

31

32

Considerations in Selecting an Antimicrobial Drug

Identifying the Agent

• Identify the microorganism causing the infection.

• Identification of infectious agent should be attempted as soon as possible.

• Test the microorganism’s susceptibility (sensitivity) to various

• Specimens should be taken before antimicrobials are

drugs in vitro when indicated.

initiated.

• The overall medical condition of the patient

33

34

35

36

Testing for Drug Susceptibility Minimum inhibitory concentration [MIC] – The smallest concentration of antibiotic that inhibits the growth of organism

Liquid media (dilution) allows MIC estimation Solid media (diffusion) – Disk diffusion (Kirby‐Bauer) – E‐tests – Allows MIC estimation

Beta lactamase production: quick screening method

119

Dilution in liquid broth

Kirby‐Bauer disc testing Antibiotic‐impregnated discs placed on an agar plate at the

• Tubes containing increasing antibiotic concentrations

interface between test organism and susceptible control organism

• Incubation during 18 hr at 37°C

Resulting zones of inhibition compared, use of controls

• Tedious

Susceptibility is inferred (standard tables)

Bacterial growth

0 (Control) 0,25

Inhibition

0,50

1

2

4

8

mg/l 37

38

E‐test

Reading E‐tests Ciprofloxacin for Yersinia pestis

Plastic strips with a predefined gradient of – One antibiotic – One antifungal Only one manufacturer One strip per antibiotic Wide range of antibiotics Easy to use Storage at ‐20°C Short shelf life, expensive

Resistant > 4 ug/ml

Intermediate 1-4 ug/ml

Susceptible < 1 Upper reading 39

40

Interpretation

Critical points in quality assurance

The main concept is the “clinical categorisation"

1. Culture media: Muller‐Hinton

• Strains are sorted according to level of Minimal Inhibitory

2. Reagents: disks

Concentration (MIC) versus reference breakpoints

3. Size of the inoculums

• c and C are the minor and major breakpoints Susceptible

Intermediate

4. Incubation condition 5. Control with reference strains

Resistant

6. Reading inhibition diameters (accurate measurement) MIC <

c

≤ MIC <

C

≤ MIC

7. Knowledge of staff 41

42

120

Common interpretation problems


Results depends on the technique used

An agar gel that is too thick leads to smaller zones

Many factors influence results – Lack of standardization of the inoculums – Thickness and quality of the culture media – Quality and conservation of the disks – Quality control with standardized strains – Condition and duration of incubation 43

44



Problem with the size of the inoculums Contamination with another organism Solution: • Use McFarland 0.5 photometer • Scale ‐> same tubes 45

46


Common interpretation problems Problems with E‐test reading

Bad manipulation Inoculation of the Muller Hinton – Swabbing – Not by flooding

47

48

121


General Characteristics of the Staphylococci • Common inhabitant of the skin and mucous membranes


• Spherical cells arranged in irregular clusters


• Gram‐positive • Lack spores and flagella • May have capsules • 31 species

The Cocci of Medical Importance

2

Staphylococcus aureus • Grows in large, round, opaque colonies • Optimum temperature of 37oC • Facultative anaerobe • Withstands high salt, extremes in pH, and high temperatures • Carried in nasopharynx and skin • Produces many virulence factors 3

4

Virulence factors of S. aureus Enzymes: • Coagulase – coagulates plasma and blood; produced by 97% of human isolates; diagnostic • Hyaluronidase – digests connective tissue • Staphylokinase – digests blood clots • DNase – digests DNA • Lipases – digest oils; enhances colonization on skin • Penicillinase – inactivates penicillin 5

6

122

Virulence factors of S. aureus Toxins: • Hemolysins (α, β, γ, δ) – lyse red blood cells • Leukocidin – lyses neutrophils and macrophages • Enterotoxin – induce gastrointestinal distress • Exfoliative toxin – separates the epidermis from the dermis • Toxic shock syndrome toxin (TSST) ‐ induces fever, vomiting, shock, systemic organ damage 7

8

Staphylococcal Disease

Epidemiology and Pathogenesis

Range from localized to systemic • Present in most environments frequented by humans

• Localized cutaneous infections – invade skin through wounds, follicles, or glands

• Readily isolated from fomites

– Folliculitis – superficial inflammation of hair follicle; usually resolved with no

• Carriage rate for healthy adults is 20‐60%.

complications but can progress – Furuncle – boil; inflammation of hair follicle or sebaceous gland progresses

• Carriage is mostly in anterior nares, skin, nasopharynx, intestine.

into abscess or pustule

• Predisposition to infection include: poor hygiene and nutrition, tissue

– Carbuncle – larger and deeper lesion created by aggregation and

injury, preexisting primary infection, diabetes, immunodeficiency.

interconnection of a cluster of furuncles – Impetigo – bubble‐like swellings that can break and peel away; most common

• Increase in community acquired methicillin resistance ‐ MRSA

in newborns 9

10

Staphylococcal Disease • Systemic infections – Osteomyelitis – infection is established in the metaphysis; abscess forms – Bacteremia ‐

primary origin is bacteria from another

infected site or medical devices; endocarditis possible

11

12

Staphylococcal Disease • Toxigenic disease – Food intoxication – ingestion of heat stable enterotoxins; gastrointestinal distress – Staphylococcal scalded skin syndrome – toxin induces bright red flush, blisters, then desquamation of the epidermis – Toxic shock syndrome – toxemia leading to

shock and

organ failure 13

14

Other Staphylococci Coagulase‐negative staphylococcus; frequently involved in nosocomial and opportunistic infections • S. epidermidis – lives on skin and mucous membranes; endocarditis, bacteremia, UTI • S. hominis – lives around apocrine sweat glands • S. capitis – live on scalp, face, external ear • All 3 may cause wound infections by penetrating through broken skin. • S. saprophyticus – infrequently lives on skin, intestine, vagina; UTI

15

16

17

18

Identification of Staphylococcus in Samples • Frequently isolated from pus, tissue exudates, sputum, urine, and blood

• Cultivation, catalase, biochemical testing, coagulase

Clinical Concerns and Treatment

Prevention of Staphylococcal Infections • Universal precautions by healthcare providers to prevent

• 95% have penicillinase and are resistant to penicillin and ampicillin.

nosocomial infections • Hygiene and cleansing

• MRSA – methicillin‐resistant S. aureus – carry multiple resistance

• Abscesses have to be surgically perforated.

• Systemic infections require intensive lengthy therapy.

19

20

21

22

23

24

General Characteristics of Streptococci • Gram‐positive spherical/ovoid cocci arranged in long chains; commonly in pairs • Non‐spore‐forming, nonmotile • Can form capsules and slime layers • Facultative anaerobes • Do not form catalase, but have a peroxidase system • Most parasitic forms are fastidious and require enriched media. • Small, nonpigmented colonies • Sensitive to drying, heat and disinfectants • 25 species

Streptococci • Lancefield classification system based on cell wall Ag – 17 groups (A,B,C,….) • Another classification system is based on hemolysis reactions. ‐hemolysis – A,B,C,G and some D strains  –hemolysis – S. pneumoniae and others collectively called viridans

Human Streptococcal Pathogens

β‐hemolytic S. pyogenes

• S. pyogenes

• Most serious streptococcal pathogen

• S. agalactiae

• Strict parasite

• Viridans streptococci

• Inhabits throat, nasopharynx, occasionally skin

• S. pneumoniae • Enterococcus faecalis

25

26

27

28

Virulence Factors of β ‐hemolytic S. pyogenes Produces surface antigens: – C‐carbohydrates – protect against lysozyme – Fimbriae ‐ adherence – M‐protein – contributes to resistance to phagocytosis – Hyaluronic acid capsule – provokes no immune response

Virulence Factors of β ‐hemolytic S. pyogenes

Virulence Factors of β ‐hemolytic S. pyogenes

Extracellular toxins:

Extracellular enzymes

Streptolysins – hemolysins; streptolysin O (SLO) and streptolysin S (SLS) – both cause cell and tissue injury

Streptokinase – digests fibrin clots

Pyogenic toxin (erythrogenic) – induces fever and typical red Hyaluronidase – breaks down connective tissue

rash Superantigens

–

strong

monocyte

and

lymphocyte

DNase – hydrolyzes DNA

stimulants; cause the release of tissue necrotic factor 29

30


Scope of Clinical Disease

• Humans only reservoir

Skin infections • Impetigo (pyoderma) – superficial lesions that break and form highly

• Inapparent carriers

contagious crust; often occurs in epidemics in school children; also

• Transmission – contact, droplets, food, fomites

associated with insect bites, poor hygiene, and crowded living conditions • Erysipelas – pathogen enters through a break in the skin and eventually

• Portal of entry generally skin or pharynx

spreads to the dermis and subcutaneous tissues; can remain superficial • Children predominant group affected for cutaneous and throat infections

or become systemic

Throat infections

• Systemic infections and progressive sequelae possible if untreated

• Streptococcal pharyngitis – strep throat 31

32

33

34

Long‐Term Complications of Group A Infections

Scope of Clinical Disease Systemic infections

• Rheumatic fever – follows overt or subclinical pharyngitis in children;

• Scarlet fever – strain of S. pyogenes carrying a prophage that codes for

carditis with extensive valve damage possible, arthritis, chorea, fever

pyrogenic toxin; can lead to sequelae

• Acute glomerulonephritis – nephritis, increased blood pressure,

• Septicemia

occasionally heart failure; can become chronic leading to kidney failure

• Pneumonia • Streptococcal toxic shock syndrome

35

36

Group B: Streptococcus agalactiae

Group D Enterococci and Groups C and G Streptococci

• Regularly resides in human vagina, pharynx and large intestine

• Group D: – Enterococcus faecalis, E. faecium, E. durans

• Can be transferred to infant during delivery and cause severe infection

– normal colonists of human large intestine

– most prevalent cause of neonatal pneumonia, sepsis, and meningitis

– cause opportunistic urinary, wound, and skin infections,

– 15,000 infections and 5,000 deaths in US

particularly in debilitated persons – Pregnant women should be screened and treated.

• Groups C and G: • Wound and skin infections and endocarditis in debilitated people

– common animal flora, frequently isolated from upper respiratory; pharyngitis, glomerulonephritis, bacteremia 37

38

39

40

Identification • Cultivation and diagnosis ensure proper treatment to prevent possible complications. • Rapid diagnostic tests based on monoclonal antibodies that react with C‐ carbohydrates • Culture using bacitracin disc test, CAMP test

Treatment and Prevention

• Groups A and B are treated with penicillin.

• Sensitivity testing needed for enterococci

• No vaccines available

41

42

α‐Hemolytic Streptococci: Viridans Group

Viridans Group

• Large complex group

• Bacteremia, meningitis, abdominal infection, tooth abscesses

– Streptococcus mutans, S. oralis, S. salivarus, S. sanguis, S. milleri, S. mitis

• Most serious infection – subacute endocarditis – blood‐borne bacteria

• Most numerous and widespread residents of the gums and teeth, oral

settle and grow on heart lining or valves

cavity and also found in nasopharynx, genital tract, skin

• Persons with preexisting heart disease are at high risk.

• Not very invasive; dental or surgical procedures facilitate entrance

• Colonization of heart by forming biofilms

43

44

Viridans Group

Streptococcus pneumoniae: The Pneumococcus

• S. mutans produce slime layers that adhere to teeth, basis for plaque.

• Causes 60‐70% of all bacterial pneumonias

• Involved in dental caries

• Small, lancet‐shaped cells arranged in pairs and short chains

• Persons with preexisting heart conditions should receive prophylactic

• Culture requires blood or chocolate agar.

antibiotics before surgery or dental procedures.

• Growth improved by 5‐10% CO2 • Lack catalase and peroxidases – cultures die in O2

45

46

S. pneumoniae • All pathogenic strains form large capsules – major virulence factor. • Specific soluble substance (SSS) varies among types. • 84 capsular types have been identified • Causes pneumonia and otitis media

47

48

Epidemiology and Pathology • 5‐50% of all people carry it as normal flora in the nasopharynx; infections are usually endogenous. • Very delicate, does not survive long outside of its habitat • Young children, elderly, immune compromised, those with other lung diseases or viral infections, persons living in close quarters are predisposed to pneumonia • Pneumonia occurs when cells are aspirated into the lungs of susceptible individuals. • Pneumococci multiply and induce an overwhelming inflammatory response. • Gains access to middle ear by way of eustachian tube

49

50

Cultivation and Diagnosis

Treatment and Prevention • Traditionally treated with penicillin G or V

• Gram stain of specimen – presumptive identification • Increased drug resistance • α hemolytic; optochin sensitivity

• Two vaccines available for high risk individuals: – Capsular antigen vaccine for older adults and other high risk

• Quellung test or capsular swelling reaction

individuals‐effective 5 years – Conjugate vaccine for children 2 to 23 months

51

52

Genus Neisseria

Family Neisseriaceae

• Gram‐negative, bean‐shaped, diplococci • Gram‐negative cocci

• None develop flagella or spores.

• Residents of mucous membranes of warm‐blooded animals

• Capsules on pathogens • Pili

• Genera include Neisseria, Moraxella, Acinetobacter.

• Strict parasites, do not survive long outside of the host • 2 primary human pathogens:

• Aerobic or microaerophilic

– Neisseria gonorrhoeae

• Oxidative metabolism

– Neisseria meningitidis

• Produce catalase and cytochrome oxidase • Pathogenic species require enriched complex media and CO2.

53

130

54

Neisseria gonorrhoeae: The Gonococcus • Causes gonorrhea, an STD • Virulence factors: – pili, other surface molecules for attachment; slows phagocytosis – IgA protease – cleaves secretory IgG

55

56

57

58

59

60

Epidemiology and Pathology • Strictly a human infection • In top 5 STDs • Infectious dose 100‐1,000 • Does not survive more than 1‐2 hours on fomites

Gonorrhea Infection is asymptomatic in 10% of males and 50% of females. • Males – urethritis, yellowish discharge, scarring and infertility • Females – vaginitis, urethritis, salpingitis (PID) mixed anaerobic abdominal infection, common cause of sterility and ectopic tubal pregnancies • Extragenital infections – anal, pharygeal, conjunctivitis, septicemia, arthritis

131

Gonorrhea in Newborns • Infected as they pass through birth canal • Eye inflammation, blindness • Prevented by prophylaxis immediately after birth

61

62

63

64

Diagnosis and Control • Gram stain – Gram‐negative intracellular (neutrophils) diplococci from urethral, vaginal, cervical, or eye exudate – presumptive identification • 20‐30% of new cases are penicillinase‐producing PPNG or tetracycline resistant TRNG • Combined therapies indicated • Recurrent infections can occur. • Reportable infectious disease

Neisseria meningitidis: The Meningococcus


Virulence factors:

• Prevalent cause of meningitis; sporadic or epidemic • Human reservoir – nasopharynx; 3‐30% of adult population; higher in

– Capsule

institutional settings

– Pili

• High risk individuals are those living in close quarters, children 6months‐3

– IgA protease

years, children and young adults 10‐20 years. • Disease begins when bacteria enter bloodstream, pass into cranial

– Endotoxin

circulation, and multiply in meninges

• 12 strains; serotypes A, B, C cause most cases

• Very rapid onset; neurological symptoms; endotoxin causes hemorrhage and shock; can be fatal 65

66

132

67

68

Clinical Diagnosis


• Gram stain CSF, blood, or nasopharyngeal sample

• Treated with IV penicillin G, chloramphenicol • Prophylactic

treatment

of

family

members,

medical

• Culture for differentiation personnel, or children in close contact with patient • Rapid tests for capsular antigen

• Vaccines exist for group A and C.

69

70

Other Gram‐negative Cocci and Coccobacilli • Genus Branhamella – Branhamella catarrhalis – found in nasopharynx: significant opportunist in cancer, diabetes, alcoholism

• Genus Moraxella – Bacilli; found on mucous membranes

• Genus Acinetobacter – Gram‐negative bacilli; nonliving reservoir; source of nosocomial infections 71

133


Training Program for Health Institute Graduates Laboratory Technician

The Gram‐Negative Bacilli of Medical Importance 2

Aerobic Gram‐Negative Nonenteric Bacilli

Aerobic Gram‐Negative Nonenteric Bacilli

• Large, diverse group of non‐spore‐forming bacteria • Pseudomonas and Burkholderia – an opportunistic pathogen

• Wide range of habitats – large intestines (enteric), zoonotic, respiratory, soil, water

• Brucella and Francisella – zoonotic pathogens

• Most are not medically important; some are true pathogens, some are opportunists

• Bordetella and Legionella – mainly human pathogens

• All have a lipopolysaccharide outer membrane of cell wall – endotoxin

3

4

Pseudomonas: The Pseudomonads

Pseudomonas aeruginosa

• Small gram‐negative rods with a single polar flagellum • Free living – Primarily in soil, sea water, and fresh water; also colonize plants and animals

• Important decomposers and bioremediators • Frequent contaminants in homes and clinical settings • Use aerobic respiration; do not ferment carbohydrates • Produce oxidase and catalase • Many produce water soluble pigments 5

6

134

Pseudomonas Aeruginosa

Skin rash from Pseudomonas

• Common inhabitant of soil and water • Intestinal resident in 10% normal people • Resistant to soaps, dyes, quaternary ammonium disinfectants, drugs, drying • Frequent contaminant of ventilators, IV solutions, anesthesia equipment • Opportunistic pathogen

7

8

Pseudomonas (left) and Staphylococcus (right)

Pseudomonas Aeruginosa • Common cause of nosocomial infections in hosts with burns, neoplastic disease, cystic fibrosis • Complications include pneumonia, UTI, abscesses, otitis, and corneal disease • Endocarditis, meningitis, bronchopneumonia • Grapelike odor • Greenish‐blue pigment (pyocyanin) • Multidrug resistant • Cephalosporins, aminoglycosides, carbenicillin, polymixin, quinolones, and monobactams 9

10

Related Gram‐Negative Aerobic Rods

Burkholderia

• Genera Burkholderia, Acinetobacter, Stenotrophomonas

• Burkholderia cepacia Burkholderia cepacia – active in biodegradation of a variety of substances; opportunistic

• Similar to pseudomonads

agent in respiratory tract, urinary tract, and occasionally skin

• Wide variety of habitats in soil, water, and related environments

infections; drug resistant • B. pseudomallei – generally acquired through penetrating

• Obligate aerobes; do not ferment sugars

injury or inhalation from environmental reservoir; wound

• Motile, oxidase positive

infections, bronchitis and pneumonia, septicemia • Opportunistic 11

12

135

Acinetobacter and Stenotrophomonas

Brucella and Brucellosis

• Acinetobacter baumanii – nosocomial and community

• Tiny gram‐negative coccobacilli • 2 species:

acquired infections; wounds, lungs, urinary tract, burns,

– Brucella abortus (cattle)

blood; extremely resistant – treatment with combination

– Brucella suis (pigs)

antimicrobials

• Brucellosis, malta fever, undulant fever, and Bang disease – a zoonosis

• Stenotrophomonas maltophilia – forms biofilms; contaminant

transmitted to humans from infected animals

of disinfectants dialysis equipment, respiratory equipment,

• Fluctuating pattern of fever – weeks to a year

water dispensers, and catheters; clinical isolate in respiratory

• Combination of tetracycline and rifampin or streptomycin

soft tissue, blood, CSF; high resistance to multidrugs

• Animal vaccine available • Potential bioweapon 13

Agglutination titer test for brucellosis

14

Francisella Tularensis and Tularemia • Causes tularemia, a zoonotic disease of mammals endemic to the northern hemisphere, particularly rabbits • Transmitted by contact with infected animals, water and dust or bites by vectors • Headache, backache, fever, chills, malaise, and weakness • 10% death rate in systemic and pulmonic forms • Intracellular persistence can lead to relapse • Gentamicin or tetracycline • Attenuated vaccine • Potential bioterrorism agent 15

16

Bordetella Pertussis

Bordetella Pertussis

• Minute, encapsulated coccobacillus

• Virulence factors – Receptors that recognize and bind to ciliated respiratory epithelial

• Causes pertussis or whooping cough, a communicable childhood affliction

cells – Toxins that destroy and dislodge ciliated cells

• Acute respiratory syndrome

• Loss of ciliary mechanism leads to buildup of mucus and blockage of the • Often severe, life‐threatening complications in babies

airways • Vaccine – DTaP – acellular vaccine contains toxoid and other Ags

• Reservoir – apparently healthy carriers • Transmission by direct contact or inhalation of aerosols 17

18

Prevalence of pertussis in the United States

Legionella Pneumophila and Legionellosis • Widely distributed in water • Live in close association with amoebas • 1976 epidemic of pneumonia afflicted 200 American Legion members attending a convention in Philadelphia and killed 29 • Legionnaires disease and Pontiac fever • Prevalent in males over 50 • Nosocomial disease in elderly patients • Fever, cough, diarrhea, abdominal pain, pneumonia fatality rate of 3‐30% • Azithromycin 19

20

Enterobacteriaceae Family Appearance of Legionella pneumophila

• Enterics • Large family of small, non‐spore‐forming gram‐negative rods • Many members inhabit soil, water, decaying matter, and are common occupants of large bowel of animals including humans • Most frequent cause of diarrhea through enterotoxins • Enterics, along with Pseudomonas sp., account for almost 50% of nosocomial infections

21

22

Bacteria that account for the majority of hospital infections

• Facultative anaerobes, grow best in air • All ferment glucose, reduce nitrates to nitrites, oxidase negative, and catalase positive • Divided into coliforms (lactose fermenters) and non‐coliforms (non‐lactose fermenters) • Enrichment, selective and differential media utilized for screening samples for pathogens

23

24

137

Isolation media for enterics

Biochemical traits for separating enteric genera

25

26

Antigenic Structures and Virulence Factors

api, biochemical testing of enterics

Complex surface antigens contribute to pathogenicity and trigger immune response: • H – flagellar Ag • K – capsule and/or fimbrial Ag • O – somatic or cell wall Ag – all have • Endotoxin • Exotoxins 27

28

Antigenic structures in gram‐negative enteric rods

Coliform Organisms and Diseases

29

30

138

Escherichia Coli: The Most Prevalent Enteric Bacillus

Pathogenic Strains of E. Coli • Enterotoxigenic E. coli causes severe diarrhea due to heat‐labile toxin and

• Most common aerobic and non‐fastidious bacterium in gut

heat‐stable toxin – stimulate secretion and fluid loss; also has fimbriae

• 150 strains

• Enteroinvasive E. coli causes inflammatory disease of the large intestine

• Some have developed virulence through plasmid transfer,

• Enteropathogenic E. coli linked to wasting form infantile diarrhea

others are opportunists

• Enterohemorrhagic E. coli, O157:H7 strain, causes hemorrhagic syndrome and kidney damage

31

Escherichia coli

32

Rapid identification of E. coli O157:H7

• Pathogenic strains frequent agents of infantile diarrhea – greatest cause of mortality among babies • Causes ~70% of traveler’s diarrhea • Causes 50‐80% UTI • Coliform count – indicator of fecal contamination in water

33

Other Coliforms

34

A capsule stain of Klebsiella pneumoniae

Clinically important mainly as opportunists • Klebsiella pneumoniae – normal inhabitant of respiratory tract, has large capsule, cause of nosocomial pneumonia, meningitis, bacteremia, wound infections, and UTIs • Enterobacter sp. – UTIs, surgical wounds • Citrobacter sp. – opportunistic UTIs and bacteremia • Serratia marcescens – produces a red pigment; causes pneumonia, burn and wound infections, septicemia and meningitis

35

36

Serratia marcescens

Noncoliform Lactose‐Negative Enterics

• Proteus, Morganella, Providencia

• Salmonella and Shigella

37

38

Opportunists: Proteus and Its Relatives

Wavelike, swarming pattern of Proteus vulgaris

Proteus, Morganella, Providencia – ordinarily harmless saprobes in soil, manure, sewage, polluted water, commensals of humans and animals – Proteus sp. – swarm on surface of moist agar in a concentric pattern – Involved in UTI, wound infections, pneumonia, septicemia, and infant diarrhea – Morganella morganii and Providencia sp. involved in similar infections

•

All demonstrate resistance to several antimicrobials 39

40

Salmonella and Shigella

Typhoid Fever and Other Salmonelloses

• Well‐developed virulence factors, primary pathogens, not

• Salmonella typhi – most serious pathogen of the genus;

normal human flora

cause of typhoid fever; human host

• Salmonelloses and Shigelloses

• S. cholerae‐suis – zoonosis of swine

– Some gastrointestinal involvement and diarrhea but often affect

• S. enteritidis – includes 1,700 different serotypes based on

other systems

variation on O, H, and Vi • Flagellated; survive outside the host • Resistant to chemicals – bile and dyes 41

42

140

Typhoid Fever

Prevalence of salmonelloses

• Bacillus enters with ingestion of fecally contaminated food or water; occasionally spread by close personal contact; ID 1,000‐10,000 cells • Asymptomatic carriers; some chronic carriers shed bacilli from gallbladder • Bacilli adhere to small intestine, cause invasive diarrhea that leads to septicemia • Treat chronic infections with chloramphenicol or sulfatrimethoprim • 2 vaccines for temporary protection

43

44

Animal Salmonelloses

The Phases Of Typhoid Fever

• Salmonelloses other than typhoid fever are called enteric fevers, Salmonella food poisoning, and gastroenteritis • Usually less severe than typhoid fever but more prevalent • Caused by one of many serotypes of Salmonella enteritidis; all zoonotic in origin but humans can become carriers – Cattle, poultry, rodents, reptiles, animal, and dairy products – Fomites contaminated with animal intestinal flora

45

46

The appearance of the large intestinal mucosa in Shigella

Shigella and Bacillary Dysentery • Shigellosis – incapacitating dysentery • S. dysenteriae, S. sonnei, S. flexneri, and S. boydii • Human parasites • Invades villus of large intestine, does not perforate intestine or invade blood • Enters Peyer’s patches instigate inflammatory response; endotoxin and exotoxins • Treatment – fluid replacement and ciprofloxacin and sulfatrimethoprim

47

48

141

The Enteric Yersinia Pathogens

Nonenteric Yersinia Pestis and Plague

• Yersinia enterocolitica – domestic and wild animals, fish, fruits,

• Nonenteric

vegetables, and water

• Tiny, gram‐negative rod, unusual bipolar staining and

– Bacteria enter small intestinal mucosa, some enter lymphatic and

capsules

survive in phagocytes; inflammation of ileum can mimic appendicitis

• Virulence factors – capsular and envelope proteins protect

• Y. pseudotuberculosis – infection similar to Y. enterocolitica, more

against phagocytosis and foster intracellular growth

lymph node inflammation

– Coagulase, endotoxin, murine toxin

49

50

Gram‐stain of Yersinia pestis

Yersinia Pestis • Humans develop plague through contact with wild animals (sylvatic plague) or domestic or semidomestic animals (urban plague) or infected humans • Found in 200 species of mammals – rodents, without causing disease • Flea vectors – bacteria replicates in gut, coagulase causes blood clotting that blocks the esophagus; flea becomes ravenous 51

52

Infection cycle of Yersinia pestis

Pathology of Plague • ID 3‐50 bacilli • Bubonic – bacillus multiplies in flea bite, enters lymph, causes necrosis and swelling called a bubo in groin or axilla • Septicemic – progression to massive bacterial growth; virulence

factors

cause

intravascular

coagulation

subcutaneous hemorrhage and purpura – black plague • Pneumonic – infection localized to lungs, highly contagious; fatal without treatment 53

54

142

The bubo, classic sign of bubonic plague • Diagnosis depends on history, symptoms, and lab findings from aspiration of buboes • Treatment: streptomycin, tetracycline, or chloramphenicol • Killed or attenuated vaccine available • Prevention by quarantine and control of rodent population in human habitats

55

56

Oxidase‐Positive Nonenteric Pathogens

Pasteurella Multocida

• Pasteurella multocida

• Zoonotic genus; normal flora in animals • Opportunistic infections

• Haemophilus influenzae

• Animal bites or scratches cause local abscess that can spread • H. aegyptius

to joints, bones, and lymph nodes • Immunocompromised are at risk for septicemia and

• H. ducreyi

complications • H. parainfluenzae

• Treatment: penicillin and tetracycline

• H. aphrophilus 57

58

Haemophilus

Haemophilus • Tiny gram‐negative pleomorphic rods

• H. influenzae – acute bacterial meningitis, epiglottitis, otitis media, sinusitis, pneumonia, and bronchitis

• Fastidious, sensitive to drying, temperature extremes, and disinfectants

– Subunit vaccine Hib

• None can grow on blood agar without special techniques – chocolate agar

• H. aegyptius – conjunctivitis, pink eye • Require hemin, NAD, or NADP

• H. ducreyi – chancroid STD • Some species are normal colonists of upper respiratory tract or vagina (H.

• H. parainfluenzae and H. aphrophilus – normal oral and

parainfluenzae, H. ducreyi)

nasopharyngeal flora; infective endocarditis

• Others are virulent species responsible for childhood meningitis, and chancroid

59

60

Meningitis in the United States

Acute conjunctivitis

61

62

144

Medically Important Gram‐Positive Bacilli


Three general groups:


1.

Endospore‐formers Bacillus, Clostridium


2.

Non‐endospore‐formers Listeria, Erysipelothrix

3.

Irregular shaped and staining properties Corynebacterium,

Proprionibacterium,

Mycobacterium,

Actinomyces, Nocardia

The Gram‐Positive Bacilli of Medical Importance 2

Spore‐forming Bacilli

Genus Bacillus

Genus Clostridium

3

4

General Characteristics of the Genus Bacillus

Bacillus anthracis

• Gram‐positive, endospore‐forming, motile rods

• Large, block‐shaped rods

• Mostly saprobic

• Central spores that develop under all conditions except in the living body

• Aerobic and catalase positive

• Virulence factors – polypeptide capsule and exotoxins

• Versatile in degrading complex macromolecules

• 3 types of anthrax:

• Source of antibiotics

– Cutaneous – spores enter through skin, black sore‐ eschar; least dangerous

• Primary habitat is soil

– Pulmonary –inhalation of spores

• 2 species of medical importance:

– Gastrointestinal – ingested spores

– Bacillus anthracis – Bacillus cereus 5

6

145

Control and Treatment • Treated with penicillin, tetracycline, or ciprofloxacin • Vaccines – Live spores and toxoid to protect livestock – Purified toxoid; for high risk occupations and military personnel; toxoid 6X over 1.5 years; annual boosters

7

8

Bacillus cereus

The Genus Clostridium

• Common airborne and dustborne; usual methods of disinfection and

• Gram‐positive, spore‐forming rods

antisepsis are ineffective • Anaerobic and catalase negative

• Grows in foods, spores survive cooking and reheating

• 120 species

• Ingestion of toxin‐containing food causes nausea, vomiting, abdominal cramps and diarrhea; 24 hour duration

• Oval or spherical spores produced only under anaerobic conditions

• No treatment • Synthesize organic acids, alcohols, and exotoxins

• Increasingly reported in immunosuppressed

• Cause wound infections, tissue infections, and food intoxications

9

10

Gas Gangrene • Clostridium perfringens most frequent clostridia involved in soft tissue and wound infections ‐ myonecrosis • Spores found in soil, human skin, intestine, and vagina • Predisposing factors – surgical incisions, compound fractures, diabetic ulcers, septic abortions, puncture wounds, gunshot wounds

11

12

146

Virulence Factors

Pathology

• Virulence factors

• Not highly invasive; requires damaged and dead tissue and anaerobic conditions

– toxins –

• Conditions stimulate spore germination, vegetative growth and release of

• alpha toxin – causes RBC rupture, edema and tissue

exotoxins, and other virulence factors.

destruction

• Fermentation of muscle carbohydrates results in the formation of gas and further destruction of tissue.

– collagenase – hyaluronidase – DNase 13

14

Treatment and Prevention • Immediate cleansing of dirty wounds, deep wounds, decubitus ulcers, compound fractures, and infected incisions • Debridement of disease tissue • Large doses of cephalosporin or penicillin • Hyperbaric oxygen therapy • No vaccines available

15

16

Clostridium difficile‐Associated Disease (CDAD)


• Normal resident of colon, in low numbers

• Mild uncomplicated cases respond to fluid and electrolyte replacement and withdrawal of antimicrobials.

• Causes antibiotic‐associated colitis

• Severe

– Relatively non‐invasive; treatment with broad‐spectrum antibiotics

infections

treated

with

oral

vancomycin

or

metronidazole and replacement cultures

kills the other bacteria, allowing C. difficile to overgrow

• Produces enterotoxins that damage intestines

• Increased precautions to prevent spread

• Major cause of diarrhea in hospitals • Increasingly more common in community acquired diarrhea

17

18

Pathology

Tetanus

• Spores usually enter through accidental puncture wounds, • Clostridium tetani

burns, umbilical stumps, frostbite, and crushed body parts.

• Common resident of soil and GI tracts of animals

• Anaerobic environment is ideal for vegetative cells to grow

• Causes tetanus or lockjaw, a neuromuscular disease

and release toxin.

• Most commonly among geriatric patients and IV drug

• Tetanospasmin – neurotoxin causes paralysis by binding to

abusers; neonates in developing countries

motor

nerve

endings;

blocking

the

release

of

neurotransmitter for muscular contraction inhibition; muscles contract uncontrollably • Death most often due to paralysis of respiratory muscles 19

20

Treatment and Prevention • Treatment aimed at deterring degree of toxemia and infection and maintaining homeostasis • Antitoxin therapy with human tetanus immune globulin; inactivates circulating toxin but does not counteract that which is already bound • Control infection with penicillin or tetracycline; and muscle relaxants • Vaccine available; booster needed every 10 years

21

22

Clostridial Food Poisoning

Botulinum Food Poisoning

• Clostridium botulinum – rare but severe intoxication usually

• Botulism – intoxication associated with inadequate food

from home canned food

preservation

• Clostridium perfringens – mild intestinal illness; second most

• Clostridium botulinum – spore‐forming anaerobe; commonly

common form of food poisoning worldwide

inhabits soil and water

23

24

Pathogenesis • Spores are present on food when gathered and processed. • If reliable temperature and pressure are not achieved air will be evacuated but spores will remain. • Anaerobic conditions favor spore germination and vegetative growth. • Potent toxin, botulin, is released. • Toxin is carried to neuromuscular junctions and blocks the release of acetylcholine, necessary for muscle contraction to occur. • Double or blurred vision, difficulty swallowing, neuromuscular symptoms

25

Infant and Wound Botulism •


Infant botulism – caused by ingested spores that germinate

• Determine presence of toxin in food, intestinal contents or feces

and release toxin; flaccid paralysis •

26

• Administer antitoxin; cardiac and respiratory support

Wound botulism – spores enter wound and cause food

• Infectious botulism treated with penicillin

poisoning symptoms • Practice proper methods of preserving and handling canned foods; addition of preservatives.

27

28

Gram‐Positive Regular Non‐Spore‐Forming Bacilli

Clostridial Gastroenteritis • Clostrium perfringens • Spores contaminate food that has not been cooked thoroughly enough to destroy spores. • Spores germinate and multiply (especially if unrefrigerated). • When consumed, toxin is produced in the intestine; acts on epithelial cells, acute abdominal pain, diarrhea, and nausea • Rapid recovery

Medically important: • Listeria monocytogenes • Erysipelothrix rhusiopathiae

29

30

Listeria monocytogenes • • • • • •

Non‐spore‐forming Gram‐positive Ranging from coccobacilli to long filaments 1‐4 flagella No capsules Resistant to cold, heat, salt, pH extremes and bile Virulence attributed to ability to replicate in the cytoplasm of cells after inducing phagocytosis; avoids humoral immune system

31

32

Epidemiology and Pathology

Diagnosis and Control

• Primary reservoir is soil and water; animal intestines • Can contaminate foods and grow during refrigeration • Listeriosis ‐ most cases associated with dairy products, poultry, and meat • Often mild or subclinical in normal adults • Immunocompromised patients, fetuses and neonates; affects brain and meninges

• • • •

Culture requires lengthy cold enrichment process. Rapid diagnostic tests using ELISA available Ampicillin and trimethoprimsulfamethoxazole Prevention – pasteurization and cooking

– 20% death rate 33

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Erysipelothrix rhusiopathiae

Gram‐Positive Irregular Non‐Spore‐Forming Bacilli

• Gram‐positive rod widely distributed in animals and the environment • Primary reservoir – tonsils of healthy pigs • Enters through skin abrasion, multiples to produce erysipeloid, dark red lesions • Penicillin or erythromycin • Vaccine for pigs

Medically important genera: • Corynebacterium • Proprionibacterium • Mycobacterium • Actinomyces • Nocardia

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Corynbacterium diptheriae • Gram‐positive irregular bacilli • Virulence factors assist in attachment and growth.

• Pleomorphic; stain unevenly • 20 genera; Corynebacterium, Mycobacterium, and Nocardia greatest clinical significance • All produce catalase, possess mycolic acids, and a unique peptidoglycan.

– diphtherotoxin – exotoxin • 2 part toxin – part B binds and induces endocytosis; part A arrests protein synthesis

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Epidemiology and Pathology • Reservoir of healthy carriers; potential for diphtheria is always present • Most cases occur in non‐immunized children living in crowded, unsanitary conditions. • Acquired via respiratory droplets from carriers or actively infected individuals

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Epidemiology and Pathology 2 stages of disease: 1. Local infection inflammation

–upper

respiratory

tract

– sore throat, nausea, vomiting, swollen lymph nodes; pseudomembrane formation can cause asphyxiation

2. Diptherotoxin production and toxemia – 41

target organs primarily heart and nerves 42

Diagnostic Methods • • • •


Pseudomembrane and swelling indicative Stains Conditions, history Serological assay

• Antitoxin • Penicillin or erythromycin • Prevented by toxoid vaccine series and boosters

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Genus Proprionibacterium • • • • • •

Mycobacteria: Acid‐Fast Bacilli

Propionibacterium acnes most common Gram‐positive rods Aerotolerant or anaerobic Nontoxigenic Common resident of sebaceous glands Causes acne

• • • • • • •

Mycobacterium tuberculosis M. leprae M. avium complex M. fortuitum M. marinum M. scrofulaceum M. paratuberculosis

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46

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Genus Mycobacterium Gram‐positive irregular bacilli Acid‐fast staining Strict aerobes Produce catalase Possess mycolic acids and a unique type of peptidoglycan • Do not form capsules, flagella or spores • Grow slowly

• • • • •

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Mycobacterium tuberculosis

Epidemiology of Tuberculosis

• Tubercle bacillus • Produces no exotoxins or enzymes that contribute to infectiousness • Virulence factors ‐ contain complex waxes and cord factor that prevent destruction by lysosomes or macrophages

• Predisposing factors include: inadequate nutrition, debilitation of the immune system, poor access to medical care, lung damage, and genetics. • Estimate 1/3rd of world population and 15 million in U.S. carry tubercle bacillus; highest rate in U.S. occurring in recent immigrants • Bacillus very resistant; transmitted by airborne respiratory droplets

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Course of Infection and Disease

Primary TB

• Only 5% infected people develop clinical disease • Untreated, the disease progresses slowly; majority of TB cases contained in lungs • Clinical tuberculosis divided into:

• Infectious dose 10 cells • Phagocytosed by alveolar macrophages and multiply intracellularly • After 3‐4 weeks immune system attacks, forming tubercles, granulomas consisting of a central core containing bacilli surrounded by WBCs – tubercle • If center of tubercle breaks down into necrotic caseous lesions, they gradually heal by calcification.

– primary tuberculosis – secondary tuberculosis (reactivation or reinfection) – disseminated tuberculosis

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Secondary TB • If patient doesn’t recover from primary tuberculosis, reactivation of bacilli can occur. • Tubercles expand and drain into the bronchial tubes and upper respiratory tract. • Gradually the patient experiences more severe symptoms. – violent coughing, greenish or bloody sputum, fever, anorexia, weight loss, fatigue

• Untreated, 60% mortality rate 53

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Extrapulmonary TB

Diagnosis

• During secondary TB, bacilli disseminate to regional lymph nodes, kidneys, long bones, genital tract, brain, and meninges. • These complications are grave.

1. In vivo or tuberculin testing Mantoux test – local intradermal injection of purified protein derivative (PPD); look for red wheal to form in 48‐72 hours‐ induration; established guidelines to indicate interpretation of result based on size of wheal and specific population factors

2. X rays 3. Direct identification of acid‐fast bacilli in specimen 4. Cultural isolation and biochemical testing

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Management and Prevention of TB • 6‐24 months of at least 2 drugs from a list of 11 • One pill regimen called Rifater (isoniazid, rifampin, pyrazinamide) • Vaccine based on attenuated bacilli Calmet‐Guerin strain of M. bovis used in other countries

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Mycobacterium leprae: The Leprosy Bacillus

Epidemiology and Transmission of Leprosy

• 6‐24 months of at least 2 drugs from a list of 11 • One pill regimen called Rifater (isoniazid, rifampin, pyrazinamide) • Vaccine based on attenuated bacilli Calmet‐Guerin strain of M. bovis used in other countries

• Endemic regions throughout the world • Spread through direct inoculation from leprotics • Not highly virulent; appears that health and living conditions influence susceptibility and the course of the disease • May be associated with specific genetic marker

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Course of Infection and Disease

Diagnosing

• Macrophages phagocytize the bacilli, but a weakened macrophage or slow T cell response may not kill bacillus. • Incubation from 2‐5 years; if untreated, bacilli grow slowly in the skin macrophages and Schwann cells of peripheral nerves • 2 forms possible:

• Combination of symptomology, microscopic examination of lesions, and patient history • Numbness in hands and feet, loss of heat and cold sensitivity, muscle weakness, thickened earlobes, chronic stuffy nose • Detection of acid‐fast bacilli in skin lesions, nasal discharges, and tissue samples

– Tuberculoid – superficial infection without skin disfigurement which damages nerves and causes loss of pain perception – Lepromatous – a deeply nodular infection that causes severe disfigurement of the face and extremities

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Infections by Non‐Tuberculosis Mycobacteria (NTM)


• M. avium complex – third most common cause of death in AIDS patients • M. kansaii – pulmonary infections in adult white males with emphysema or bronchitis • M. marinum – water inhabitant; lesions develop after scraping on swimming pool concrete • M. scrofulaceum – infects cervical lymph nodes • M. paratuberculosis – raw cow’s milk; recovered from 65% of individuals diagnosed with Crohn’s disease

• Treatment by long‐term combined therapy • Prevention requires constant surveillance of high risk populations. • WHO sponsoring a trial vaccine

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Actinomycetes: Filamentous Bacilli • Genera Actinomyces & Nocardia are nonmotile filamentous bacteria related to mycobacteria. • May cause chronic infection of skin and soft tissues • Actinomyces israelii – responsible for diseases of the oral cavity, thoracic or intestines ‐ actinomycoses • Nocardia brasiliensis causes pulmonary disease similar to TB.

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Formation and Physiology • ~First recognized by Cotugno in 1764, CSF is the third major fluid of the body.


• Physiologic System 1. To supply nutrients to the nervous system 2. To remove metabolic wastes 3. To produce a mechanical barrier to cushion the brain and spinal cord against trauma.


• Meninges Layers 1. Dura mater 2. Arachnoid mater 3. Pia mater

- outer layer - middle layer - inner layer

Cerebrospinal Fluid (CSF) Culture 2

Aim of the test • CSF flows through the subarachnoid space between the arachnoid and pia mater

 Diagnosis of the bacteria or fungal meningitis by microscopic examination and culture with identification and susceptibility test of the isolated organism

• 20 ml of fluid produced every hr in choroids plexus and reabsorbed by arachnoid villi

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Infection of CSF Common bacterial pathogen Haemophilus influenzae Salmonella (rare) Neisseria meningitidis Brucella (rare) Streptococcus pneumoniae Treponema pallidum (rare) Group A & B streptococci Listeria monocytogenes Gram negative bacilli Viruses Enteroviruses(coxsackieviruses Herpes simplex virus A and B, echoviruses) Mumps virus Arboviruses (togavirus,bugavirus and other) Parasite Free living amoebae Toxoplasma (rare) Toxoplasma gondii Strongyloides strecoralis Entamoeba histolytica

CSF Examination

Microbiology Testing

Cell Count

Biochemical Testing

Microbes that cause chronic meningitis M. tuberculosis

Blastomyces dermatitides

Cryptococcus neoformans

Candida spp.

Coccidoides immitis

Nocardia

Histoplasma capsulatum

Actinomyces

glucose protein and globulin

Note: CSF is a sterile body fluid and does not contain any commensals; however, care should be taken not to contaminate the specimen with skin normal flora during collection

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Specimen collection •

Who will collect the specimen:

•

Methods and precautions:

–

– –

•

• Time relapse before processing the sample - CSF is an emergency specimen and should be processed immediately.

Only physicians.

lumbar puncture Ventricular puncture

CSF usually collected in three sterile tubes – – –

•

Specimen transport

Tube 1 – used for chemical and serologic test (tubes are frozen) Tube 2 – used for microbiology lab ( room temp.) Tube 3 – used for hematology (cell count) ( refrigerated)

• Storage - Room Temperature, Do not refrigerate.

Quantity of specimen: Mini. 5-10 ml of CSF is recommended for culture.

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CSF processing Visual detection of Etiologic agent

Criteria of specimen rejection

 Non sterile container and the general causes for rejection as mislabeling, un-labeling, insufficient amount.  Specimen more than 30 minutes.

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10

Macroscopic inspection

Initial Processing  centrifugation of all specimens greater than 1 mL in volume for at least 15 minutes at 1500x g.

 Report the appearance:  clear, slightly turbid, cloudy or definitely purulent (looking like pus),  contains blood  contains clots

 The supernatant is removed to a sterile tube, leaving approximately 0.5 mL of fluid in which to suspend the sediment before visual examination or culture.  The supernatant can be used for to test for the presence of antigens or for chemistry evaluations.

– Normal c.s.f. Appears clear and colourless.

Note: if the specimen turbid it is not

necessary to make the initial processing as mentioned above.

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Microscopic examination

Staining of CSF:

Gram stain of N. meningitidis in CSF with associated PMNs.

After thoroughly mixing the sediment heaped drop is placed on the surface on a sterile slide. The sediment should never spread out on the slide surface, because this increases the difficulty of finding small numbers of microorganisms, the drop of sediment is allowed to air dry, and heated or methanol fixed and stained by gram stain, and another slide stained by Methylene blue in parallel.

N. meningitidis may occur intracellularly or extracellularly in PMN leukocytes and will appear as gram‐negative, coffee‐bean shaped diplococci. 13

14

Gram stain of S. pneumoniae with WBCs

Gram stain of H. influenzae

S. pneumoniae may occur intracellularly or extracellularly and will appear as gram‐ positive, lanceolate diplococci, sometimes occurring in short chains.

H. influenzae are small, pleomorphic gram‐negative rods or coccobacilli with random arrangements. 15

16

Wet mount preparation

India ink stain:

India ink stain: the large polysaccharide capsule of cryptococcus neoformans allows these organisms to be visualized After mixing the CSF and ink to make a smooth suspension, a coverslip is applied to the drop and the preparation is examined under high power magnification for characteristics encapsulated yeast cells and by oil immersion.

Wet mount: amoebas are best observed by examining thoroughly mixed sediment as a wet preparation under phase contrast microscopy or light microscopy with the condenser closed slightly. Amoebas are recognized by typical slow, methodical movement in one direction by advancing pseudopodia. 17

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Culture

Preliminary identification H. Influenzae On Chocolate agar

After vortexing the sediment and preparing smears Several drops of the sediment shoud be inoculated to each medium. Plates should be incubated at 37oC for at least 72 hours. The broth should be incubated in air for at least 5 days. These media will support the growth of almost all bacterial pathogens and several fungi. 19

20

Satellitism phenomenon H. influenzae both V and X factors

H. parainfluenzae only V factor

Sheep blood agar contains hemin but not NAD, Several bacterial species including Staphylococcus aureus, produce NAD as a metabolic byproduct,therfore tiny colonies of Haemophilus spp. May be seen growing on blood agar very close to colonies that can produce V factor.

21

Streptococcus pneumoniae

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streptococcus agalactiae

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Susceptibility testing

Post specimen processing Interfering factors

 Patient on antibiotic therapy.  Improper sample collection.

Result reporting

 Results of the microscopy and all positive cultures of CSF are reported immediately to the treating physician.

Turn around time

 Gram stain result is reported within 30 minutes of specimen receipt  Positive Culture results = 3‐ 5 days  Negative Culture results = 2‐3 days

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CSF processing Direct detection of Etiologic agent  Latex agglutination testing (antigen detection)

Bacteria – N. meningitidis groups A, B, C, Y and W135 – E. coli K1 antigen – H. influenzae type b – S. pneumoniae – S. agalactiae

Cryptococcus neoformans

 Rapid diagnostic tests (RDTs) RDT for meningococcal meningitis RDT for pneumococcal meningitis

 Molecular methods

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What is a Blood Culture? • A blood culture is a laboratory test in which blood is injected into bottles with culture media to determine whether microorganisms have invaded the patient’s bloodstream.


Blood Culture 2

Need for Blood Culture?

Proof in Blood borne Infection

“No microbiological test is more essential to the clinician than the blood culture. The finding of pathogenic microorganisms in a patient’s bloodstream is of great importance in terms of diagnosis, prognosis, and therapy.”

• A clinically suspected infection is ultimately confirmed by isolation or detection of the infectious agent. Subsequent identification of the microorganism and antibiotic susceptibility tests further guide effective antimicrobial therapy. • Bloodstream infection is the most severe form of infection and is frequently life‐threatening, and blood culture to detect circulating microorganisms has been the diagnostic standard.

‐ L. Barth Reller, Clin. Infect. Diseases, 1996

3

4

Diseases. • Depending on the clinical situation, bacteremia may be:

• Blood is normally sterile

– Transient

• The presence of bacteria in the bloodstream may represent:

 Comes and goes  Usually occurs after a procedural manipulation (ex. Dental procedures)

(1) a transient bacteremia; (2) bacteremia secondary to infection at a primary site, such as the lungs; or (3) an infection of the bloodstream, known as septicemia or sepsis.

– Intermittent  Can occur from abscesses at some body site that is “seeding” the blood

 Septicemia is a serious disease characterized by chills, fever, prostration, and the presence of bacteria and/or their toxins in the bloodstream.

– Continuous Bacteremia  Infective endocarditis 5

6

Most likely pathogens. • Primary Bacteremia:  blood stream bacterial invasion with no preceding or simultaneous site of infection with the same microorganism

• Secondary Bacteremia:  isolation of a microorganism from blood as well as other site(s)

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8

Clinical specimens. • The general rule is to collect two or three blood specimens, each inoculated with at least 10 to 20 mL of blood, per 24‐hour period.

• Venous blood  infants: 0.5 – 2 ml  children: 2 – 5 ml  adults: 5 – 10 ml

• Specimens should be collected at intervals no closer than 3 hours, to demonstrate that the bacteremia is continuous.

• Requires aseptic technique

• Specimen collection should be done before initiation of antimicrobial therapy. If antimicrobial therapy is to be initiated immediately, two specimens should be collected from different sites.

• Multiple blood specimens should be drawn to detect intermittent bacteremia or to confirm or rule out contaminated cultures.

• Blood collected through peripheral or indwelling central venous catheters is often contaminated with members of the indigenous microflora of the skin. 9

10

Specimen preservation.

Contaminants. • During collection, blood specimens frequently become contaminated with members of the indigenous microflora of the skin.

Transport time at room temperature should be less than 2 hours.

• Coagulase‐negative staphylococci, especially S. epidermidis, are especially common contaminants.

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Gram stain information.

Culture media.

• Gram staining of blood specimens at the time of collection is rarely of value.

• Traditional blood culture systems o o

Conventional broth method Biphasic methods

o

Lysis centrifugation methods

–

• It can be helpful when bacteria are observed in hematology smears, as in cases of meningococcemia, Streptococcus pneumoniae infection, or other infections in which the concentration of bacteria in the bloodstream is very high (104 bacteria/mL or higher).

1st used for recovery of Brucella spp from blood.

• Modern blood culture systems o o

most often are two‐bottle systems support the growth of most aerobes or anaerobes that could be present in the blood.

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14

15

16

characteristics of continuous‐monitoring blood culture systems

Screening tests.

Laboratory diagnosis.

• Traditional blood culture systems:

• Positive blood cultures are: 1. Gram stained:

– bottles were examined by clinical microbiology laboratory (CML) professionals at regular intervals‐at least once daily‐ for evidence of bacterial growth, such as hemolysis, bubbles, or turbidity.

• If bacteria are observed, a preliminary report (often telephonic) should be sent to the clinician stating the cellular morphology, cellular arrangement, and Gram reaction of the organism(s) observed.

• The newer systems: – detect gas production or other metabolic activities of any microorganisms present. – Detection techniques include infrared spectroscopy, color change in an indicator, and pressure measurement. 17

18

3. Further workup:

2. Inoculated onto appropriate media: • The types of media inoculated are most often based on the Gram

• depends on the type(s) of organisms that grow on these media.

stain observations. • It is important to keep in mind that more than one pathogen may

• Anaerobic media should be inoculated if the cellular morphology

be present.

is suggestive of a particular species of anaerobe, or when only the anaerobic bottle is positive. • When no organisms are observed on Gram stain, a routine media is inoculated, such as blood agar, chocolate agar, and MacConkey agar (MAC) • Some CMLs routinely inoculate media to be incubated anaerobically. 19

20

Factors affecting isolation of causative organisms:

Safety precautions

• The possible types of bacteremia (presence of bacteria in blood) • Specimen collection methods • Blood volumes • Number & timing of blood cultures • Previous antimicrobial therapy • Interpretation of results • Type of patient population being served by the laboratory.

• Do not puncture the site twice as this may cause infection. • When injecting blood into culture bottles be careful not to prick your fingers. • Needles should not be recapped, but discarded in a safety container. • Follow all precautions necessary for prevention of blood borne disease (standard precautions) • NOTE: working with blood cultures, keep the cultures within a Biosafety Cabinet or behind a shield, or wear a face mask and always wear gloves, lab coat because blood cultures contain material from patients that may harbor blood –born pathogens. Follow all precautions necessary for prevention of blood‐born disease (standard precautions). 21

22

Intravenous catheter tips

Principle. • When colonization of an indwelling catheter is suspected of being the focus of septicemia, the catheter may be cultured to determine its status. • The number of CFU of bacteria on the catheter directly relates to whether it is the source of infection or not. If the isolate is identical to that of blood culture it is most properly the source of infection. • A semi quantitative culture technique of Maki et al is used to distinguish between low density colonization (contamination) and significant colonization and real infection.

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Specimen collection and transport.

Culture procedure.

• The skin around the catheter is carefully disinfected with alcohol and then iodine preparation. • Catheter is removed. • A short section (approximately 5cm), including the area just beneath the skin is cut off. • Catheter tip is sent to the microbiology laboratory in sterile container without liquid. • Tips should be cultured within 2 hrs of collection to avoid desiccation of microorganisms.

• Use sterile forceps to transfer catheter tip from transport container to a blood agar plate. • Using light pressure, roll catheter tip back and forth across agar surface at least 4 times. It is essential that the tip has good contact with the surface of the agar. If the tip is bent and hard to roll, use forceps to pick up tip, and rub all surface onto agar. • Incubate plate overnight at 35‐37ºC in CO2 incubator • After incubation count colonies.

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26

Interpretation of culture. • A positive culture (equal to more than ≥15 correlates well with the catheter tip serving source of infection. • Identify and perform susceptibility testing on organism that produces ≥15 CFU • More than 2 organisms in quantities > 15 generally represent contamination of the tip.

Reporting results CFU) as a

• For < 15 CFU reported as: no growth. or < 15CFU without identification or susceptibility.

each

• Report > 15 CFU, with the identification and susceptibility pattern ,ward the report as follows: (significant colonization > 15 CFU).

CFU

• Three different species with > 15 CFU isolated, generally represents contamination of the tip during catheter withdrawal and reported as: 3 different species isolated and this properly represents contamination). 27

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The Urinary System


• Upper urinary tract: – Paired kidneys – A ureter for each kidney


• Lower urinary tract: – Urinary bladder – Urethra Urine Culture 2

Urinary Tract Infection (UTI)

Terms relating to infectious disease of urinary tract

• an infection of one or more structures in the urinary system. • are characterized as being either Upper or Lower based on anatomic location of the infection.

3

4

Factors predispose to urinary tract infection:

Symptoms of UTIs

• Are more common in women

• Typical features of cystitis and urethritis include:

• Incomplete bladder emptying:

    

Abrupt onset of frequency of micturition (urination) Scalding pain in the urethra during micturition (dysuria) Lower back pain, abdominal pain and tenderness over bladder Suprapubic pain during and after voiding Intense desire to pass more urine after micturition due to spasm of inflamed bladder (urgency)  Urine that may appear cloudy and have an unpleasant smell  Presence of blood in the urine (haematuria)  Cystitis has more acute onset and severe symptoms

 Bladder outflow obstruction  Neurological problems (e.g. multiple sclerosis, diabetic neuropathy)  Gynaecological abnormalities (e.g. uterine prolapse)

• Foreign bodies:

 Urethral catheters  Ureteric stent  stone

Systemic symptoms suggestive of pyelonephritis:

• Loss of host defences:

 Fever above 38.3°C  Loin pain  may be indication for hospitalization

 Atrophic urethritis and vaginitis in post‐menopausal women  Diabetes mellitus

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Symptoms continued..

Possible pathogens

• Prostatitis is suggested by:

I‐ BACTERIA

 Pain in the lower back, perirectal area and testicles  High fever, chills and symptoms similar to bacterial cystitis  Inflammatory swelling of prostate, which can lead to urethral obstruction  Urinary retention, which can cause abscess formation or s eminal vesiculitis

 Gram positive – Staphylococcus saprophyticus – Haemolytic streptococci  Gram negative – Escherichia coli – Proteus species – Pseudomonas aeruginosa – Klebsiella strains – *Salmonella Typhi – *Salmonella Paratyphi – *Neisseria gonorrhoeae *These species are not primarily pathogens of the urinary tract, but may be found in urine.

Also Mycobacterium tuberculosis, Leptospira interrogans, Chlamydia, Mycoplasma and Candida species.

7

8

• E. coli is the commonest urinary pathogen causing 60–90% of infections. Some strains are more invasive, e.g. capsulated strains are able to resist phagocytosis, other strains are more adhesive.

II‐ PARASITES

• Candida urinary infection is usually found in diabetic patients and those with immunosuppression.

– Schistosoma haematobium, Trichomonas vaginalis, and occasionally Enterobius vermicularis, Wuchereria bancrofti and Onchocerca volvulus.

• UTIs caused by Pseudomonas, Proteus, Klebsiella species and S. aureus, are associated with hospital‐acquired infections, often following catheterization or gynaecological surgery.

– Finding intestinal parasites in urine indicates faecal contamination.

• Proteus infections are also associated with renal stones. • S. saprophyticus infections are usually found in sexually active young women.

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Probabilities of UTI in case of negative urine culture

Commensals

• M. tuberculosis is usually carried in the blood to the kidney from another site of infection. It is often suspected in a patient with chronic fever when there is pyuria but the routine culture is sterile.

• The urethra however may contain a few commensals and also the perineum (wide variety of Gram positive and Gram negative organisms) which can contaminate urine when it is being collected.

• The bladder and urinary tract are normally sterile.

• With female patients, the urine may become contaminated with organisms from the vagina.

• Pyuria with a negative urine culture may also be found when there is infection with Chlamydia trachomatis, Ureaplasma, or N. gonorrhoeae, or when a patient has taken antimicrobials.

• Vaginal contamination is often indicated by the presence of epithelial cells (moderate to many) and a mixed bacterial flora. • Most urine specimens will contain fewer than 10*4 contaminating organisms per ml providing the urine has been collected with care to minimize contamination and the specimen is examined soon after collection (Why?) before the commensals have had time to multiply significantly.

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Micro flora that colonizes urethra: • • • • • • • • • •

Clinical specimen

Coagulase negative staphylococci Viridians (except aerococcus uriae) & non‐hemolytic streptococci Lactobacilli Diphtheroids (except Corynebacterium urealyticum) Non pathogenic Neisseria species Gram negative bacilli (Enterobacteriaceae) Anaerobic cocci, propionibacterim spp Anaerobic gram negative cocci and bacilli Commensal Mycobacterium spp Occasional yeast

• The value of laboratory testing procedures and interpretation is dependent on the quality of the specimen collected. • Prevention of contamination by normal vaginal, perineal, and anterior urethral flora is the most important consideration for collection of a clinically relevant urine specimen. • Good patient education is essential (collection after instruction given to patients by healthcare worker). 13

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Clinical specimen (Methods of collection)  Clean voided mid stream urine (MSU) – – – – –

Adhesive bag:

most common method of urine collection more convenient least traumatic normal flora can contaminate the specimen not suitable in children

• Suitable for infant or children • a urinary collection bag (plastic bag with an adhesive seal on one end) is attached over the labia in girls or a boy's penis to collect the specimen

• Follow the Broomhall et al method By tapping just above the pubis with two fingers place on suprapubic region after 1 hour of feed, tapping on at the rate of 1 tap/second for a period of 1 minute, if not successful tapping is repeated once again. The child spontaneously pass the Urine and to be collected in a sterile container

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16

Catheter collection:

• Suprapubic aspirate of urine (SPA)

• In‐out catheter: should be restricted to those patients who are unable to produce a midstream sample and performed with aseptic technique.

‐ Gold standard test for children and for cases difficult to interpret

• Indwelling catheters: Specimens aspirated through the soft rubber connector between the catheter & the collecting tubing not from catheter bag.

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Collection containers • Collect urine in sterile wide mouth container. • Should be placed in biohazard labeled specimen transport bags. The specimen in one compartment and the request form in the second.

Properly labeled container at least with: Name‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ ID‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ Source‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ Doctor‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ Date/hour‐‐‐‐‐‐‐‐‐‐‐‐‐‐

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Acceptable Volumes

Specimen Transport

• ≥1 ml • Volumes less than ≤ 1 ml call the ward and request additional sample. • If unable to obtain additional sample, inform the ward that the specimen will not be cultured. • Document on problem log book and store specimen in refrigerator (problematic specimens to be held for 1 week).

• Transport urine to the laboratory as soon as possible after collection. • Culture within 2 hours after collection • If any delay refrigerate at 4ºC & culture within 24 hours.

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Specimen Transport

Specimen Transport

Dip‐slide technique:

• • • •

• • • • • •

Commercially available slide Covered on each side with culture medium Then immersed in freshly voided urine Sent to laboratory for incubation 35°C Semi quantitative Suitable for general practices that are situated distance from a laboratory

23

Boric acid with final concentration 1.8% in the urine. It will preserve urine count Also white blood cells Few bacterial strains are inhibited by boric acid.

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Specimen handling in the laboratory

Unsuitable Procedures

• Specimens should be refrigerated immediately upon receipt in the laboratory unless they are processed at once.

• Routine culture of urine in any broth medium • Culture of centrifuged sediment • Routine culture for anaerobes (done only on Suprapubic aspirates). • Inoculate urine in multiple different media • Culture of specimens delayed > 2 hrs without refrigeration • Culture of foleys catheter tips • Direct, unstandardaized antimicrobial susceptibility except in emergency

• Specimens which are received more than 2 h after collection without evidence of refrigeration should be refrigerated, and a request for a repeat specimen should be telephoned. • When information on collection time and method is not provided, same procedure should be followed or comment on the final report should be included. 25

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Laboratory Examination of Urine 1.

Describe the appearance of the specimen: –

Report: – Colour of specimen – Whether it is clear or cloudy (turbid)

• Note: – Normal freshly passed urine is clear and pale yellow to yellow depending on concentration. – When left to stand, a cloudiness may develop due to the precipitation of urates in an acid urine or phosphates and carbonates in an alkaline urine. – Urates may give the urine a pink‐orange colour. Note: Other changes in the colour of urine can be caused by the ingestion of certain foods, herbs, and drugs especially vitamins. 27

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Report the following 2. Examine the specimens microscopically: •

1.

      

Bacteria (report only when the urine is freshly passed):

Urine is examined microscopically as a wet preparation to detect:

 

Significant pyuria, i.e. WBCs in excess of 10 cells/μl of urine Red cells Casts Yeast cells Trichomonas vaginalis motile trophozoites Schistosoma haematobium eggs Bacteria (providing the urine is freshly collected)

Large cellular cast

Note: In a urinary infection, protein and nitrite are often found in the urine.

•

 

29

Usually seen as rods, but sometimes cocci or streptococci. Bacteriuria is usually accompanied by pyuria (pus cells in urine).

With E. coli infections, the urine is markedly acid. An alkaline urine is found with Proteus infections.

Large cellular cast, pus cells, red cells, and bacteria (bacilli in background) in urine sediment as seen with the 40 objective.

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2. White blood cells (pus cells): 

These are round, 10–15 m in diameter, cells that contain granules



In urinary infections they are often found in clumps. In urine sediments, white blood cells (WBC) are usually reported as: •

Few: Up to 10 WBCs/HPF (high power field, i.e. using 40 objective)

•

Moderate number: 11–40/HPF

•

Many: More than 40 WBC/HPF

•

Note: A few pus cells are normally excreted in urine. Pyuria is usually regarded as significant when moderate or many pus cells are present, i.e. more than 10 WBC/μl.

•

Bacteriuria without pyuria may occur in diabetes, enteric fever, bacterial endocarditis, or when the urine contains many contaminating organisms.

•

Pyuria with a sterile routine culture may be found with renal tuberculosis, gonococcal urethritis, C. trachomatis infection, and leptospirosis, or when a patient with urinary infection has been treated with antimicrobials.

Urine sediment showing pus cells (larger granulated cells) and red cells as seen with the 40X objective.

Pus cells

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3. Red blood cells: 

These are smaller and more refractile than white cells. They have a definite outline and contain no granules.



They are usually reported as few, moderate or many in number per high power field.



When the urine is isotonic, they have a ringed appearance.

•

When the urine is hypertonic, i.e. more concentrated than the fluid inside the red cells, fluid will be drawn out of the cells and they will appear smaller than normal and often crenated (spiky).

•

When haematuria (red cells in urine) is due to glomerulonephritis (inflammation of the glomeruli of the kidneys), the red cells often vary in size and shape (dysmorphic).

•

 Haematuria may be found in urinary schistosomiasis (usually with proteinuria), bacterial infections, acute glomerulonephritis, sickle cell disease, leptospirosis, infective endocarditis, calculi (stones) in the urinary tract, malignancy of the urinary tract, and haemorrhagic conditions.  Note: The finding of red cells in the urine of women may be due to menstruation.

Red cells

In sickle cell disease, sickled red cells can sometimes be seen in the urine.

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The following casts can be found in urine: 1.

Hyaline casts, which are colourless and empty. They are associated with damage to the glomerular filter membrane. A few may be seen following strenuous (hard) exercise or during fever. Waxy casts, which are hyaline casts that have remained in the kidney tubules a long time. They are thicker and denser than hyaline casts, often appear indented or twisted, and may be yellow in colour. They usually indicate tubular damage and can sometimes be seen in renal failure.

4. Casts: 

These can usually be seen with the 10 objective provided the condenser iris is closed sufficiently to give good contrast.

2.



They consist of solidified protein and are cylindrical in shape (Why?) because they are formed in the kidney tubules.

3.

Cellular casts, which contain white cells or red cells – –

Hyaline cast in urine as seen with the 40 objective.

–

4.

Red cell casts appear orange red. They indicate haemorrhage into the renal tubules or glomerular bleeding. White cell casts are found when there is inflammation of the kidney pelvis or tubules. Yellow‐brown pigmented casts may be seen in the urine of jaundiced patients.

Different casts which may be found in urine.

Granular casts, which contain irregular sized granules originating from degenerate cells and protein. They are also associated with renal damage. 36

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6. Yeast cells:

5. Epithelial cells: •

These are easily seen with the 10 objective. They are nucleated and vary in size and shape.

•

They are usually reported as few, moderate, or many in number per low power (10 objective) field.

•

It is normal to find a few epithelial cells in urine. When seen in large numbers, however, they usually indicate inflammation of the urinary tract or vaginal contamination of the specimen.

 These can be differentiated from red cells by their oval shape and some of the yeasts usually show single budding.  If in doubt, run a drop of dilute acetic acid under the cover glass. Red cells will be haemolyzed by the acid, but not yeast cells.  Yeast cells are usually reported as few, moderate, or many per HPF.  They can be seen in the urine of women with vaginal candidiasis, and occasionally in specimens from diabetics and those with immunosuppression.

Epithelial cells, red cells and occasional pus cell in urine sediment as seen with the 10 objective.

Yeast cells and pseudohyphae of Candida albicans in urine sediment as seen with the 40 objective

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7. Trichomonas vaginalis: •

8. Eggs of Schistosoma haematobium:

T. vaginalis is found in the urine of women with acute vaginitis (occasionally seen in the urine of men).

•

The trichomonads are a little larger than white cells and are usually easily detected in fresh urine (Why?) because they are motile.

•

They move by flagella and an undulating (wavy) membrane.

• Recognized by their large size (about 145 X 55 μm) and spine at one end. • The urine will contain red cells and protein.

Yeast cells and Trichomonas vaginalis in urine sediment as seen with the 40 objective

Egg of Schistosoma haematobium and red cells in urine sediment as seen with the 40 objective

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Culture the urine specimen

9. Others:

Semi‐quantitative method: • Calibrated platinum loop that delivers 0.001 ml of urine • Disposable, sterile, plastic calibrated loop 1μl

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Materials

Procedure

• CLED agar (cystine lactose electrolyte deficient media) *support the growth of many uro‐pathogen and *inhibit the swarming of proteus spp. *differentiate between: lactose (yellow)& non lactose (blue, grey, green) organisms, contain Bromothymol blue indicator. *support the growth of certain staphylococci, streptococci & Candida. • Blood agar (BAP). • Muller Hinton agar (for sensitivity) • Calibrated disposable loop 0.001 µl

• Well mixed urine • Use a calibrated loop vertically (0.001 µl ). • Inoculate the surface of CLED agar and BAP • making a straight line down the center of the plate, then a series of close perpendicular streaks through the original line. • Incubate ‐ aerobic atmosphere at 35°C‐37°C for 24 hour

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Culture

Appearance of some urinary pathogens on CLED agar • E. coli: Yellow (lactose‐fermenting) opaque colonies often with slightly deeper coloured centre. • Klebsiella species: Large mucoid yellow or yellow‐white colonies. • Proteus species: Transluscent blue‐grey colonies. • P. aeruginosa: Green colonies with rough periphery (characteristic colour). • E. faecalis: Small yellow colonies. • S. aureus: Deep yellow colonies of uniform colour. • S. saprophyticus and other coagulase negative staphylococci: Yellow to white colonies.

• Examine and report the cultures CLED agar culture • Look especially for colonies that could be:  Escherichia coli (perform indole and beta‐glucaronidase tests for rapid identification.  Proteus species.  Pseudomonas aeruginosa.  Klebsiella strains.  Staphylococcus aureus.  Staphylococcus saprophyticus.  Enterococcus faecalis

• Note: Contaminating organisms usually produce a few colonies of mixed growth. Most urinary infections show growth of a single type of organism although mixed infections can occur especially in chronic infections or following catheterization or gynaecological surgery. 47

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Reporting bacterial numbers

Interpretation of bacterial counts

 Count the approximate number of colonies.  Estimate the number of bacteria, i.e. colony‐forming units (CFU) per ml of urine. Report the bacterial count as:

• A bacterial count of 105 organisms/ml or more from a fresh ‘clean‐catch’ urine specimen, indicates a urinary infection. • A count of 104–105/ml, could mean infection or contamination. A repeat specimen is indicated. • A count of less than 104/ml is nearly always due to contamination unless the urine was cultured after antimicrobial treatment had been started. • It is important, however, to interpret culture counts in relation to the patient’s clinical condition. • UTIs with lower culture counts are often obtained from catheterized patients or those with urinary obstruction.

1. Less than 10 000 organisms/ml (104/ml), not significant. 2. 10 000–100 000/ml (104–105/ml), doubtful significance (suggest repeat specimen). 3. More than 100 000/ml (105/ml), significant bacteriuria.

 Example •

If 25 E. coli colonies are counted and a ml loop was used, the approximate number of CFU per 1/500ml of urine: 500x25=12 500

 Such a count would be reported as: 10 000–100 000 E. coli/ml

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Antimicrobial susceptibility testing • Perform susceptibility testing on urines with significant bacteriuria, particularly from patients with a history of recurring UTI. • Cultures from patients with a primary uncomplicated UTI may not require a susceptibility test.

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Respiratory Tract Infections • Respiratory system divided into upper and lower tracts


Respiratory Tract Infections 2

Lower respiratory tract infections:

Lower respiratory tract infections

(LRT) involve: • trachea • bronchial tree (bronchi & bronchioles) • lung tissue, or both.

Infections of the (LRT) may involve: • trachea (tracheitis) • bronchial tree (bronchitis, and bronchiolitis) • lung tissue (alveolitis and pneumonia), or both.

Purpose: for ventilation, gas exchange Presenting symptoms usually: Cough, chest pain, dyspnea and varying degrees of sputum production 3

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Flora of respiratory tract

Presenting symptoms usually: • • • • • • • •

Non specific mechanisms can protect LRT form infection:

Fever Cough Chest pain Dyspnea Varying degrees of sputum production Changes on chest x rays Respiratory distress, may require mechanical ventilation Pts on risk (aging population + immunocompromising conditions).

*Nasal hairs *Convoluted passages *Mucus lining nasal turbinate's *Secretory IgA & non specific antibacterial substances (lysozyme). WBCs, macrophages *Reflexes: such as coughing, sneezing, & swallowing. Any particles escaped from airflow turbulence & mucociliary sweeping activity enter alveoli, macrophages ingest them & carry them to the lymphatic's. *in addition to normal flora of nasopharynx & oropharynx help to prevent colonization of the upper respiratory tract by pathogenic microorganisms (under certain situation for unknown reasons‐or due to previous damage by viral infection, loss of some host immunity, or physical damage to respiratory endothelium).

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Types of specimens received: • Sputum (early‐morning • • • •

Endotracheal and Tracheostomy secretions

Sterile wide mouth container tightly fitted screw cap lid

Aspirates obtained through (ETT)/ tracheostomy reflects the colonizing flora of the trachea.

sputum samples should be obtained) This is the most commonly obtained specimen Non invasive methods to investigate the lower respiratory disease. Sputum should be collected under supervision before starting antibiotics. Blood culture should always be collected. 7

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Bronchoscope

Broncho‐alveolar lavage fluid (BAL): Involves injection of 30 to50 ml of physiologic saline through a fiber optic bronchoscope, the saline aspirated and submitted for smear preparation and culture. (This technique is useful in diagnosis pneumonia in intubated patients undergoing ventilation

• Up to one‐third of patients with bacterial pneumonia may be unable to produce a sputum specimen, even under optimal conditions using fiber optic Bronchoscopy for obtaining trans‐bronchial biopsy and Bronchial brush (particularly in patients with lung abscesses or other suspected deep pulmonary infections).

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Sputum Collection Approaches used to improve the quality of the specimen obtained include: • Patient should be standing, or sitting upright in bed • Take deep breath to full the lungs, and empty in one breath , coughing hard and deeply as possible. • Obtaining the specimen prior to antibiotic treatment • Rinsing the mouth prior to expectoration • No food for one to two hours prior to expectoration

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Sputum Collection

Sputum

• Sputum > 2 ml should not be processed unless obviously purulent • Only 1 sample per 24 hr submitted • Scoring system should be used to reject specimen that re oral contamination • Inoculation of the culture media immediately after the specimen is obtained or immediately after prompt transport to the microbiology lab

Timing • *The optimal timing for sputum specimen collection is the first morning sample (accumulated secretions during sleep) containers • *Specimens should be collected in a sterile wide‐ mouth container or respiratory trap apparatus, without any preservative, with a tightly fitted screw‐ cap lid. 13

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Transport

Transport

• All specimens should be transported to the laboratory promptly & processed as soon as possible after collection within 1‐2 hrs or refrigerated till it is processed later on the same day. • Prolonged storage at 4°C may diminish the quantity of more fragile organisms as Haemophilus.

• Handle all samples using universal precautions.

• All manipulation of lower respiratory tract specimens should be performed in a biohazard safety cabinet because of the possibility of occult Tuberculosis.

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Suboptimal specimens • *Excessive delay in transport‐specimens received >2 hrs

NB: *the request form should clearly mention the unusual organisms to be looked for like: • Corynebacterium diphtheriae • Bordetella pertussis • Legionella species, Nocardia species

after collection will be processed with the comment: (Specimen delayed in transport, interprets results with caution). • *specimens received improperly or unlabelled. Call ward to see if another sample can be collected, if not, the nurse must come to the laboratory and positively identify the specimen and personally re‐label the specimen. Include in your file comment that the specimen was re‐labeled by nurse name in the laboratory. • *leaking sputum samples are rejected & ward notified to recollect, if not possible, process specimen in the hood using gloves. 17

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Induced sputum • Diagnosis of these infections frequently is complicated by the contamination of specimens with upper respiratory tract (URT) secretions during collection. Because the organisms which commonly cause LRT are the same as those which normally colonize the URT (through which sputum must pass during expectoration). • Techniques for the examination of sputum must be able to distinguish between the two sources of the organisms; the laboratory should ensure that an appropriate specimen is processed. • The specimen must be microscopically examined both to assess its quality and to look for organisms associated with an inflammatory cell response.

• Patients who are unable to produce sputum may be assisted by respiratory therapy technician. • By allowing the patient to breath aerosolized droplets of a solution of sodium chloride for 10 min . • such sp may avoid the need for a more invasive procedures such as bronchoscopy or needle aspiration in many cases.

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*Important • •

Gram stained • All expectorated sputum specimens are screened for oropharyngeal contamination. • Select a purulent portion of the specimen –gently roll the material evenly on a slide then do gram stain • Examine the film for the presence of:

Samples were processed immediately Microscopic examination: Gram stained to determine the presence of: 1. Squamous epithelial cells indicate oropharyngeal contamination. 2. WBCs indicate lower respiratory tract infection. 3. Predominant organisms associated with PMN´s to identify the most likely pathogen.

•

– Squamous epithelial cells (SEC) and – Polymorph nuclear leucocytes using low power (×10) in at least 20‐40 representatives fields and then take the average.

• Examine the slide under oil immersion and report the predominant and different morphological types of organisms seen in areas of inflammation.

Sputum planted to blood, chocolate and MacConkey agar. 21

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Sputum gram stained

Interpretation of gram stain

Good quality specimens • Quantify no & types of inflammatory cells • Note the presence of bronchial epithelial cells • Concentrate on areas with WBCs when looking for organisms • Determine if there is a predominant organism(>10 per oil immersion field) *Semiquantitate and report organism with descriptive *If no predominant organism is present, report ‘‘mixed gram positive and gram negative flora” 25

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Reporting microbial observations

Rejection criteria for sputum

Type/number of organisms/HPF • Gram positive cocci • Gram negative cocci • Gram negative rod • Gram positive rod *PF: (low power field)x10 (examine 10‐20 fields) *HPF:(high power field) oil immersion

1. Sputum: ≥10 SECs/LPF Note: If the number of WBCs is 10 times the number of SECs & there is 3 to 4+ of a single morphotype of bacteria, accept the specimen for culture.

BAC/HPF

None

Few

Moderate

Many

0

1‐5

6‐30

>30

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Rejection criteria for tracheal aspirates

Reporting

2.

1. ‘Smear contains ≥10 Squamous epithelial cell per low power field, suggestive of poor quality ; culture not performed. Please recollect if clinically indicated’.

3.

Tracheal aspirates from adults: ≥10 SECs/LPF or no organisms seen. Tracheal aspirates from pediatric patients: no organisms seen.

Note: if no organisms are seen in a specimen with numerous 4+ WBCs and cellular debris (Pseudomonas and Haemophillus can be missed in such smears, because they can not be distinguished among the cellular debris).

2. ‘Smear is negative for bacteria after examination of 40 fields; culture not performed. Contact laboratory if further studies are clinically indicated’.

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Follow up

Note

Notify the caregiver that the specimen will not be cultured.

1. Neutropenic patient are cultured regardless the presence of WBCs. 2. Don’t reject sputum and endotracheal aspirates for Legionella or AFB, or specimens from cystic fibrosis patients as purulent cell responses are not typically elicited.

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Culture Media

Incubation

Purulent portion of the sputum is cultured directly on to the plates and then make a thin smear on a microscopic slide

• Incubate plates in 5% CO2 at 35°C for 18‐24 hrs, All cultures are kept for a minimum of 2 days before discarding plates and finalizing the culture.

• Sheep Blood agar • Chocolate agar • MacConkey agar • Sabauroud dextrose agar plate ( if any yeast cells or pseudohyphae are seen in the gram stain) • *For transtracheal aspirates add anaerobic culture also. 33

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Interpretation of Culture Media •

• All plates are examined after 24 hrs. incubation, if there is no growth, reincubate for another 24hrs

Look for predominant growth of a potential lower respiratory tract pathogens include:

‐Streptococcus pneumonia ‐Haemophilus influenzae ‐Branhamella catarrhalis ‐Beta hemolytic streptococcus ‐Pseudomonas, Klebsiella, and other gram negative bacilli ‐Staph aureus

•

Normal respiratory flora that can be isolated from expectorated sputum which is mixed with oropharyngeal secretions, consists of: ‐Alpha and gamma ‐streptococci ‐Diphtheroids ‐Neisseria species nonpathogenic ‐Coagulase negative staph

‐Aspergillus, Yeast, fungus 35

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Interpretation of Culture Media

Examine for and always report • • • • • • • • • •

Streptococcus pyogens Group B strespstsococci in pediatric population Bordetella spp Yerisnia pestis Nocardia spp Bacillus anthracis Cryptococcus neo Molds, not considered saprophytic contaminants Neisssersia gonorrhoeae Francisella tularensis

• Correlate the amount and type of organisms grown on culture with the presence of WBCs and corresponding morphotypes seen on gram stain • Potential pathogens present in amounts less than the normal flora, should be considered part of the normal flora *streptococcus pneumoniae *haemophilus influenza 37

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Interpretation of Culture Media

Reporting Results

• Report growths of potential pathogens in the third and fourth quadrants ( however, a flexible approach is essential, and on occasions low numbers of organisms may be reported).

A‐If none of the above organisms are isolated report as (no pathogens isolated) B‐If any pathogen isolated, it is reported in a semiquantitative manner as follow: Scant growth in 1st quadrant only Few growth in 1st and 2nd quadrant Moderate growth in 1st, 2nd and 3rd quadrant Heavygrowth in all 4 quadrants

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Use the following as a guideline for cultures with multiple potential pathogens from sputum & endotracheal suction: • 1 or 2 potential pathogens, full identification with sensitivity seen as predominant on gram stain. • 3 or more potential pathogens + with or ‐‐ without normal flora report preliminary identification without sensitivities e.g [many normal flora including many streptococcus pneumonia, haemophilus spp., pseudomonas.etc] • 3 or more coliforms + with normal flora do not perform identification or sensitivity and reporting as [many normal flora including mixed coliforms]. • 3 or more coliforms‐‐ without normal flora do not perform identification and sensitivity, report as [Mixed coliforms, no normal flora present]. 42

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Interpretation of Culture Media Transtracheal aspirates, bronchial lavage and brush cultures (Deep respiratory specimens) should be sterile, with no normal respiratory flora However the specimen may be contaminated with oropharyngeal flora during collection. If normal flora is present, list organisms with a normal flora & list the potential pathogens separately. e.g. many normal flora consisting of: Alpha hemolytic streptococci Coagulase negative staphylococci Many pseudomonas aeruginosa Isolate, identify, and perform antibiotic sensitivity on the predominant growth

• Alpha strep ‐rule out S. pneumonia • Yeast‐rule out Cryptococcus neoformans only • S aureus, gram negative bacilli *
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Bronchial alveolar lavage (BAL) or bronchial washing The methodology of choice for the bronchoscopic diagnosis of pneumonia especially ventilator associated pneumonia.

Storage

Processing Procedure Centrifuge the specimen at 3000 rpm for 10 mins.

Specimen should be processed within 2 hrs of collection otherwise refrigeration should be utilized. Prolonged storage at 4°C may diminish the quantities of the more fastidious and fragile bacteria.

Vortex the sediment for 10 to 20 seconds. Culture the sediment on Blood agar plate‐‐‐‐‐‐‐‐‐‐ incubate in CO2 at 35°‐37°C Blood agar plate‐‐‐‐‐‐‐‐‐‐incubate anaerobically for 48 hours at 37°C Chocolate agar plate‐‐‐‐‐incubate in CO2 MacConkey agar plate‐‐‐incubate aerobically at 37°C Report as either (no pathogen isolated) or / scanty or / moderate or / heavy growth of‐‐‐‐‐‐‐‐‐

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Quantitative examination of (BAL)

Processing Procedure

Principle: Colony counts in excess of 10*4cfu/ml discriminate between colonization and infection

Centrifuge the specimen at 3000 rpm for 10 mins. Vortex the sediment for 10 to 20 seconds. • Using a sterile loop select loopful of the suspension for gram‐ staining • Dilute the original suspension with 9.0ml sterile saline to make 1:10 dilution • Using calibrated loop 0.01 of the diluted sample and steak the plates in the fashion of a urine colony count • Incubate the plates at 35‐37ºC for a full 24 hrs and read • Greater than 10 colonies represents 104 cfu/ml • Identify any colony morphptypes present in numbers >10*4 cfu/ml and submit them for appropriate susceptibility‐testing

Materials: Blood agar plate‐‐‐‐‐‐‐‐‐‐ incubate in CO2 at 35°‐37°C Chocolate agar plate‐‐‐‐‐incubate in CO2 MacConkey agar plate‐‐‐incubate aerobically at 37°C

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Reporting Report the gram stain results in terms of the presence of pus cells, epithelial cells and bacterial. A semi‐quantitative grading system is sufficient. • Report with identification and sensitivities, any isolates occurring in numbers >10*4 cfu/ml. • Report negative results as <10*4 cfu/ml of respiratory organisms isolated or as no pathogens isolated *culture of anaerobes in BAL specimens is not routinely recommended.

Causes of pneumonia acquired outside or inside hospital

Media • • • •

• Bacterial causes:

Blood agar‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐CO2 Chocolate agar‐‐‐‐‐‐‐‐‐‐‐‐‐‐CO2 MacConkey agar‐‐‐‐‐‐‐‐‐‐‐aerobic Blood agar anaerobic set up only if anaerobic infection is suspected, e.g. – – – –

– – – – – – – –

Transtracheal aspiration, Bronchial brushing, Lung abscess, and Fibrosis of lung

S.pneumoniae, H.influenza, S.pyogenes, B.catarrhalis, S. aureus, Anaerobes, Occasionally aerobic gram negative rods (kleb.). Pseudomonas aeruginosa

• Atypical pneumonia – Mycoplasma pneumonia, chlamydia, legionella pneumophila.

• Tuberculosis 53

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Parasitology

184

Kingdom of Saudi Arabia – Ministry of Health General Directorate of Training and Scholarship Training Program for Health Institute Graduates Parasitology

Medical Parasitology Lab - Slide 8 – Collection of Fecal Specimen * Urine will destroy the amoebic trophozoites and dirt will interfere with examination - Slide 12 – Preservation methods for fecal specimens Bayer’s solution - This technique was found to be very useful, particularly for cyst. - Slide 16 – SAF Need to expand out CB, IHK into actual words for the students. What is CB and IHK? - Slide 92 – Zinc Sulphate technique * A sedimentation technique is also recommended to detect these infections. - Slide 132 – Reagents and method * A 10 vol. (volume) H2O2 solution means that 1 volume will give 10 volumes of oxygen at NTP on complete degradation. - Slide 135 – False Reactions 1.1- Aspirin and NSAIDs other than acetaminophen should not be taken for 7 days prior to specimen collection to prevent possible gastrointestinal Irritation. 1.2.3- To avoid this it is best if the patient does not eat these food for at least 1 day before the test specimen is obtained. 2- Vitamin C and iron supplements containing vitamin C should be avoided for 3 days prior to collections, because ascorbic acid is a strong reducing agent that interferes with the peroxidase reaction. - Slide 136 – Interpretation Need to restrict consumption of red meat etc before doing this test. - Slide 141 – Diarrhea The total fecal osmolality is close to the serum osmolality (290 mOsm/kg) Stool PH: Human feces is normally 7-8 - Slide 151 – Procedures Neutral Fat Stain Procedure : 60 large orange-red droplets indicates malabsorption. Split Fat Stain Procedure: 100 orange-red droplets measuring 6–75 m indicates malabsorption

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Topics 1.

Introduction Sample Collection and Preservation Methods Wet mount Preparation

• •


  


2. 3.

Normal saline 0.85% Iodine BMB

Artifacts Concentration Techniques • •

4.

Modified Formal‐ Ether Sedimentation technique Acid‐ Ether Sedimentation technique

Flotation Techniques • • •

By using Sheather’s solution By using Sodium Chloride solution By using Zinc Sulphate

Medical Parasitology Lab 2

Topics (cont.)

General Lab Objectives

5. Staining of parasites 6. Detecting of Blood Parasites

1. To familiarize the student with the most widely used techniques for detection of parasites. 2. To be able to identify the parasite stages. 3. To teach the student, how to deal with risk samples.

•

Thick and thin Blood smear

7. Counting of Helminthes Eggs in Feces 8. Chemical Tests • • • •

Fecal PH test Testing feces for Occult Blood Fecal fat test Stool reducing sugar test 3

4

What is the stool or feces?

Fecal Specimen

1. Waste residue of indigestible material (cellulose during the previous 4 days) 2. Bile pigments and electrolyte. 3. Intestinal secretions, including mucus. 4. Leukocytes that migrate from the bloodstream 5. Epithelial cells that have been shed. 6. Bacteria and Inorganic material (10‐20%) chiefly Calcium and Phosphates. Undigested and unabsorbed food

• Fecal specimen are examined for protozoa, helminthes larvae or eggs. • The stages of protozoa found in stool samples are trophozoites and cysts or oocysts. • The stages of helminthes usually found in the stool samples are eggs and larvae, though whole adult worms or segment of worms may also be seen. • Adult worms and segment of tape worms are usually visible to naked eye, but eggs, larvae, cyst, oocyst and trophozoites can be seen only with the microscope. • In order to see these structure, the fecal material must be properly collected and examined. 5

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Number of Specimens and Collection Time

Collection of Fecal Specimen

• No technique is 100% successful in detecting parasites by single stool examination • At least three serial stools must be examined before a patient can be considered free from infections • Because of the intermittent passage of certain parasites, the possibility of finding organisms is increased by examining multiple specimens. • It is suggested that 3 different stool motions should be examined both pretreatment and post treatment (to ensure eradication of documented pathogenic protozoa).

• Because of fragile nature of many intestinal parasites, and the need to maintain their morphology for accurate identification, reliable microscopic diagnosis can not made unless the stool is collected properly. • The stool specimen must be enough for satisfactory examination of fresh feces uncontaminated by urine, dirt, water or other body secretion such as menstrual blood. • If the sample is too small (less than the size of a walnut or similar) or contaminated with urine, it should not be accepted. Ask the patient to pass another specimen.

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Collection of Fecal Specimen (cont.)


• Collect the specimen in a clean dry screw‐capped container • Collect the stool with a clean wooden tongue depressor or similar object. • The container should be free from antiseptics and disinfectant. • Random specimen: suitable for qualitative testing for blood and microscopic examination. • Timed specimen: for quantitative fecal testing such as fecal fat testing, because of the variability of bowel habit and the transit time required for food to pass through the digestive tract, so the most representative sample is a complete 3 day collection.

• The container with the specimen should be clearly labeled with the following: o Patient’s name and patient identifier number. o Date and time of collection. • All samples should be accompanied by a requisition form from the physician giving relevant clinical details and recent travel history. • Samples and forms from patient with a confirmed or suspected diagnosis of certain infectious diseases such as AIDS or hepatitis should be clearly labeled with “Biohazard” 9

10


Preservation methods for fecal specimens

• Most viable parasites are susceptible to desiccation or temperature variation. • If time lapse between collection and observation is considerable, i.e. more than 24 hours, it may be necessary to add some form of preservative to feces specimen to retain morphology. • Formed samples can be kept in a refrigerator at 4 ° C for a short time, but not in incubator. • Any whole worms or segments passed should be placed in a separate container

• Preservation allows fecal samples to be examined after a delay in delivery or testing. • Many methods for the preservation of stool samples and permanent staining procedures. • The most common fixatives are:     

Polyvinyl Alcohol, PVA Merthiolate Iodine Formalin, MIF Sodium acetate Acetic acid Formalin, SAF Formalin. Bayer’s solution* (Not a very well known technique – not mentioned anywhere else so suggest deleting this)

• The preservatives used have different effect on the various stages of the parasites.

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12

 Formalin

 PVA

• Formalin 4% has been used for many years as an all purpose fixative that is appropriate for helminthes eggs and larvae and for protozoan cyst. • The fixative has a long shelf life. • Concentration methods, like formalin‐ ethyl acetate concentration can be performed from the preserved stool samples without loss of concentration abilities. • The major disadvantage of formalin is that permanent staining procedures can't be performed from formalin preserved stool samples.

• This fixative is recommended for the preservation of the trophozoite and cyst stages of the intestinal protozoa, and also suitable for helminthes eggs and larvae. • The preservation of the two stages of protozoa is excellent. • The PVA is a plastic resin that serves as adhesive for the stool material. • Has a long shelf life. ( months to years ). • Concentration methods can’t performed from the specimen preserved by PVA.

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 PVA (cont.)

 SAF • Good routine fixative for protozoan cyst and trophozoites, helminthes eggs, and larvae. • Has long shelf life. ( months to years). • The preserved stool samples permits concentration techniques, most monoclonal detection kits, and permanent staining. • Unlike the PVA, the SAF fixative has poor adhesive properties when SAF preserved samples are used to prepare permanent stained smears. ( Mayer’s albumin has been recommended as an adhesive. • The combination of SAF preserved material and CB, IHK, and mod. Ziel Neelsen provides excellent staining of protozoan where staining of SAF preserved material with Trichrome gives poor results.

• The greatest advantage of this fixative is that a permanent stain can be prepared from stool specimen preserved by PVA, giving excellent result with trichrome staining. • Specimen preserved by PVA can’t be used with immunoassay kits. • Toxic, because contain mercury compound.

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 SAF (cont.)

 MIF

 Specific advantages of the use of SAF are:  SAF preserved material can be used for concentration techniques and permanent stained smears (CB, IHK).  SAF preserved material can be used for some immunoassay methods.  SAF is easy to prepare and has a long shelf life.  Unlike the PVA, the SAF fixative contain no mercury compounds. It is therefore much less toxic than PVA

• This fixative was originally developed as a screening procedure for intestinal parasites. • MIF combines preservation and staining for most kinds and stages found in feces. • It’s contains Merthiolate, Iodine, and Formalin. • The preserved material permits concentration techniques. • The major disadvantages are the short shelf life ( due to iodine) and permanent stained smears can’t be prepared from MIF preserved material. 17

18

188

Fixative used for the preservation of stool samples: an overview of the advantages and disadvantages: Formalin 4%

PVA

SAF

MIF

Toxicity

+/‐

+++ ( duo to Hg )

+/‐

+/‐

Shelf life

Long (months)

Long (months/years)

Long (months/years)

Limited

Preparation

Easy

Difficult

Easy

Easy

Quality of fixation

Egg: ++

Egg: ++

Egg: ++

Egg: ++

Cyst: ++

Cyst: +++

Cyst: ++

Cyst: ++

Troph’s: +/‐

Troph’s: +++

Troph’s: +++

Troph’s: +/‐

Possible

Not possible

Possible

Possible

Preservation of worms 1.

Cestodes  Wash in water to remove the mucus. Large tapeworms such as Taenia can be washed for several hours to relax the musculature, and can then be fixed in 10% formol saline b/w two glass slides to give flatter specimens.

2.

Trematodes  These should be treated in a similar manner to cestodes, and mounted with the ventral sucker uppermost

3.

Nematodes  Adult are washed in saline to remove mucus. Worms up to about 7 cm in length are fixed in hot(60‐70˚C) 70% alcohol, which straightens out living worms, except those which have natural curvatures at the head or the tail. Alternatively, they can be fixed in hot 5% formalin.  Large worms such Ascaris lumbricoides can be fixed and preserved in cold 5% formalin

19

Formalin ether

20

Stool Analysis

Stool Analysis (cont.)

• A stool analysis is a series of tests done on a stool (feces) sample to help diagnose certain conditions affecting the digestive tract . • These conditions can include infection (such as from parasites, viruses, or bacteria), poor nutrient absorption, or cancer.

• Laboratory analysis includes macroscopic, microscopic examination, chemical tests, and microbiologic tests. • The stool may be checked for color, consistency, weight (volume), shape, odor, and the presence of mucus and parasites stages. • The stool may be examined for hidden (occult) blood, fat, meat fibers, bile, white blood cells, and sugars called reducing substances. • The pH of the stool also may be measured. • A stool culture is done to find out if certain bacteria may be causing an infection. 21

22

Fecal Sample Examination 1.

Macroscopic Examination o o o o

2.

Color Consistency Abnormal features Adult worm or segment

1. Color: • Brown is normal color, results from the intestinal oxidation of stercobilinogen to urobilin. • Bright red to dark red to black stools occur when iron or bismuth is taken or when there is intestinal hemorrhage. • Pale yellow stools indicate the biliary obstruction, steatorrea, and also associated with diagnostic procedures that use barium sulfate. • White stools occur when there is obstructive jaundice. • Green stool may observed in patient taking oral antibiotic, because of oxidation of bilirubin to biliverdin.

Microscopic Examination o o o o o o

3.

Macroscopic Examination

WBC/HPF RBC/HPF Mucus Yeast Cyst, trophozoite, or both Larvae, egg, or both

Chemical Examination o o o o

Fecal pH test Fecal fatty acid testing Testing feces for Occult Blood Stool reducing substances testing 23

24

189

Macroscopic Examination (cont.)

Macroscopic Examination (cont.) 3.

Abnormal features: • If mucus is present writ “M”, and “B” if blood is present. • The presence of mucus coated stool is indicative for intestinal inflammation or irritation. 4. Adult worm or segments • The feces may have adult helminthes or segments present such as Ascaris lumbricoides, Entrobius vermicularis, or Taenia spp. gravid segment, these can be seen by naked eye. • And frequently motile for several days and may migrate to the top of the container.

2. Consistency: degree of moisture, will be a guide as to whether the trophozoite stage or the cyst stage of protozoa is likely to present.  Formed, write “F”  Soft , write “S”  Loose , write “L”  Watery , write “W”

25

26

Notice

Microscopic Examination of wet mount

o If several specimens are received at the same time; those containing blood and mucus should be examined first, followed by liquid specimens. These specimens are the most likely to contain amoebic trophozoites ( which die soon after being passed), and must be examined within 1 hour after passage. o Formed specimens may be examined at any time during the first day, but should not be left overnight (cyst may disintegrate). o Excessive bulky stools may indicate conditions such as giardiasis.

• Wet mount is the simplest and easiest technique for the examination of feces, and this method should be performed in all laboratories at peripheral level. • A wet mount can be prepared directly from fecal material or from concentrated specimens. • The basic types of wet mount that should be used for each fecal examination are normal saline (0.85% NaCl), iodine, and buffered methylene blue. 27

28

Microscopic Examination of wet mount (cont.)


 The Saline Wet Mount

 The Iodine Wet Mount

 Is used for the initial microscopic examination of stool specimens.  It is employed primarily to demonstrate worms eggs, larvae. Protozoan trophozoites, and cysts.  This type of mount can also reveal the presence of red blood cells and white blood cells.  If the presence of amoebic trophozoites is suspected, warm saline (37˚C) should be used.

 Is used mainly to stain glycogen and the nuclei of cysts, if present.  Cysts can usually be specifically identified in this mount.  Trophozoite can not be revealed by this type of wet mount, because iodine kill trophozoite.

29

30

190


Notice

 The Buffered Methylene Blue Wet Mount  Should be prepared each time amoebic trophozoites are seen in a saline wet mount, or when their presence is suspected.  BMB stains amoebic trophozoites, but not stain amoebic cysts, flagellate trophozoites or flagellate cysts.  BMB stain is appropriate only for fresh unpreserved specimens.  BMB stains live organism only, it isn’t used on preserved samples in which the organism have been killed  Wait for five minutes to allow the stain to penetrate the trophozoites. It will overstrain the trophozoites in 30 minutes.

Formed stool: take the portion of stool from an area to include inside and outside parts of the specimen. Stool with mucus: if mucus is present, label a second slide with the patient’s name or number. Put a drop of saline on the slide, pick up a small portion of mucus and mix with the saline. Trophozoites, if present, are sometimes more readily found in mucus than in the solid parts of the stool. Loose watery stool: if mucus is not present, pick up a small portion of the stool (any part) and mix with the saline.

31

32

Wet mount procedures

Making Direct smear Microscopy  Materials and reagents:     

Microscope slides. Cover slips. Applicator sticks. Marker or pen for labeling. Reagents:

Examine the slide on microscope: o 10X o 40X

 Saline solution (isotonic)  Lugols iodine (1% solution)  Buffered Methylene Blue (BMB) 33

34

Result of Examination

Arifacts

• If no parasites are found:

 “No ova, cysts or parasites seen”, and specify whether this result was obtained by direct examination or by a concentration method (name method used).  Never state categorically: “No parasites” • If any parasites are seen, write the scientific name of the parasite with stages • Example: Giardia lambilia cyst

35

36

191

Definition 



Blastocystis • Round or oval, sometimes with angular irregular edges, contain one large vacuole taking up almost the whole cell, the compressed cytoplasm forms a granular ring round it.

Artifacts: other things, living or artificial, present in the stool that are not parasites and could mislead the laboratory worker. Note: “Artifacts not to be mistaken for cysts”.

37

38

Blastocystis

Yeast • Oval, often with buds, often contain eccentric cluster of 3‐6 small granules. • Some related forms of yeast are rectangular, with a very clear oval cytoplasm inside: arthrospores.

39

40

Yeast

Leukocytes • Round or slightly elongated, with an irregular outline. • Contain refractile cytoplasm, clear and granular with tiny vacuoles. • Nucleus indistinct, sometimes with a star‐ shaped false karyosome. Yeast

Giardia lamblia cyst

Yeast in an iodine-stained concentrated wet mount of stool. Yeast in wet mount may be confused for Giardia lamblia cyst. 41

42

192

Pus

Bacteria

• Pus can be seen by the naked eye as opaque, greyish streaks( not transparent like mucus). • Under the microscope it appears as a mass of more or less degenerate leukocytes

43

44

Coccidia (cont.)

Coccidia • These are protozoa that may be parasite of men without causing any significant pathogenic effects, or may be found in transit in stool following the consumption of infected foods. • They appear in stool in a form resembling cyst called oocysts or sporocysts. • An elongated oval, sometimes tapered at one pole. • There three types: a. 4 sporozoites (small banana shaped rods), each containing a small round nucleus, sometimes a few large granules massed at one pole. b. One large round granular cell. c. Refractile granules completely fill the interior. 45

46

Coccidia (cont.)

Air bubbles

47

48

193

Oil droplet

Fungal spores

Fungal spore

Entamoeba histolytica/dispar cyst

Fungal spore in a wet mount of stool. Such spores may be confused for the cysts of Entamoeba spp. 49

50

Plant fiber

Strongyloides stercoralis larvae

51

52

Hair fiber

Misdiagnosis can lead to improper treatment

53

54

194

Plant cell

Plant cell

May be confused with helminthes eggs

Plant material in an iodine‐stained concentrated wet mount of stool. This material can be confused for a hookworm egg 55

56

Epithelial cells

Charcot‐Leyden crystals

58

57

Macrophages

RBCs

59

60

195

Pollen grains

Pollen grains

61

62

Starch granules

Crystals

63

64

Non‐ parasitic structure found in stool

Cont.

 Non parasitic objects may be misidentified as parasites. The differentiation of the most common pseudoparasites is as follow:

3.

1.

Protozoan cyst: may be confused with air bubbles, fat globules or yeasts.

4. 5. protozoan trophozoites and macrophages. – Trophozoites of Entamoeba histolytica/dispar must be motile and hematophagus. – Macrophages found in cases of intestinal amoebiasis are distinguishable from amoebic trophozoites by possessing a larger nucleus and, although they can haematophagus, they are only motile for a very short time. Their pseudopodia are small, blunt and granular.

6.

Ova, their general shape, except for Entrobius, is perfectly symmetrical, distinguishing them from various objects found in stools. Trichuris and Taenia ova may be confused with pollen grains. Ascaris ova may be confused with vegetable cells, the latter having smooth, thick walls but irregular shape. Strongyloides or hookworm larvae can be confused with hair or vegetable fibers. The latter are usually tapered at one end and the other being blunt and with no internal structure. – Free living nematode larvae may be found in concentrates if contaminated water is used

65

66

196

Concentration techniques

Cont. 7. Fasciola ova resemble vegetable cells. 8. Insect egg may be found in stools as spurious infection. Mite eggs may be confused with hookworm eggs. 9. Dipylidium caninum eggs sacs can look similar to vegetable cells. 10. Other structure found in stool are crystals, Charcot‐ Leyden are the breakdown products of eosinophil cells and may be present in stools or sputum. Can also indicate parasitic infection present. 11. Starch granules are sometimes seen in stool. When undigested, they appear as concentric rings and stain blue with iodine, when partially digested, they stain red. 67

68

2.

The microscopic examination of feces is required for the recognition and identification of intestinal parasites:

Concentration techniques : Advantages  Maximizes the numbers of organisms detected which may be too scanty to be seen by direct microscopy alone.  Worm eggs, larvae, and protozoan cysts may be recovered. • Disadvantages  Destroys trophozoite stages. Most concentration methods destroy trophozoites stages.  Will not pick up Enterobius vermicularis as these are lost during the process due to eggs deposited on outside of feces not throughout the feces. •

1. Direct Microscopy: • Advantages Useful for the observation of motile protozoan trophozoites. • Disadvantages May not detect ova, cysts and larvae which are present in scant numbers.

 The purpose of concentrating feces is to increase possibility to finding ova, cyst, or larvae in samples that not be able to seen by direct microscopy. 69

Concentration Methods

70

Modified Formalin‐Ethyl Acetate sedimentation

1. Sedimentation method  

Modified Formalin‐Ethyl Acetate sedimentation technique Acid‐ Ethyl Acetate sedimentation technique

Sedimentation Methods

2. Flotation method   

Saturated Salt Solution technique Sheather’s Sugar Centrifugal Flotation technique Zinc Sulphate Centrifugal Flotation technique

71

72

Materials and Method

Modified Formalin‐Ethyl Acetate Sedimentation • Formalin‐ Ethyl acetate method is the recommended concentration procedures. • Most types of worm eggs (round worms, tapeworms, schistosomes, and other fluke eggs), larvae, and protozoan cysts may be recovered by this method. • Advantages: 1. Speed: one sample can be processed in 5 minutes. 2. Broad spectrum: it will recover most ova, cyst and larvae. 3. The morphology of most parasites is retained for easy identification. • Disadvantages: 1. Requires several pieces of apparatus which does not make it an easy. 2. The preparation contains some debris. 3. Formalin is an irritant. 4. Hymenolepis nana and Fasciola spp. do not concentrate well.

 Libra  Applicator stick  Glass centrifugal tubes  Beaker  Wire sieve  Vortex or whirlimixer  Centrifuge.  Reagent: Reagent I: 10% formalin solution in distilled water. Reagent II: ethyl acetate. 73

74

Procedures

Procedures (cont.)

1. Emulsify 1 gm. of feces in 7 ml of 10% formalin in a centrifuge tube. 2. Strain the suspension through a brass wire sieve, and collect in beaker. 3. Pour the filtrate into a 15 ml boiling tube and add 3 ml of ethyl acetate, then mix well 15 sec on vortex or whirlimixer or 1 min by hand. 4. Transfer the ethyl acetate ‐ formalin suspension back into the washed centrifuge tube, and centrifuge at 3,000 rpm for 1 min.

5. Loosen the fatty layer and debris at the top of the tube with an applicator stick and invert the tube quickly to discard the supernatant. 6. On righting the tube, a few drops only should remain with the sediment, mix the sediment well and transfer one drops onto a glass slide and cover it with coverslip. 7. Scan the whole coverslip using 10x objective, turning into 40x for confirmation of identification of parasites. 75

76


Acid‐ ethyl acetate sedimentation technique

       

Sedimentation Methods

Libra Applicator stick Glass centrifugal tubes Beaker Wire sieve Vortex or whirlimixer Centrifuge. Reagent:  Reagent I: 15% Hydrochloric acid.

77

78

198

Procedures

Procedures (cont.)

1. Mix thoroughly 1 gm. feces with 3 ml of 15% of hydrochloric acid and then mix well. 2. Add and additional 5‐6 ml of 15% HCl and mix. 3. Strain the suspension through a wire sieve into beaker. 4. Place suspension in a glass centrifuge tube and make up to the 10 ml with distilled water. 5. Add 4 ml of ethyl acetate, stopper the tube and shake vigorously 20 ‐30 sec using vortex.

6. Centrifuge 2‐3 min at 1500 rpm, the suspension now will be layered. 7. Loosen plug of debris with applicator stick and immediately pour off liquid. 8. Transfer one drops onto a glass slide and cover it with coverslip. 9. Scan the whole coverslip using 10x objective, turning into 40x for confirmation of identification of parasites. 79

80

Flotation Method

Concentration Methods 1. Flotation method

Concentration Techniques

  

Saturated Salt Solution technique Sheather’s Sugar Centrifugal Flotation technique Zinc Sulphate Centrifugal Flotation technique

81

82

Flotation technique

Saturated Salt Solution

 These method use the high specific gravity of a solution to float the lighter ova and cyst. They can be improved by centrifugation.

Flotation Techniques

• Advantage: • Easy to perform . • Disadvantage: • Delay in examination can result in distortion. • Larvae and some fluke eggs do not concentrate. • Frequent checking of specific gravity.

83

84

199


Saturated Salt solution technique

1.

 Advantages:

2. 3. 4. 5. 6. 7.

Boil granular sodium chloride in excess in water to produce a saturated solution which when cooled has a specific gravity of 1.18 ‐ 1.2. Half fill a wide‐ mounted flat bottomed container with the saturated salt solution. Emulsify 1gm of feces in the solution and strain it to remove the debris from the surface. Pour the filtrate into meniscus and fill it to the top with saturated salt solution. Lay a glass slide or large coverslip over the top, avoiding any bubbles being trapped. Leave for 20 min before quickly inverting the slide. Scan for ova using the 10x objectives.

– It is cheap preparation using simple apparatus. – It concentrates nematode ova well.

 Disadvantages: – It doesn’t concentrate cysts.

85

86


Sheather’s Sugar Centrifugal Flotation Technique

 Sheather’s sugar solution: – Table sugar ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 500gm – Distilled water ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 320ml – Phenol crystal ( melt in hot water bath) ‐‐‐‐‐ 6.5gm

87

88

Procedures

Sheather’s Sugar solution technique

1. Soften 1gm of feces with water to a soft. 2. Strain the aqueous suspension through a wire sieve. 3. Mix 1 part aqueous suspension with 2 part of Sheather's sugar solution. 4. Pour into a centrifuge tube, centrifugation 1500 rpm for 10 minutes. 5. Pour the supernatant into a meniscus and add a sufficient solution to bring the meniscus to the top. 6. Place a coverslip and wait for 10 minutes. 7. Examine under microscope.

 Advantages: – Reveals most nematode eggs and protozoan cyst.

 Disadvantages: – Flukes eggs and tape worm eggs are not demonstrate well. – Also most nematode larvae are not demonstrate well.

89

90

200

Zinc Sulphate method

Zinc Sulphate technique  Advantages: – Zinc sulphate centrifugal flotation technique is useful for the recovery of protozoan cysts and helminthes eggs.

 Disadvantages: – Large trematode eggs, some tape worm eggs, and infertile Ascaris lumbricoides eggs are not concentrated by this method.

91

92


Staining of Parasites

 Zinc sulphate solution with specific gravity 1.18. – Mix 330gm dry zinc sulphate in 670ml distilled water. – Use the hydrometer or densitometer to adjust specific gravity around 1.18

93

94

Protozoa in wet mount

Continued

A. Saline wet mount: • In saline wet mount, trophozoites and cyst of amoeba, flagellates and ciliate may be seen. • Cyst will appear as round or oval, refractile structure. • Trophozoite of amoeba may be round or irregular, but trophozoite of flagellate are usually pyriform (elongated, pear‐ shaped). • In freshly passed feces (the stool must not be more than 1hour old), motile trophozoites may be seen. • Motility can be very helpful in identifying species, especially in case of flagellates.

• Organism may be detected with the low power (10x) objective, but a high power (40x) dry objective will be necessary to identify reliable the structure as a cyst or trophozoite. • With the high power dry objective, you can see motility, inclusions like erythrocytes and yeast in amoebic trophozoites, chromatoid bodies in amoebic cyst. • Also, we can see the shape and structural detail (sucking discs, spiral grooves, or filaments) of flagellate trophozoites and cysts. 95

201

Continued

Continued

• However, it’s necessary to regulate carefully the microscope illumination so that the objects appear clearly. • Too much or too little light will interfere with your observations. • It’s also necessary to focus up and down to see all the layer of the specimen. • Remember to examine the whole coverslip area in a systematic manner to reduce the chances of overlooking organisms.

B. Iodine wet mount: • Iodine wet mount are examined for amoebic and flagellate cysts but not trophozoites because it’s killed by iodine. • In the iodine wet mount, cytoplasm of the cyst will stain yellow or light brown, and nuclei will stain dark brown. • Cyst stained with iodine can be detected with 10x objective, but they are not refractile as in saline mount. 40x used to see the characteristics of the cysts. • In iodine stained cysts of Entamoeba, the arrangement of peripheral chromatin and the position of karyosome can be seen. If the peripheral chromatin isn’t present, the cyst is not an Entamoeba spp. These peripheral chromatin bodies stain light yellow. • Sometimes, young cysts contain glycogen, this stains dark brown with iodine.

97

98

Continued

Continued

 Buffered Methylene Blue wet mount: • If you see amoebic trophozoites, or structures that resemble trophozoites, you should prepare and examine BMB mount. • After 5‐10 minutes of staining, the trophozoite sometimes remain motile, but often curl up in BMB preparation. For that do not confuse curled trophozoites with cysts do not stain with BMB solution. • In the trophozoites, the nucleus and inclusion (RBC, yeast) will stain dark blue while the cytoplasm will stain light blue.

• Occasionally, some trophozoite will not stain, so you should look for well‐ stained organisms. • Look for peripheral nuclear granules (granules in membrane around the nucleus), if these are present, the trophozoite is an Entamoeba ssp. and you must identify the species.

99

100

Helminthes in wet mount

Continued

• Eggs may be easily detected and identified in saline mounts. • They should not be stained (stains may interfere with identification). • Most eggs are large enough to be recognized with the low power (10x) objective, but a few small eggs will require a high power dry lens (40x) objective. • In saline mounts, larvae may be present, and it’s easily recognized with 10x objective.

• In saline mounts, larvae of Strongyloides stercoralis may be seen, but Hookworm larvae are not usually present if the sample is fresh. • If the sample is old and contain Hookworm infection, then larvae may be present. • SO, it may be necessary to distinguish between these two species. Activity Find the ways to differentiate between species in old samples. 101

102

202

Counting helminthes eggs in feces

Counting of Helminthes Eggs – Stoll’s Method

• The intensity of an intestinal helminthes infection may sometimes be indicated by the concentration of its eggs in feces. • Eggs counts can be value in epidemiological surveys. • The approximate number of eggs per gram of feces can be calculated by using formal ether technique. • When a more accurate count is required the Stoll’s method can be used.

103

104

Stoll’s technique procedures

Continued

1. 2.

8.

3. 4. 5.

6. 7.

Weigh 3 grams of feces in a screw cap container. Add 42 ml of water to give (1/15) dilution of the feces. – If the feces are formed specimen, use sodium hydroxide 0.1mol/l solution instead of water. Using a rod, break up the feces and mix it with the water. Cap the container and shake hard to complete in mixing. Using a graduated plastic bulled pipette, or a Pasteur pipette marked to measure the required volume, pick 150 μl of the suspension and transfer this slide. Cover the slide with long coverslip if available, or two squares coverslips. Examine systematically the entire preparation, using 10x objective. Include the count any eggs laying outside the edges of the coverslip because these are contained in 150 μl sample.

Multiply the number of eggs counted by 100 to give the number of eggs per gram of feces. – If the specimen isn’t formed, the following additional calculation is necessary to give the number of eggs per gram. • Fluid specimen ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ *5 • Unformed watery specimen ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ *4 • Unformed soft specimen ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ *3 • Semiformed specimen ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ *2 9. Calculate the number of eggs per day, by multiplying the number of eggs per gram by the total weight of 24hrs fecal specimen. 10. Calculate the number of burden worm by dividing the number of eggs per gram on number of eggs the parasite laying per day.

105

106

Interpretation of results

Detection of Blood Parasites

The Center For Disease Control, Atlanta Protozoa

Helminthes

Rare

2‐5 / coverslip

2‐5 / coverslip

Few

1 / 5‐10 HPF

1‐2 / LPF

1‐2/ HPF or 1/ 2‐3 HPF

1 / 5‐10 HPF

Several / HPF

Several / LPF

Moderate Many

Thick & Thin Blood Smear

107

108

203

Blood Examination

Continued

• The most commonly used technique for blood examination is stained blood films. • Geimsa stain is usually used to stain the films. • Delafild’s haematoxylin stain is used for microfilariae. • Either thick or thin films may be used depending on the circumstances. • The thick film is more sensitive in detecting parasite and also saves time in examination.

• Therefore, both thick and thin films must always be prepared when searching for plasmodia and trypanosomes. • If a precise identification can not be made from thick film, the thin film will be available. • Thick films should be used when searching for microfilariae. • The most economical use of slides is achieved by making a combination thick and thin slide. (not recommended due to differing drying times) • However, combination films must dry thoroughly 8‐10 hrs. to overnight before they can be satisfactorily stained. • Slides for malaria should be stained in the same day.

– Good film thickness can be gauged by being able to just still read printing through the film when slide placed on a printed page.

• The thin film technique cause very little distortion of the parasite, and permits species identification when it may not be possible in thick films, but many fields must be examined to detect parasite when they are few in number. 109

110

Continued

Examination of Thick & Thin blood smear

• The thin films will dry quickly and can be stained as soon as they are dry, and examine for parasites. • If parasites are not seen in the thin film, stain the thick film using Field’s stain, and examine for parasites. • Direct wet mounts of fresh whole blood (or centrifuged blood) are usually used for detection of microfilariae and trypanosomes, this only gives evidence of infection and stained films are necessary for confirmation of species present. • In areas where malaria, trypanosomes, and/or microfilariae may all present, both wet and stained films should be prepared and examined. • If neither trypanosomes nor microfilariae occur in region, only stained smears need to be made for detection of plasmodia.

• For optimum staining, the thick and thin films should be made on separate slides and different concentrations used for staining. • When it’s done, good quality staining of thick film is of primary importance. Best results are obtained if the blood smear have dried overnight. • Fixation of thin blood film done by adding 3 drops of methanol, or dipping it in a container of methanol for few seconds, with prolonged fixation it may be difficult to demonstrate Schuffner’s dots and Maurer’s dots. • To permit dehemoglobinization, thick film should not be fixed; therefore avoid exposure to methanol or methanol vapor

111

Reading of Thick Film

Reading of Thin Film

• Focus on film with 10x objective and search for microfilariae. They are easily detected with 10x objective. • If microfilariae are present, switch to oil‐ immersion objective and identify the species. • Also, look for malaria parasites with oil‐ immersion objective, at least 100 fields should be examined. • Microscopy of thick film should reveal the following features: 1. The background should be clean, free from debris, with a pale mottled‐ gray color derived from the lysed erythrocytes. 2. Leukocyte nuclei are stained a deep, rich purple. 3. Malaria parasite are well defined with deep‐ red chromatin and pale purplish blue cytoplasm.

• Focus with the 10x objective on the thin terminal end of the film where the RBCs are in one layer, put oil drop on the slide and switch to the oil‐ immersion objective. • When examining fro malaria parasites and trypanosomes, at least 200 fields should be examined. • Microscopy of thin film should reveal the following features: 1. The background should be clean and free from debris; erythrocytes are stained a pale greyish pink. 2. Neutrophil leukocytes have deep purple nuclei and well defined granules. 3. Malaria parasite are well defined with deep‐ red chromatin and pale purplish blue cytoplasm. 4. Like plasmodia, the cytoplasm of trypanosomes stain blue, the nucleus and kinetoplast stain red or purple. 113

112

114

204

Comparison

Identification of malarial parasites • In thin films, look at the appearance of the parasite and the appearance of the RBCs containing the parasites. 1. The appearance of the parasites 2. The appearance of the RBC containing the parasites:  Size & Shape: Is the parasitized cell the same size as the blood cell without parasite or Is it enlarged?  Stippling: Is the RBC filled with pink or red staining dots? • Schuffner’s stippling in the “ghost” of the erythrocyte can some times be seen at the edges of the film and indicate infection with Plasmodium vivax or P. ovale,. • Maurer’s dots show as stippling in erythrocytes containing the larger ring forms of Plasmodium falciparum.

Thick smear

Thin smear

Lysed RBCs, many layer

Fixed RBCs, single layer

( large volume ) 0.25 μl blood/100 fields

( small volume ) 0.005 μl blood/100 fields

Good screening test ( positive or negative )

Good species differentiation

Save time in examination

Requires more time to read

Low density infection can be detected Low density infections can be missed as blood elements more concentrate 115

(more sensitive)

Blood Protozoa

(less sensitive)

116

Trypanosoma Trypomastigotes Microfilariae

Trypanosoma Plasmodium falciparum

Blood Parasite Leishmania

Plasmodium vivax Plasmodium Plasmodium ovale

Plasmodium malariae 117

118

119

120

Leishmania promastigotes

205

121

122

Ring form

P. vivax

P. malariae

P. ovale

P. falciparum

123

124

Trophozoite form

P. vivax

P. malariae

Schizonts form

P. ovale

P. falciparum

P. vivax

P. malariae

125

206

P. ovale

P. falciparum

126

Gametocyte form

Species Differentiation On Thin Films Feature

Enlarged infected RBC

Infected RBC shape

P. vivax

P. ovale

Stippling infected RBC

Trophozoite shape

Chromatin dot

P. malariae

P. falciparum

Mature schizont

127

P. Falciparum

P. vivax

P. ovale

P. malariae

‐

+

+

‐

round

round, distorted

oval, fimbriated

round

Maurer’s clefts

Schuffner's spots

Schuffner's dots

none

small ring, applique

large ring, amoeboid

large ring, compact

small ring, compact

often double

single

large

single

rare, 12‐30 merozoites

12‐24 merozoites

4‐12 merozoites 6‐12 merozoites ( rosette ) ( scattered )

128

Fecal Occult Blood Test “ FOBT” • Hematemesis: bleeding into the gastrointestinal tract may be rapid with the vomiting of blood. • Melaena: the passage of blood through the rectum. • When the bleeding is chronic with only small amounts of blood being passed in the feces. • If the blood or it’s products is not recognized in the feces, it is referred to Occult blood (hidden Blood).

Fecal Chemical Tests Testing for Fecal Occult Blood

129

130

Principle of the Test

Reagents and method

• Chemical tests to detect occult blood are based on the principle that hemoglobin and it’s derivatives react in a similar way to peroxidase enzymes (pseudo‐ peroxidase activity). • Chromogen such as guaiac, O‐ toluidine, 4‐ aminophenazone or benzidine will be used as indicator for oxidation reaction. • Hemoglobin and its derivatives catalyze the transfer of oxygen from hydrogen peroxide to Guaiac, oxidation of the colorless chromogen produces a blue color.

•

•

Hemoglobin + H2O2 + Guaiac Pseudo-peroxidase Oxidized guaiac + H2O O2

Blue color 131

Reagents: – Acetic acid 10% v/v – Alcohol 95% v/v – 4‐aminophenazone (4‐aminoantipyrine) – Hydrogen Peroxide (H2O2) 10 vol. solution* Working 4‐aminophenazone reagent: – The amounts given are sufficient for 1 test with positive and negative controls. – Prepare fresh as follows: • Alcohol 95% v/v ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 15 ml • Acetic acid 10% v/v ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 1ml • 4‐aminophenazone ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 0.4g • Dissolve the 4‐aminophenazone in alcohol solution and immediately before use add the acetic acid. Mix well 132

207

Procedures

Continued

1. 2.

6.

Dispense about 7ml of distilled water into a test tube Add a sample of feces about 1gm., use a glass or plastic rod to emulsify the feces. 3. Allow the fecal particles to settle or centrifuge the emulsified specimen. 4. Take 3 completely clean tubes and label them as : – T: Patient's test. – Neg.: Negative control. – Pos.: Positive control. 5. Add into each tube as follow: – T ‐‐‐‐‐‐ 5ml supernatant fluid from emulsified feces. – Neg. ‐‐‐‐‐‐ 5ml distilled water. – Pos.: ‐‐‐‐‐‐5ml distilled water in which 5ul of whole blood has been mixed.

Add 5ml of working 4‐aminophenazone reagent on top of the fluid in each tube .Do Not Mix 7. Add 10 drops of the 10 vols. Hydrogen peroxide solution. Do Not Mix, allow to stand for 1 minute. 8. Look for the appearance of a blue color where 4‐aminophenazone reagents meets the sample or control solutions. – Neg. control: this should show no color change. – Pos. control: this should show a positive reaction.

133

Color

Result

No color change

Negative

Pale blue

Positive +

Dark blue

Positive + +

Blue – black

Positive + + +

False Reactions

Interpretation

 False positive: 1 1. Aspirin and anti‐inflammatory medications. 2. Red meat (contain Myoglobin) and fish. 3. Green vegetables (Melons), and Horseradish. 4. Menstrual and hemorrhoid contamination. 5. Some intestinal bacteria that produce peroxidase enzymes.  False negative: 2 1. Vitamin C greater than 250 mg/dl 2. Iron supplements containing vitamin C

• The commonest cause of positive occult blood tests in tropical and other developing countries are Hookworm infections, peptic ulcer, and bleeding from esophagus or liver cirrhosis. • Other causes include carcinoma in gastrointestinal tract, erosive gastritis duo to alcohol or drugs, or swallowed blood from nosebleeds. • If the test is negative but there is high clinical suspicion, a further two specimen should be tested to detect bleeding which be intermittent.

134

135

136

137

138

Commercial Methods • Therefore, to prevent false‐positive reactions, the sensitivity of the test must be increased. • Many commercial testing kits are available for occult blood testing with guaiac reagent. • The kits contain guaiac impregnated filter paper, to which the fecal specimen and hydrogen peroxide are added. • Two or three filter paper areas are provided for application of material taken from different areas of the stool, and positive and negative controls are also included.

208

Continue …... • iFOBT: The immunochemical fecal occult blood test, Hemoccult ICT, is specific for the globin portion of human hemoglobin and uses anti-human hemoglobin antibodies. • Because Hemoccult ICT is specific for human blood in feces, it does not require dietary or drug restrictions. • It is more sensitive to lower GI bleeding that could be an indicator of colon cancer or other gastrointestinal disease. • Can be used for patients who are taking aspirin and other anti-inflammatory medications.

• Fecal Chemical Tests • Fecal Fat Testing & Reducing Sugars

139

140

Diarrhea

Continued

• Diarrhea is defined as an increase in daily stool weight above 200 with increased liquidity and frequency of more than three times per day. • Diarrhea classification can be based on four factors: duration of the illness, mechanism, severity, and stool characteristics. • Diarrhea lasting less than 4 weeks is defined as acute, and diarrhea persisting for more than 4 weeks is termed chronic diarrhea. • The major mechanisms of diarrhea are secretory, osmotic, and altered motility. • The laboratory tests used to differentiate these mechanisms are fecal electrolytes (fecal sodium, fecal potassium), fecal osmolality, and stool pH.

• Secretory Diarrhea: Bacterial, viral, and protozoan infections produce increased secretion of water and electrolytes, which override the reabsorptive ability of the large intestine • Osmotic Diarrhea: Incomplete breakdown or reabsorption of food presents increased fecal material to the large intestine, resulting in the retention of water and electrolytes in the large intestine which in turn results in excessive watery stool. • Altered Motility: Altered motility describes conditions of enhanced motility (hypermotility) or slow motility (constipation).

141

142

Malabsorption & Maldigestion

Investigations of Malabsorption

• Malabsorption is defined as the impaired absorption of nutrients by the intestine. • Maldigestion is defined as the impaired digestion of food. • Maldigestion and malabsorption contribute to osmatic diarrhea. • Specific nutrient absorptive disorders:

 Investigations of Carbohydrate Malabsorption:  D‐ xylose Test  Lactose Intolerance  Stool Reducing Sugar  Investigations of Fat Malabsorption:  Fecal Fat Test  Investigations of Protein Malabsorption:  Albumin Level  α1‐ antitrypsin  Investigations of Mineral and Vitamins Malabsorption:  Stool Electrolyte (Na+, K+)

– – – –

Carbohydrate Malabsorption Fat malabsorption/Maldigestion Protein Malabsorption Mineral and Vitamin Malabsorption 143

144

209

Investigation of Carbohydrate Malabsorption

Continued

Stool Reducing Sugar • This test measures unabsorbed sugars in stool. It is used to evaluate the body's ability to digest carbohydrates, or to absorb nutrients from food and drinks. • Testing for fecal reducing substances detects congenital disaccharidase deficiencies as well as enzyme deficiencies due to nonspecific mucosal injury. • Fecal carbohydrate testing is most valuable in assessing cases of infant diarrhea and may be accompanied by a pH determination. • The copper reduction test is performed using a Clinitest tablet and one part stool emulsified in two parts water .

• A result of 0.5 g/dL is considered indicative of carbohydrate intolerance. • The Clinitest on stools can distinguish between diarrhea caused by abnormal excretion of reducing sugars and those caused by various viruses and parasites. • Sucrose is not detected by the Clinitest method because it is not a reducing sugar. • A positive result would be followed by more specific serum carbohydrate tolerance tests, the most common being the D‐xylose test for malabsorption and the lactose tolerance test for maldigestion. 145

146

Continued

Investigation of Fat Malabsorption

• The Fecal Reducing Substances test is performed in a laboratory, on a sample of stool as small as 5 grams. • Unfortunately this sample needs to be delivered to the laboratory ASAP, preferably within 1 hour. • This is because lactose (or other sugars) in the stool will normally be broken down by chemical processes within 2‐4 hours after the specimen is produced • Adults and Children : – Normal: = 0.25 g/dl – Suspicious: 0.25‐0.5 g/dl – Abnormal: > 0.5 g/dl

• Steatorrhea: is the presence of excess fat in feces, Stools may also float due to excess lipid, have an oily appearance and be especially foul‐smelling. • There is increased fat excretion, which can be measured by determining the fecal fat level. • Possible biological causes can be lack of bile acids, defects in pancreatic enzymes “maldigestion”, and defective mucosal cells “malabsorption”. • The absence of bile acids will cause the feces to turn gray or pale. • Quantitative fecal fat analysis is used as a confirmatory test for steatorrhea 147

148

Investigation of Fat Malabsorption

Qualitative Microscopy Fat Testing

• Fecal fat testing can be done by either qualitative microscopy method or quantitative methods. 1. Qualitative Microscopy Method: – Is the simplest form of the fecal fat test, a random fecal specimen is submitted to the hospital laboratory and examined under a microscope after staining with a Sudan III dye. Visible amounts of fat indicate some degree of fat malabsorption. – The staining procedure consists of two parts, the neutral fat stain and the split fat stain.

• Neutral fats stain are readily stained by Sudan III and appear as large orange‐red droplets, often located near the edge of the coverslip. • Observation of more than 60 large orange‐red droplets/HPF can be indicative of steatorrhea. • The split fat stain representing total fat content can provide a better indication. • The breakdown of neutral fats by bacterial lipase and the spontaneous hydrolysis of neutral fats may lower the neutral fat count. • Observation of more than 100 large orange‐red droplets/HPF can be indicative of steatorrhea • This also precludes the comparison of the two slide tests to determine whether maldigestion or malabsorption is causing steatorrhea 149

150

210

Procedures

Quantitative Fat Testing

• Neutral Fat Stain Procedure 1. Homogenize one part stool with two parts water. 2. Mix emulsified stool with one drop 95% ethyl alcohol on slide. 3. Add two drops saturated Sudan III in 95% ethanol. 4. Mix and coverslip. 5. Examine under high power 6. Count orange droplets per high‐power field

• Split Fat Stain Procedure

• Quantitative fecal fat analysis is used as a confirmatory test for steatorrhea. • Quantitative fecal analysis requires the collection of at least a 3‐day specimen. • The patient must also maintain a regulated intake of fat (100 g/dl) prior to and during the collection period. • Refrigerating the specimen prevents any bacterial degradation. • The method routinely used for fecal fat measurement is the Van de Kamer titration, although gravimetric methods are available. • Fecal lipids are converted to fatty acids and titrated to a neutral endpoint with sodium hydroxide.

1. Mix emulsified stool with one drop of 36% acetic acid. 2. Add two drops saturated Sudan III. 3. Mix and coverslip. 4. Heat gently almost to boiling. 5. Examine under high power. 6. Count and measure the orange droplets per high‐ power field 151

152

Continued

Continued

• The fat content is reported as grams of fat or the coefficient of fat retention per 24 hours.

• The acid steatocrit is a reliable tool to monitor a patient’s response to therapy and screen for steatorrhea in pediatric populations.

( Dietary Fat – Fecal Fat )

• The coefficient of fat =

Dietary Fat

Fatty layer length in cm

X 100

• Normal values based on a 100 g/dl intake are 1 to 6 g/dl or a coefficient of fat retention of at least 95%. • Although the Van de Kamer titration is the gold standard for fecal fat, the acid Steatocrit is a rapid test to estimate the amount of fat excretion. • It is similar to the microhematocrit test.

• Acid steatocrit % = (Fatty layer length in cm + solid layer X100 length)

• An acid steatocrit value less than 31% was considered normal while a value greater than 31% indicated steatorrhea in adults. • Acid steatocrit is higher in infants and droppped with age . An acid steatocrit of less than 10% is indicative of steatorrhea in children. 153

154

Continued • Calculate the fecal fat in grams per 24 hours. • In adult: – Fecal fat in g/24 hrs.= [0.45 X acid steatocrit %] – 0.43

• In children up to 15 years: – Fecal fat in g/24 hrs.= [0.1939 X acid steatocrit %] – 0.2174

155

211


Parasitology • The study of eucaryotic parasites, protozoa and helminths • Cause 20% of all infectious diseases • Less prevalent in industrialized countries; increasingly common in AIDS patients


The Parasites of Medical Importance “Protozoa”

2

Typical Protozoan Pathogens • Single‐celled, animal‐like microbes, most having some form of motility • Estimated 100,000 species, approximately 25 are important pathogens • Life cycles vary – Most propagate by simple asexual cell division of the active feeding cell (trophozoite). – Many undergo formation of a cyst. – Others have a complex life cycle that includes asexual and sexual phases. 3

4

Entamoeba histolytica and Amebiasis • Alternates between a large trophozoite, motile by means of pseudopods and a smaller nonmotile cyst • Trophozoite has a large nucleus and lacks most other organelles. • Humans are the primary hosts. • Ingested • Carried by 10% of world population

Infective Amebas

5

6

212

Entamoeba histolytica • Cysts are swallowed and arrive at the small intestine; alkaline pH and digestive juices stimulate cysts to release 4 trophozoites. • Trophozoites attach, multiply, actively move about and feed. • Asymptomatic in 90% of patients • Ameba may secrete enzymes that dissolve tissues and penetrate deeper layers of the mucosa. • Causing dysentery, abdominal pain, fever, diarrhea and weight loss 7

8

Entamoeba histolytica

Amebic Infections of the Brain

• Life‐threatening manifestations are: hemorrhage, perforation, appendicitis, and tumorlike growths, amebomas. • May invade liver and lung • Severe forms of disease result in 10% fatality rate. • Effective drugs are iodoquinol, metronidazole, and chloroquine

• Caused by Naegleria fowleri and Acanthamoeba • Ordinarily inhabit standing water • Primary acute meningoencephalitis is acquired though nasal contact with water or traumatic eye damage. • Infiltration of brain is usually fatal.

9

10

11

12

An Intestinal Ciliate: Balantidium coli • An occupant of the intestines of domestic animals such as pigs and cattle • Acquired by ingesting cyst‐containing food or water • Trophozoite erodes intestine and elicits intestinal symptoms. • Healthy humans are resistant. • Rarely penetrates intestine or enters blood • Treatment – tetracycline, iodoquinol, nitrimidazine or metronidazole

Trichomonads: Trichomonas species • • • •

The Flagellates

Small, pear‐shaped 4 anterior flagella and an undulating membrane Exist only in trophozoite form 3 infect humans:

– T. vaginalis – T. tenax – T. hominis

13

14

Trichomonas vaginalis Causes an STD called trichomoniasis Reservoir is human urogenital tract 50% of infected are asymptomatic. Strict parasite, cannot survive long outside of host 3 million cases yearly, a top STD Female symptoms – foul‐smelling, green‐to‐yellow discharge; vulvitis; cervicitis; urinary frequency and pain • Male symptoms – urethritis, thin, milky discharge, occasionally prostate infection • Metronidazole • • • • • •

15

16

17

18

Giardia lamblia and Giardiasis • Pathogenic flagellate • Unique symmetrical heart shape with concave ventral surface that acts like a suction cup • Cysts are small, compact, and multinucleate. • Reservoirs include beavers, cattle, coyotes, cats, and humans. • Cysts can survive for 2 months in environment. • Usually ingested with water and food • ID 10 to 100 cysts

Hemoflagellates: Vector‐Borne Blood Parasites • Obligate parasites that live in blood and tissues of human host • Cause life‐threatening and debilitating zoonoses • Spread in specific tropical regions by blood‐sucking insects that serve as intermediate hosts • Have complicated life cycles and undergo morphological changes • Categorized according to cellular and infective stages

• Cysts enter duodenum, germinate, travel to jejunum to feed and multiply • Causes giardiasis – diarrhea, abdominal pain • Diagnosis is difficult because organism is shed in feces intermittently. • Treatment: quinacrine or metronidazole • Agent is killed by boiling, ozone, and iodine

19

20

Trypanosoma species and Tropanosomiasis • Distinguished by their infective stage; trypomastigote is an elongate, spindle‐shaped cell with tapered ends, eel‐like motility • 2 types of trypanosomiasis:

– T. brucei – African sleeping sickness – T. cruzi – Chagas disease – endemic to Central and South America

21

22

Trypanosoma brucei and African Sleeping Sickness • Spread by tsetse flies • Harbored by reservoir mammals • Two variants of disease caused by 2 subspecies:

• Chronic disease symptoms are sleep disturbances, tremors, paralysis and coma. • Trypanosomes are readily demonstrated in blood, spinal fluid or lymph nodes. • Treatment before neurological involvement melarsoprol, eflornithine • Control involves eliminating tsetse fly.

– T.b.gambiense – Gambian strain; West Africa – T.b. rhodesiense – Rhodesian strain; East Africa

• Biting of fly inoculates skin with trypomastigotes, which multiplies in blood and damages spleen, lymph nodes and brain.

23

24

Trypanosoma cruzi • Causes Chagas disease • Reduviid bug (kissing bug) is the vector. • Infection occurs when bug feces is inoculated into a cutaneous portal. • Local lesion, fever, and swelling of lymph nodes, spleen, and liver • Heart muscle and large intestine harbor masses of amastigotes. • Chronic inflammation occurs in the organs (especially heart and brain). • Treatment nifurtimox and benzonidazole 25

26

Leishmania species and Leishmaniasis • Leishmaniasis ‐ zoonosis transmitted among mammalian hosts by female sand flies that require a blood meal to produce eggs • Endemic to equatorial regions • Promastigotes are injected with sand fly bite, convert to amastigote and multiply; if macrophage is fixed the infection is localized; systemic if macrophage migrates.

27

28

• Cutaneous‐oriental sore, Baghdad boil ‐ localized ulcerated sore • Espunda – skin and mucous membrane infection of the head; chronic infection • Systemic‐visceral ‐ high intermittent fever; weight loss, enlarged spleen, liver, and lymph nodes – Kala azar is the most severe and fatal form if untreated.

29

30

216

Apicomplexan parasites

Plasmodium: The Agent of Malaria

• Sporozoans • Lack locomotor organelles in the trophozoite state • Alternate between sexual and asexual phases and between different animal hosts • Most form specialized infective bodies that are transmitted by arthropod vectors, food, water, or other means.

• Dominant protozoan disease • Obligate intracellular sporozoan • 4 species: P. malariae, P. vivax, P. falciparum and P. ovale • Female Anopheles mosquito is the primary vector; blood transfusions, mother to fetus • 300‐500 million new cases each year • 2 million deaths each year

– Plasmodium – Toxoplasma – Cryptosporidium 31

32

2 distinct phases of malarial parasite development: • Sexual phase – mosquito host

• asexual phase – human host

– Mosquito draws infected RBCs; gametes fertilize forming diploid cell which forms sporozoites in stomach. – Sporozoites lodge in salivary glands; available to infect human host

– Infected female mosquito injects asexual sporozoite which localizes in liver; it then undergoes schizogony generating 2,000‐40,000 merozoites which enter circulation in 5‐16 days depending on species. – Merozoites attach to and enter red blood cells, convert to trophozoites and multiply; red cell bursts releasing merozoites that differentiate into gametes.

33

34

Plasmodium • Symptoms include episodes of chills‐fever‐sweating, anemia, and organ enlargement. • Symptoms occur at 48‐72 hour intervals as RBCs rupture; interval depends on species. • P. falciparum most malignant type; highest death rate in children • Diagnosis by presence of trophozoite in RBCs, symptoms • Increasing drug resistance • Therapy is chloroquine, quinine, or primaquine. 35

36

217

Coccidian Parasites

Toxoplasma gondii and Toxoplasmosis • Intracelllular apicomplexan parasite with extensive distribution • Lives naturally in cats that harbor oocysts in the GI tract • Acquired by ingesting raw meats or substances contaminated by cat feces • Most cases of toxoplasmosis go unnoticed except in fetus and AIDS patients who can suffer brain and heart damage. • Treatment: pyrimethamine and sulfadiazine

• Zoonotic in domestic animals and birds

37

38

Sarcocystis and Sarcocystosis • Sarcocystis – parasites of cattle, swine, and sheep • Domestic animals are intermediate hosts; they pick up infective cysts while grazing on grass contaminated with human feces. • Humans are infected when the meat is consumed. • Symptoms include diarrhea, nausea, and abdominal pain. • No specific treatment

39

Cryptosporidium: A Newly Recognized Intestinal Pathogen • • • • • • • •

40

Isospora belli and Coccidiosis

An intestinal pathogen Infects a variety of mammals, birds, and reptiles Exists in tissue and oocyst phases 1990s – 370,000 cases in Milwaukee, WI due to contaminated water; filtration required for removal Ingestion of oocysts which give rise to sporozoites that penetrate intestinal cells Causes gastroenteritis, headache, sweating, vomiting, abdominal cramps, diarrhea AIDS patients may suffer chronic persistent diarrhea. No effective drugs

Intracellular intestinal parasite with oocyst stage Transmitted in fecally contaminated food or drink Infection usually asymptomatic or self‐limited Symptoms include malaise, nausea and vomiting, diarrhea, fatty stools, abdominal cramping, and weight loss. • Treat with sulfadiazine and pyrimethamine, when required

• • • •

41

42

Cyclospora cayetanensis and Cyclosporiasis

Babesia species and Babesiosis

• Emerging protozoan pathogen; causes cyclosporiasis • Oral‐fecal transmission; fresh produce and water • Oocysts enter small intestine and invade the mucosa. • Symptoms of watery diarrhea, stomach cramps, bloating, fever, muscles aches • Diagnosis can be complicated. • Treatment: trimethoprim and sulfamethoxazole

• First protozoan found to cause a disease – redwater fever of cattle • First protozoan found to be associated with a vector ‐ tick • Human babesiosis ‐ relatively rare zoonosis • Associated with infected rodents • Infection resembles malaria.

43

44

219


Parasitology • The study of eucaryotic parasites, protozoa and helminths • Cause 20% of all infectious diseases • Less prevalent in industrialized countries; increasingly common in AIDS patients


The Parasites of Medical Importance “Helminth”

2

A Survey of Helminth Parasites • Adults are large, multicellular animals with specialized tissues and organs. • Adult worms mate and produce fertilized eggs that hatch; larvae then mature in several stages to adults. • The sexes may separate or are hermaphroditic. • Adults live in the definitive host. • Eggs and larvae may develop in the same host, the external environment of the intermediate host. • A transport host experiences no parasitic development. • Four basic patterns of life and transmission 3

4

5

6

Helminths • Pathology arises from worms feeding on and migrating through tissues, accumulation of worms and worm products. • Diagnosis based on blood cell count (eosinophilia), serological tests; eggs, larvae, or adult worms in feces; sputum, urine, blood, or tissue biopsies. • Antihelminthic drugs suppress a helminthic metabolic process that differs from the human process, inhibit the worm’s movement, prevent it from holding position, and act locally in the intestine.

220

Nematode (Roundworm) Infestations

Ascaris lumbricoides

• Most abundant animal groups; 50 species that affect humans • Enlongated, cylindrical worms with protective cuticles, circular muscles, a complete digestive tract, and separate sexes • Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularis, hookworms, Strongyloides stercoralis, Trichinella spiralis, filarial worms

• • • •

A large intestinal roundworm Most cases in the U.S. occur in the southeastern states Indigenous to humans Ascaris spends its larval and adult stages in humans; release embryonic eggs in feces, and are spread to other humans; food, drink, or contaminated objects • Ingested eggs hatch into larvae and burrow through the intestine into circulation and travel to the lungs and pharynx and are swallowed. • Adult worms complete cycle in intestines and reproduce – 200,000 eggs/day. 7

8

Ascaris lumbricoides

Trichuris trichiura and Whipworm Infection

• Worms retain motility, do not attach. • Severe inflammatory reactions mark the migratory route. • Allergic reactions can occur. • Heavy worm loads can retard physical and mental development.

• • • •

Whipworm Humans sole host Trichuriasis has its highest incidence in the tropics. Eggs hatch in intestines, larvae attach, penetrate the outer wall and develop into adults. • Females lay 3,000‐5,000 eggs daily. • Worms can pierce capillaries, cause localized hemorrhage, and allow bacteria to leave intestine. • Heavy infestations can cause dysentery, rectal prolapse – can be fatal in children. 9

10

Enterobius vermicularis and Pinworm Infection

Hookworms

• Pinworm or seatworm • Enterobiasis most common worm disease of children in temperate zones • Eggs are picked up from surroundings and swallowed. • After hatching in the small intestine, they develop into adults. • Anal itching occurs when mature females emerge from intestine to release eggs. • Self‐inoculation is common. • Tape test – used to pick up eggs in anal area

• Characteristic curved ends and hooked mouths • Necator americanus and Ancylostoma duodenale • Humans shed eggs in feces, which hatch into filariform larvae and burrow into the skin of the lower legs. • Larvae travel from blood to lungs, proceed up bronchi and throat and are swallowed. • Worms mature and reproduce in small intestine and complete, the cycle. • May cause pneumonia, nausea, vomiting, cramps and bloody diarrhea • Blood loss is significant – anemia. 11

12

Strongyloides stercoralis and Strongyloidiasis • Threadworm • Tiny roundworms completes life cycle in humans or moist soil. • Larvae penetrate skin and migrate to lungs, are swallowed and complete development in the intestine. • Can reinfect the same host without leaving the body • Heavy worm loads can cause pneumonitis and eosinophilia, bloody diarrhea, liver enlargement, bowel obstruction and malabsorption. 13

14

15

16

Trichinella spiralis and Trichinosis • Life cycle entirely within mammalian host • Acquired from eating undercooked pork or bear meat • Larvae migrate from intestine to blood vessels, muscle, heart, and brain, where it forms cysts • First symptoms –flulike, diarrhea • Second symptoms – muscle and joint pain, shortness of breath, pronounced eosinophilia • No cure after larva have encysted

Wucherereia bancrofti and Bancroftian Filariasis

Tissue Nematodes • Complete their life cycle in human blood, lymphatics, or skin • Filarial worms; elongate, filamentous bodies, spread by biting arthropods • Cause chronic, deforming disease • Wuchereria bancrofti – elephantiasis • Onchocerca volvulus – river blindness • Loa loa – eye worm

• Tropical infection spread by mosquitoes • Vector deposits larvae which move into lymphatics and develop into adults. • Chronic infection causes blockage of lymphatic circulation and elephantiasis, massive swelling in the extremities.

17

18

222

Onchocerca volvulus and River Blindness

Loa loa: The African Eye Worm

• Transmitted by biting black flies • Larvae develop into adults in subcutaneous tissues. • Adult females migrate via the blood to the eyes, provoking inflammatory reactions. Affects optic nerve. • Co‐infection with Wolbachia bacteria causes river blindness. • Treatment: tetracycline and ivermectin

• Spread by bite of small flies • Temperature‐sensitive worm migrates around/under the skin and may enter the eye. • Treatment – pull worm from a small hole in conjunctiva or diethylcarbamazine

19

20

Trematodes or Flukes

Blood Flukes: Schistosomes

• Flatworms with ovoid leaflike bodies • Have digestive, excretory, neuromuscular, and reproductive systems • Lack circulatory and respiratory systems • Animals such as snails or fish are usually the intermediate hosts and humans are the definitive hosts.

• Schistosomiasis ‐ prominent parasitic disease • Schistosoma mansoni, S. japonicum, S. haematobium • Adult flukes live in humans who release eggs into water; early larva (miracidium) develops in freshwater snail into a 2nd larva (cercaria). • This larva penetrates human skin and moves into the liver to mature; adults migrate to intestine or bladder and shed eggs, giving rise to chronic organ enlargement. 21

22

• Zoonotic Liver flukes: • Opisthorchis (Chlonorchis) sinesis ‐ cycles between mammals and snails and fish; humans are infected by eating inadequately cooked fish containing cercaria, larvae crawl into bile duct, mature and shed eggs into feces; snail are infected. • Fasciola hepatica‐cycles between herbivores, snails, and aquatic plants; humans are infected by eating raw aquatic plants; fluke lodges in liver.

Lung fluke: • Paragonimus westermani – cycles between carnivorous animals, snails, and crustaceans; humans infected by eating undercooked crustaceans; intestinal worms migrate to lungs.

23

24

223

Cestode (Tapeworm) Infestations

Taenia saginata

• Flatworms • Long, very thin, ribbonlike bodies composed of sacs (proglottids) and a scolex that grips the intestine • Each proglottid is an independent unit adapted to absorbing food and making and releasing eggs. • Taenia saginata • Taenia solium

• • • •

Beef tapeworm Very large, up to 2,000 proglottids Humans are the definitive host. Animals are infected by grazing on land contaminated with human feces. • Infection occurs from eating raw beef in which the larval form has encysted. • In humans, larva attaches to the small intestine and becomes an adult. • Causes few symptoms; vague abdominal pain and nausea; proglottids in stool 25

26

Taenia solium • Pork tapeworm • Infects humans through ingesting cysts or eggs • Eggs hatch in intestine, releasing tapeworm larva that migrate to all tissues and encyst. • Most damaging if they lodge in heart muscle, eye, or brain • May cause seizures, psychiatric disturbances

27

28

The Arthropod Vectors of Infectious Disease Insects • Mosquitoes – require an aquatic habitat; females take blood meal transmitting disease: malaria, filariasis, zoonoses • Fleas – highly motile, flattened bodies; feed on warm‐blooded animals; carry zoonotic diseases: plague, murine typhus • Lice – small, soft; attach to head and body hair feeding inconspicuously on blood and tissue fluid; release feces that contaminate wound; epidemic typhus, relapsing fever • Flies – tsetse fly, sand fly

• Arthropods – exoskeleton and jointed legs; includes arachnids and crustaceans; many must feed on blood and tissue fluid of host during life cycle; ectoparasites • Those of medical importance transmit infectious microbes in the process of feeding – biological vectors

29

30

224

Arachnids • Ticks – cling on vegetation and attach to host on contact; larvae, nymph and adults get blood meal by piercing skin of host – hard ticks – Dermacentor, Ixodes – small compact, rigid bodies; transmit rickettsial, borrelial, and viral diseases – soft or argasid ticks – Ornithodoros‐ flexible outer bodies; transmit relapsing fever

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225


Clinical Chemistry

226


Introduction • The function of the Clinical Biochemistry Lab is to perform quantitative and qualitative tests on variable body fluids as well as on feces, tissues and calculi to diagnose certain diseases, monitor their progress or their response to treatment and to screen for a disease in seemingly healthy individuals. • This requires certain technologies for applications of suitable procedures of sound analytical methods.


Technology in Clinical Chemistry (1) 2

Photometry

Spectrophotometry

• Many determinations in the clinical chemistry are based on measurement of radiant energy emitted, transmitted, absorbed or reflected under controlled conditions. The measurement of the luminous intensity of light or the amount of luminous light is called photometry. • The instruments depending on this photometric principle include:

• Spectrophotometers are instruments used to measure the intensity of light from the UV range through the visible light. • Most spectrophotometric measurements in clinical chemistry are made in the visible region (400 – 700 nm) where the normally visible colors occur. • A substance (to be measured by colorimetric method) must either have a characteristic color or participate in a reaction which produces such a color. The amount of color produced should be proportional to the amount of substance being measured.

– Spectrophotometers, flame emission spectrophotometers and atomic absorption spectrophotometers. – Fluorescence, phosphorescence & chemiluminescence techniques. – Fluorometry, turbidimetry and nephelometry depending on the principles of fluorescence and light scattering measurements. 3

4

Basic Components of Spectrophotometer

Types of Spectrophotometers 1. Single‐beam spectrophotometers: o

In this instrument, the light from the lamp travels along only one pathway to the cell compartment (sample holder) and all samples (blank, standard & tests) are read in the same position.

2. Double‐beam spectrophotometers: o

o

These instruments operate like single‐beam spectrophotometers except they are designed to compensate for possible variations in intensity of the light source. In this type, the light from the monochromator is split into two beams; one beam is used as reference (directed through the reference cuvet), the other for reading (transmitted directly through the sample cuvet). So, any change in light intensity affects both cuvettes simultaneously and thus is canceled out.

6

5

227

Double‐beam Spectrophotometers

Double‐beam Spectrophotometers

1. Double‐beam‐in‐space spectrophotometer:

2. Double‐beam‐in‐time spectrophotometer:

7

8

Atomic Emission Spectrophotometry (Flame Photometry)

Flame Photometer

• In the ground state of an atom, the electrons occupy the lowest energy level. • In order to move from its energy level in the ground state to an exited state, the energy of an electron must be raised by the absorption of an amount of energy exactly equivalent to that involved in the transition. • The electrons in the higher energy orbits are unstable and tend to return to lower energy orbits (ground state). • In doing so, the energy previously absorbed is released as quanta of light, the wave lengths of which are characteristic of the substance, thus giving rise to the emission spectrum, e.g. sodium is identified by yellow color, potassium produces a violet color while lithium imparts a red color to a flame.

9

10

Atomic Absorption Spectrophotometry

Atomic Absorption Spectrophotometer

• Atomic Absorption Spectrophotometry (AAS) is the measurement of the absorption of light by free metallic atoms. • It uses the heat of a flame to dissociate molecules to free atoms, primarily at their lowest energy level (ground state). • AAS measures the absorption of light of a unique wave length by atoms in the ground state. The unique wave length absorbed corresponds to the particular line spectrum for that element. • With the proper light source, a particular cation can be analyzed in a mixture of many cations. 11

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228

Fluorometry

Fluorometer

• Fluorometry is the technique for the measurement of fluorescence emitted from certain substances. • It is used in the clinical chemistry laboratory for certain classes of compounds, particularly when great sensitivity is required e.g. drugs, hormones, vitamins, amino acids and porphyrins. • In fluorescence, the exciting radiation is electromagnetic usually within UV or visible range. • The absorbed energy causes molecules of the substance involved to pass into an excited state. • After a part of the energy has been lost, the molecule returns to its ground state by re‐emission of a quantum of energy smaller than that absorbed, thus the emitted radiation is of a lower frequency and hence larger wave length. • In fluorescence, the excited state persists for less than 6‐10 seconds. 13

14

Chemiluminescence

Simple Luminometer

• Luminescence is the phenomenon of emission of light by an electronically excited molecule on decay to ground state. • In chemiluminescence, the energy responsible for excitation arises as a result of a chemical reaction. • In chemiluminescence, the molecule emitting the light is very unstable chemically and thus it only exists transiently during the reaction. • The emitted photons of an excited molecule can be quantified accurately by using luminometer. • Chemiluminescence provides a sensitive and rapid method for measuring a wide range of substances of biological and clinical interest e.g. certain steroids, hormones, drugs and immunoglobulins. 15

16

Electrophoresis

Diagram of Electrophoresis Chamber

• Electrophoresis is the migration of charged particles in a liquid medium under the influence of an electric field. • A charged particle placed in an electrical field migrates towards the anode (+) or cathode (‐), depending on the net charge carried by the particle. The rate of migration in a porous medium varies with its net charge and the strength of the electrical field. • The electrophoresis apparatus is designed so that the circuit between the two poles is bridged by the support medium holding the sample, and the current flow is partially carried by the components of the sample. 17

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229

Factors Affecting Electrophoresis

Types of Electrophoresis

1. Sample:

1. 2. 3. 4. 5.

–

Charge ‐ Size – Shape

2. Electric field: –

Current – Voltage – Resistance ‐ Heat

3. Buffer: –

Moving boundary electrophoresis. Zone electrophoresis. Iso‐electric focusing. High voltage electrophoresis. Two‐dimensional electrophoresis.

Composition – Concentration – pH

4. Support media: –

Adsorption ‐ Electro‐osmosis ‐ Molecular sieving

19

20

Blotting Techniques • Southern Blotting: – This technique is widely used in molecular biology for identifying a particular DNA sequence; determining the presence, position, and number of copies of a gene in a genome; and typing DNA.

• Northern Blotting: – This technique is used to separate and detect RNAs and RNA fragments instead of DNAs or DNA fragments.

• Western Blotting: – It is a method used to separate, detect, and identify one or more proteins in a complex mixture. 21

230


Electrochemistry Several types of analytical methods are based on electrochemical phenomenon.


• Applications of electrochemistry:


1. Determination of pH of solutions by pH meter. 2. Determination of electrolytes by ion selective electrode (ISE). 3. Determination of arterial blood gases by blood gases analyzer.

Technology in Clinical Chemistry (2) 2

pH Meter

Ion‐Selective Electrodes (ISE)

• pH meter is the electrometric method which is now commonly used for the determination of the pH of solutions. • This method depends on using a glass electrode to measure the pH of unknown solution against the calomel reference electrode.

• A range of ion‐selective electrodes have been developed and use more specific media instead of glass. In all cases the basic design of the electrode is the same. • All ISE are similar in their principles of operation. The difference arises in the mechanism by which a particular electrode achieves its selectivity for the desired ion. • According to the physical state of the membrane, ISE can be classified into: 1.

Solid state electrode (solid membrane): •

2.

e.g. Sodium electrode

Liquid membrane electrode: •

e.g. potassium electrode

3

4

Gas Sensors

Enzyme Electrodes

Except for the oxygen electrode (which is an amperometric), the other gas‐sensing electrodes used in clinical chemistry are potentiometric sensors separated from the fluid being analyzed by a thin, gas permeable membrane. 1. pCO2 Electrode:

• These electrodes are biosensors which consist of immobilized material in contact with a transducer which will turn the biochemical signal into an electrical signal. • The substance (substrate for the enzyme) to be measured diffuses to the enzyme where a product is formed and then the electrode is affected. • Enzyme electrodes normally have to be kept refrigerated to keep the life time as long as possible; even so, few have a life time of over a month.

•

This is essentially a pH electrode system containing a calomel and glass electrode.

2. pO2 Electrode:

– Glucose oxidase electrode: • It is used for detecting glucose. – Urease electrode: • It is a glass electrode that is sensitive to NH4+ and is covered with urease polymerized in a polyacrylamide matrix. • It has been used for determination of urea concentration.

• The oxygen electrode is not strictly an ISE. The electrode system is based on the electrochemical reduction of oxygen (amperometry). • The pO2 electrode is used to measure the oxygen pressure of blood and other body fluids.

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231

Chromatography

Chromatography

• Chromatography is an analytical procedure by which a multi‐component mixture is separated into its constituents with a differential migration phenomenon. • It is a versatile and powerful tool for the separation and quantification of many clinically relevant analytes. • The primary goal of the chromatographic process is to separate a mixture into its individual components, which are called solutes or analytes. • A chromatographic separation requires a sample to be introduced into a flowing stream of gas or liquid (mobile phase) that passes through a bed, layer, or column containing the stationary phase.

• The stationary phase may be particles of a solid or gel, or a liquid. If it is liquid, it may be distributed on solid particles. The liquid may be chemically bonded to the particles (bonded phase), or immobilized onto them (immobilized phase). • As the mobile phase carries the sample past the stationary phase, the solutes with lesser affinity for the stationary phase remain in the mobile phase and travel faster and separate from those that have a greater affinity for it. • Chromatographic separations can be used for the quantitative screening, preparation and identification, and quantitative analysis of a wide variety of chemical compounds. These include proteins, peptides, amino acids, carbohydrates, lipids, hormones and nucleic acids. Its greatest application is in the measurements of therapeutic drugs and drugs of abuse.

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8

Chromatographic Terms


Column: • A cylindrical tube for holding either the adsorbent or ion exchange resin.

• Loading: – The amount of substance applied to the paper or support.

• Development:

Stationary phase: • Usually a solid or liquid adsorbent, e.g. paper or water.

– The process of allowing the solvent to move along the column or paper.

Mobile phase: • A solvent or a gas used to separate the components.

• Multiple development:

Tanks: • Airtight containers in which development takes place.

• Polarity:

– When the development is re‐run, a number of times in a solvent system to improve resolution. – A polar compound is one that is held by the stationary phase whereas a non‐polar compound tends to move forwards in the mobile phase.

Origin: • The point of application of substance to chromatogram. 9

10


Separation Mechanisms in Chromatography

• Solvent front:

1.

Surface adsorption: o

– The level at which the elution fluid (eluent) has reached.

• Relative fraction (RF):

2.

– A ratio of the distance the solute has traveled from the point of origin to the distance traveled by the solvent front.

Partition: o

3.

• Elution: • Resolution:

4.

• Location:

5.

– The detection of the components after development, either by using a special or general reagent, or ultraviolet light.

The solutes are separated on the basis of the differences in their molecular size.

Affinity: o

11

The principle feature underlying this form of chromatography is the attraction between oppositely charged particles.

Molecular phase and size: o

– The degree of separation of the components after development.

The separation is achieved as a consequence of the relative solubility of a substance in the two phases (stationary and mobile).

Ion exchange: o

– The use of solvent to separate components.

This is competition between the mobile phase (liquid or gas) and the solutes for adsorption sites on the support (the stationary phase which is a solid).

This affinity may be due to antigen‐antibody interaction or enzyme‐ substrate interaction.

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232

Types of Chromatography

Types of Chromatography

1.

4. Affinity chromatography:

Column chromatography: –

2.

–

Thin layer chromatography (T.L.C.): – – –

3.

It is one of adsorption chromatography in which the separation of mixtures occurs on a column of suitable adsorbent packed in a glass tube and supported either by a plug of glass or cotton wool. It is primarily adsorption chromatography in which a uniform thin layer of adsorbent on a supporting glass plate is used. High‐performance thin‐layer chromatography (HPTLC) has been applied to a system of TLC. TLC is applied in the investigations of sugars, amino acids, lipids and drugs.

5. Ion‐exchange chromatography: –

Paper chromatography: – –

Affinity chromatography is a separation mechanism involving an interaction between biochemical species, e.g. enzyme‐substrate, hormone‐receptor and antigen‐ antibody.

It is a liquid‐liquid partition chromatography where the liquid stationary phase is supported on the cellulose paper sheet. Some compounds are better resolved by using a stationary organic phase (non‐polar), and a mobile aqueous phase (polar); this is called the reversed phase chromatography.

In ion‐exchange chromatography, solutes in a mixture are separated by virtue of their difference in sign and magnitude of ionic charge.

6. Gas chromatography: –

Gas chromatography is a process by which a mixture of compounds in volatilized form is separated into its constituent parts by moving a mobile phase, which is an inert, over a stationary phase.

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14

Gas‐Liquid Chromatography

High Performance Liquid Chromatography (HPLC)

1. The stationary phase is a liquid. 2. The separation is mainly by partition between the carrier gas and the liquid phase supported on the inert material (stationary phase).

• HPLC is the most recently introduced form of chromatographic separations. • HPLC has great potential in the analysis of non‐volatile compounds of biological interest such as steroid, nucleotides and nucleosides, drugs and their metabolites, amino acids and peptides, aromatic amines, lipids, etc.

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16

Immunochemical Techniques

Nephelometry

Radial Immunodiffusion • It is a passive diffusion method, in which a concentration gradient is established for a single reactant, usually the antigen. The antibody is uniformly dispersed in the gel matrix.

• It is the detection of light energy scattered or reflected toward a detector that is not in the direct path of the transmitted light. Common nephelometers measures scattered light at right angle to the incident light. • Light scattering measurements are best applied to immuno‐assays of immunoglobulins, specific proteins and haptens such as therapeutic drugs. • Two types of nephelometry are encountered:

Electroimmunoassay • In this method, a single concentration gradient is established for the antigen when an applied voltage is used to drive the antigen from the application well into a homogenous suspension of antibody in the gel. • This produces a unidirectional migration of antigen and results in a lower limit of detection.

1.

2.

17

Static nephelometry: • The nephelometer detects light scatter by utilizing an electronic system designed to reduce interference from light scatter produced by large contaminating particles such as dust. Rate nephelometry: • This is a kinetic method in which the nephelometer has been coupled with a micro‐processing unit for the rapid determination of specific proteins by means of immunoprecipitin reactions. 18

233

Labeled Immunochemical Assays Type

Turbidimetry •It is the Measurement of the decrease in the intensity of the incident beam of light as it passes through a solution of particles due to scattering, reflectance and absorption. •Turbidimetry is carried out at 180° from the incident beam. Labeled Immunochemical Assays •These are sensitive and specific methods for accurate quantitation of biologically important compounds (such as peptides, hormones, vitamins and drugs) which may occur in biological fluids or tissues in low concentrations (in µg/ml or pg/ml). •The two main types are encountered: 1. 2.

Naked eye Turbidity Nephelometry Naked eye, Spectrophoto‐ metry, Particle counting

Not required

Not required

Particle immunoassay

Blood cells, artificial particles (gelatin, latex)

Not required

Radioisotopes (125I, 3H)

Required

Photon counting

~ 5 pg/ml

Enzymes

Required

Spectrophoto‐ metry, Fluoro‐ metry, Photon counting

~ 0.1 pg/ml

Fluorophores

Required

Photon counting

~ 5 pg/ml

Chemiluminascent compounds such as luminal derivatives, acridinium esters

Required

Photon counting

~ 5 pg/ml

Enzyme immunoassay Fluorescent immunoassay Chemiluminascent immunoassay 19

B/F separation Signal detection Sensitivity

Precipitation immunoassay

Radioimmunoassay

Competitive immunoassay (limited reagent assays). Non‐competitive immunoassay (excess reagent, two‐site, sandwich assays).

Labels (Reporter groups)

~ 10 µg/ml

~ 5 ng/ml

Enzyme Immunoassay (EIA)

ELISA – Competitive Method

• Enzyme immunoassay uses enzymes as labels and is widely used nowadays. • Enzymes can amplify signals depending on the turnover of enzyme catalytic activity. • Enzyme Linked Immuno‐Sorbent Assay (ELISA) is one of EIA techniques. • The principle in ELISA involves heterogeneous methodology in which bound and free materials must be separated using a series of washing steps. • Three major methods of ELISA are commonly used.

• Its principle involves competition of patient antigen and labeled antigen for antibody binding sites, where an antibody to the analyte is covalently bound to the tube or well. • There is an inverse relation between the concentration of patient antigen and the enzyme activity.

1. 2. 3.

20

Competitive method. Indirect method. Double‐antibody sandwich method. 21

22

ELISA – Indirect Method

ELISA – Double‐antibody Sandwich Method

• Antigen to the analyte immunoglobulin is covalently bound to the inside of the tube or well. • The enzyme activity is directly proportional to the concentration of the analyte.

• Antibody to the analyte antigen is covalently bound to the wall of reaction vessel (well). • Activity is directly proportional to the analyte concentration.

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234

Automation

Terms in Automation

• Automation is the replacement of human manipulative effort and facilities in the performance of a given process by mechanical and instrumental devices that are regulated by feedback of information so that an apparatus is self‐monitoring or self‐adjusting. • This term has been applied in the field of clinical chemistry to describe the process by which an analytical instrument performs many tests with only minimal involvement of an analyst. • One of the benefits of automation is a reduction in the variability of results and errors of analysis by eliminating tasks that are repetitive and monotonous for a human that can lead to boredom or inattention.

• Analyzer configuration: The format in which analytical instruments are configured. Automated instruments are configured by the manufacturer either as open or closed systems. • Batch analysis: A type of analysis in which many specimens are processed in the same analytical run. • Centralized testing: A mode of testing where specimens are transported to a central or core facility for analysis. • Continuous‐flow analysis: A type of analysis in which each specimen in a patch passes through the same continuous stream and is subjected to the same analytical reactions as every other specimen and at the same rate. 25

26

Terms in Automation

Terms in Automation

• Discrete analysis: A type of analysis in which each specimen in a patch has its own physical and chemical space separate from every other specimen.

• Parallel analysis: A type of analysis in which all specimens are subjected to a series of analytical processes at the same time in a parallel fashion.

• Discretionary multiple‐channel analysis: A type of analysis in which specimens in sequence can be analyzed by any one or more of the available processes.

• Point of care testing: A mode of testing where the analysis is performed at the site where health care is provided (e.g. bedside testing).

• Multiple‐channel analysis: A type of analysis in which each specimen is subjected to multiple analytical processes so that a set of test results is obtained on a single specimen.

• Random‐access analysis: A type of analysis in which any specimen can be analyzed by any available process in or out of sequence with other specimens and without regard to their initial order. 27

28

Terms in Automation • Sequential analysis: A type of analysis, in which any specimen in a batch enters the analytical process one after another, and each result or set of results emerges in the same order as the specimens are entered. • Single‐channel analysis: A type of analysis in which each specimen is subjected to a single process so that only results for a single analyte are produced. • Specimen throughput rate: The rate at which an analytical system processes specimens. 29

235


Units of Measurement • •


• • •


•

•

A meaningful measurement is expressed with both a number and a unit. All quantitative measurements must be expressed in clearly defined units that are accepted and understood by all scientists. The system International Units (SI Units) have replaced the old system of reporting and measurements that can be safely understood anywhere nowadays. This system has functioned as the international authority for measurements. The International Unit (U) is used to express enzyme activity in U/L. An International Unit of enzyme activity is the amount of enzyme which under defined assay conditions will catalyze the conversion of 1 µmol of substrate per minute. . The following formula is used for the interconversion between the conventional unit (mg/dl) and the SI unit (mmol/l):

When the molecular weight of a substance cannot be accurately determined (e.g. albumin), the results are expressed in gm/l.

Units and Calculations in Clinical Chemistry 2

Interconversion between SI and Conventional Units

Interconversion between SI and Conventional Units

Conventional Unit

Chemical substance

SI Unit

Glucose

mmol/l

X

18

0.0555 X

mg/dl

Chemical substance Ammonia

µmol/l

X

1.7

0.5872 X

Urea

mmol/l

X

6

0.16666 X

mg/dl

Iron

µmol/l

X

5.6

0.1791 X

µg/dl

Creatinine

µmol/l

X 0.0113

88.49

mg/dl

Bilirubin

µmol/l

X 0.0588

17.1

X

mg/dl

Uric acid

µmol/l

X 0.0169

59.172 X

mg/dl

Total protein

gm/l

X

0.1

10

X

g/dl

Sodium

mmol/l

X

1

1

X

meq/dl

Albumin

gm/l

X

0.1

10

X

g/dl

Potassium

mmol/l

X

1

1

X

meq/dl

Immunoglobulins

gm/l

X

100

0.01

X

mg/dl

0.250

X

100

0.01

Interconversion Factor

X

SI Unit

Interconversion Factor

Conventional Unit µg/dl

Calcium

mmol/l

X

4

X

mg/dl

Total lipids

gm/l

X

mg/dl

Inorganic phosphorus

mmol/l

X

3.1

0.3229 X

mg/dl

Cholesterol

mmol/l

X 38.65

0.02586 X

mg/dl

Magnesium

mmol/l

X 2.432

0.4114 X

mg/dl

Triglycerides

mmol/l

X

0.01130 X

mg/dl

88.5

3

4

Calculations in Clinical Chemistry

Concentrations based on Volume • To calculate M, we need to know the weight of dissolved solute in gm (wtg) and its molecular weight (MW).

Concentrations based on Volume • Concentrations based on the amount of dissolved solute per unit volume are the most widely used in biochemistry laboratories. • Molar solutions are the solutions which contain one gram molecule of the substance per liter. • One gram molecule (g mole) is the molecular weight of the substance expressed in grams e.g.:

w tg MW

= m o le s

• Mole = 103 millimole = 106 micromole = 109 nanomole. • A one M solution contains Avogadro's number of molecules per liter. • Avogadro's number = number of molecules per g‐mole. = number of molecules per g‐mole. = number of atoms per g‐atom. = number of ions per g‐ion. = 6.023 X 1023

– Molar solution of NaOH = 23 + 16 + 1 = 40 – Molar solution of H2SO4 = 1X2 + 32 + 16X4 = 98 – Molar solution of H3PO4 = 1X3 + 31 + 16X4 = 98 Molarity (M) = The number of molecules per liter of solution. 5

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236

Concentrations based on Volume Concentrations based on Volume • Normal solutions are solutions which contain one gram equivalent of the substance per liter.

Normal solution of H 2 SO 4

Normality (N) = the number of equivalents of solute per liter of solution

Normal solution of H3PO4 =

• To calculate N, we need to know the weight of dissolved solute in gm (wtg) and its equivalent weight (EW). wtg EW

40 1 98 = 2

Normal solution of NaOH =

= 40 = 49

98 = 32.7 3

• The molarity (M) and normality (N) are related by: N = n M

= equivalents

where n = number of active radicals (number of replaceable H+ or OH‐ per molecule, or the number of lost or gained electrons per molecule). e.g. a 0.01 M solution of H2SO4 is 0.02N. • Weight/volume percent is often used for routine laboratory solutions.

• One gram equivalent is the equivalent weight of the substance expressed in grams.

W eig ht /vo lu m e perc en t (% w /v) = the w eig h t in g o f a s olute pe r 10 0 m l of so lu tio n

7

Concentrations based on Volume Milligram percent is often used in clinical laboratories.

8

Concentrations based on Volume The concentration of many commercial acids are given in terms of % w/w.

M illigran percent (mg %) = the weight in m g of a solute per 10 0 m l of solution

Weight/weight percent (% w/w) = the weight in g of a solute per 100 g of solution

A one M solution of a nondissociable solute is one Osmolar, while a one M solution of a dissociable salt is n Osmolar where n is the number of ions produced per molecule.

In order to calculate the volume of stock solution required for a given preparation, we must know its density (p) or specific gravity (SG). wtg = vol ml X p g/ml X % (as decimal)

where: • wtg = weight of pure substance required in g. • volml = volume o stock solution needed in ml. • pg/ml = density of the substance. • % = fraction of total weight that is pure substance.

Thus, a 0.03 M solution of KCl is 0.06 Osmolar.

9

10

Concentrations based on Volume • Molal solutions are the solutions which contain one gram molecule of the substance per 1000 grams of solvent. Molality (m) = the number of moles of solute per 1000 g of solvent

• Molality is used in certain physicochemical calculations e.g. calculation of boiling‐point elevation and freezing‐point depression. 11

237


Blood Sugar • What is blood sugar?


– Glucose is the main blood sugar


Blood Glucose Tests 2

What are the Normal Values of Blood Glucose Concentration?

What are Sources of Blood Glucose 1. Dietary carbohydrates.

• The normal fasting (8 – 12 hours after meal) plasma glucose level is 70 – 100 mg/dl.

2. Liver glycogen (glycogenolysis). • The postprandial (2 hours after meal) plasma glucose level must return to the normal fasting level or ideally just below the fasting level.

3. Non‐carbohydrate source (gluconeogenesis).

• Random plasma glucose level is up to 140 mg/dl.

3

4

Alterations in Blood Glucose Levels


• Hypoglycaemia is the decrease of fasting plasma glucose level below 40 mg/dl.

• Fasting values between 100 & 126mg/dl or random values between 140 & 200 mg/dl are called impaired glucose tolerance

• Hyperglycaemia is the rise of fasting plasma glucose level above 126 mg/dl.

• Only a small proportion of subjects with such mild impaired glucose tolerance develop diabetes mellitus later. • It is not possible to predict the outcome of this condition at the time of subject presentation. 5

6

238


Regulation of Blood Glucose

• Subjects must not be diagnosed as having diabetes mellitus merely on the basis of impaired glucose tolerance, because of the serious psychological, social and economic implications.

• Normally, blood glucose level is maintained within relatively constant range despite the various disturbing factors.

• However, because of the increased risk of vascular complications, secondary causes of impaired glucose tolerance should be excluded and the subjects should be given dietary advice and, if overweight, should be advised to lose weight.

• This homeostasis is achieved by tissue and hormonal regulation:

7

8



Tissue Regulation 1. Gastrointestinal tract (GIT) 2. Liver 3. Muscle and adipose tissue 4. Kidney

Hormonal Regulation

Insulin

Anti‐Insulin Hormones

9

10

Anti Insulin Hormones

Diabetes Mellitus

1. Adrenaline and glucagon.

• Diabetes Mellitus is a metabolic syndrome characterized by hyper‐glycaemia, glucosuria, polyuria, polydipsia and polyphagia. • It is confirmed if one of the following is present:

2. Glucocorticoids and growth hormone.

1. A fasting plasma glucose level of more than 126 mg/dl on two occasions. 2. A random plasma glucose level of more than 200 mg/dl on two occasions. 3. Both a fasting plasma glucose level of more than 126 mg/dl and a random plasma glucose level of more than 200 mg/dl.

3. Thyroxine.

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239

Diabetes Mellitus

Diabetes Mellitus

Diabetes Mellitus is usually excluded if: • A fasting plasma glucose level is less than 100 mg/dl on two occasions. • Random plasma glucose levels are less reliable for excluding than for confirming the diagnosis.

Type-I Diabetes Mellitus 1. Insulin-dependent diabetes mellitus (IDDM). 2. Juvenile diabetes. 3. Less common (10 – 20%). 4. Usually before the age of 25 years. 5. Patients are usually thin. 6. Severe type. 7. Ketoacidosis is more common. 8. No detectible circulating insulin. 9. Insulin deficiency. 10. Treated by insulin only.

Causes: 1. Insulin deficiency. 2. Resistance to insulin action. 3. Decreased the ratio between insulin and anti‐insulin hormones.

Type-II Diabetes Mellitus 1. Non-insulin-dependent diabetes mellitus (NIDDM). 2. Maturity (Adult) onset diabetes. 3. More common (80 – 90%). 4. Usually after the age of 25 years. 5. Patients are usually obese. 6. Milder type. 7. Ketoacidosis is less common. 8. High plasma insulin level. 9. Insulin resistance. 10. Treated by oral hypoglycaemic drugs and may be by insulin.

13

14

Investigations of Suspected Diabetes Mellitus

Investigations of Suspected Diabetes Mellitus

Specimen collection and storage: • Plasma or whole blood • Venous blood or capillary blood • Serum or plasma • Double void technique • Storage of 24‐h urine

Parameters for blood glucose investigations: 1. Urine glucose 2. Random blood glucose 3. Fasting blood glucose 4. Two hours post‐prandial blood glucose 5. Glucose tolerance test (GTT ) 6. Glycosylated haemoglobin (HbA1C) 7. Plasma fructosamine 8. Plasma Insulin 9. Serum C‐peptide. 15

16

Oral Glucose Tolerance Test (GTT)

Investigations of Suspected Diabetes Mellitus • Indications • Procedure • Results 1. Normal blood sugar curve 2. Renal glucosuria curve 3. Diabetic blood sugar curve (Mild diabetes ‐ Moderate diabetes ‐Severe diabetes)

4. Lag curve

17

18

240

Intravenous Glucose Tolerance Test • This test may be performed to eliminate factors related to the rate of glucose absorption as in: 1. 2. 3.

Poor absorption of orally administered glucose. Inability to tolerate a large oral glucose load. Altered gastric physiology (e.g. after gastrectomy).

• Preparation of patients is the same as for the OGTT. • The dose of glucose is 0.5 g/kg of body weight with a maximum of 35 g given as a 25 g/dl solution. • The dose is administered intravenously over 3 min ± 15 s, and blood is collected every 10 min after the mid‐ injection time for one hour. 19

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Renal Functions A. Reabsorptive and Excretory Functions: • Retain the important required substances. • Rid the body of the metabolic waste end products.


B. Regulatory Function: • Maintenance of the optimal chemical composition of body fluids. • Acid‐base homeostasis. Kidney Biochemistry 2

Renal Functions

Collection and Storage of Urine Samples

C. Endocrine Function: • Hormones released by the kidney: 1. Rennin and prostaglandins. 2. Erythropoietin. 3. Calcitriol (1,25‐dihydroxy‐cholecalciferol). • Hormones acting on the kidney: 1. Aldosterone. 2. Vasopressin (antidiuretic hormone = ADH). 3. Parathyroid hormone (PTH). • Hormones altered and inactivated by the kidney: 1. Insulin. 2. Glucagon. 3. Aldosterone.

• Single specimens of urine (random or morning samples) are used for ward examinations and qualitative tests. • Double‐voided specimen is the urine excreted during a time period after complete emptying of the bladder by 15 ‐ 30 min. • 24 h‐urine collections are preferred for quantitative tests due to the diurnal variation in the excretion of some substances. • The 24 h‐collection is as follows:

D. Metabolic Function: • Gluconeogenesis in prolonged starvation.

– At a suitable time (e.g. 8 O’clock morning), the patient empties his bladder and the urine is discarded. – All urine passed during the following 24 hours is saved in specific container. – At the same time of the next morning, the patient empties his bladder and the urine is added to the collected one. 3

4

Collection and Storage of Urine Samples

Nephrolithiasis

• For storage of urine sample, the following are possible:

• Condition characterized by the presence of renal calculi (stones). • Due to nutritional, environmental or genetic factors. • Kidney stones may be:

– It is satisfactory in most cases to use specimens collected in cool, clean containers. Urine sample can be stored for about one week in the refrigerator at 2 – 8°C. – Urine samples can be stored for many months at ‐20°C without any addition. – Concentrated hydrochloric acid (HCl), thymol or chloroform can be used for urine storage. – Acid should not be used for proteins, creatinine and steroids determination.

– Single stones which may be composed of any of: calcium oxalate, uric acid, calcium carbonate, calcium phosphate or magnesium ammonium phosphate (triple phosphate). – Mixed stones which may be composed of two or more of the mentioned constituents. – Cystine or xanthine stones which are rare and found in the inherited metabolic abnormalities: cystinuria or xanthinuria respectively.

• Both qualitative and quantitative analyses of the chemical constituents of kidney stones may be useful in establishing the etiology and in planning adequate therapy. • Radiological examinations are required to explore the degree of intrarenal calcification and papillary damage.

5

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Kidney Function Tests


I Glomerular Function Tests 1.Complete Urine Analysis:

I Glomerular Function Tests 1. Complete Urine Analysis:

A. Physical Examination: • Volume: 750 – 2000 ml / day. • Colour: pale yellow amber yellow deep yellow. • Odour: Urineferous aromatic ammoniacal. • Aspect: Clear. • Deposit: Nil. • Reaction: Acidic. • Specific gravity: 1015 – 1025.

B. Chemical Examination: • Albumin: Nil. • Blood: Nil. • Glucose: Nil. • Acetone: Nil. • Urobilinogen: Trace. • Bilirubin: Nil. 7

8



I Glomerular Function Tests 1.Complete Urine Analysis: C. Microscopic Examination: • Red blood cells: 0 – 5 / HPF. • Pus cells: 0 – 5 / HPF. • Crystals: Nil. • Casts: Nil. • Ova and parasites: Nil. • Epithelial cells: Nil in males. (amount depends on specimen collection) Few squamous epithelial cells in females. (amount depends on specimen collection)

I Glomerular Function Tests 2.Blood Urea (20 – 40 mg/dl) • Urea is the end product of protein metabolism. • Urea is synthesized in the liver from ammonia and then passes to the kidney to be excreted. • Blood urea is increased in renal failure and urinary obstruction, but it is decreased in liver cell failure. • Sometimes blood urea is represented by blood urea nitrogen (BUN) which normally ranges from 8.0 to 16.0 mg / dl. (needs to be SI units) • Blood urea is neither sensitive nor specific for kidney function because it is affected by dietary proteins, chronic constipation and gastrointestinal bleeding. 9

10



I Glomerular Function Tests 3.Plasma creatinine: (0.6 – 1.2 mg/dl in males)? SI units (0.5 – 0.9 mg/dl in females)? SI units

I Glomerular Function Tests 4. Creatinine clearance : (90 – 130 ml/min in males) (80 – 120 ml/min in females) • The most convenient method of obtaining an acceptable accurate estimation of glomerular filtration rate (GFR).

• Creatinine (creatine anhydride) is the end product of creatine metabolism. • It is formed in the muscles from creatine phosphate and then passes to the kidney to be excreted. • Plasma creatinine is increased in renal failure and urinary obstruction, but it is decreased in chronic muscle dystrophy diseases • Plasma creatinine is preferred to urea estimation as an index of renal function because creatinine is produced endogenously and is not affected by exogenous factors.

• Lower values of creatinine clearance are indicative of diminished glomerular filtration rate. • Has particular value in the general assessment of renal function especially when plasma analysis is invalid e.g. after renal dialysis.

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12



II Tubular Function Tests 1. Plasma electrolytes and minerals: •

2.

•

Measurement of urine specific gravity: (1015 – 1025) •

3.

II Tubular Function Tests: 4. Urine concentration test (Water deprivation test):

Estimation of plasma electrolytes (sodium, potassium, chloride and bicarbonate) and minerals (calcium, inorganic phosphate and magnesium) are important in assessing the tubular function of the kidney.

•

Measurement of urine osmolality: (300 – 900 mOsmol/kgH2O) • •

This is a test for renal concentration ability.

5. Vasopressin test (Pitressin test):

This measurement is a useful guide to the adequacy of the renal concentrating mechanism. This measurement is considered more valid than specific gravity measurement in assessing concentrating ability of the kidney. Ratio of urine osmolality to serum osmolality (280 – 300 mOsmol/kgH2O) should be calculated.

More pleasant for the patient than water deprivation test, and depends only on renal tubular function.

6. Urine dilution test (Water load test): •

Very simple but less sensitive than water deprivation test.

13

14


Kidney Function Tests III. Special Function Tests: 1. Urinary Microalbumin: • To compensate for variations in urine concentration in spot‐check samples, the albumin/creatinine ratio (ACR) is calculated. Microalbuminuria is defined as ACR ≥2.8 mg/mmol (male) or ≥2.0 mg/mmol(female). • The significance of microalbuminuria test is:

III Special Function Tests: 1. Urinary Microalbumin: • • •

•

Normally, albumin is not present in urine because it is filtered from the bloodstream by the kidneys. Microalbuminuria occurs when there is an abnormally high permeability for albumin in the renal glomerulus. Microalbuminuria cannot be detected by urine dipstick methods but there is specific Microalbumin urine test to determine the presence of the albumin in urine. Microalbuminuria is diagnosed from elevated concentrations (30 to 300 mg/L) on at least two occasions. An albumin level above these values is called "macroalbuminuria", or just albuminuria.

1. 2. 3.

An indicator of subclinical cardiovascular disease. Marker of vascular endothelial dysfunction. An important prognostic marker for kidney disease • In diabetes mellitus. • In hypertension.

4.

Increasing microalbuminuria during the first 48 hours after admission to an intensive care unit predicts elevated risk for acute respiratory failure, multiple organ failure, and overall mortality.

15

16



III. Special Function Tests 2. Cystatin C:

III. Special Function Tests 2. Cystatin C: (0.56 to 0.98 mg/L in males) (0.52 to 0.90 mg/L in females)

• Cystatin C or Cystatin 3 (formerly gamma trace, post‐gamma‐globulin or neuroendocrine basic polypeptide) is mainly used as a biomarker of kidney function. • In humans, all cells with a nucleus produce Cystatin C as a chain of 120 amino acids. It is found in virtually all tissues and bodily fluids. It is a potent inhibitor of lysosomal proteinases and probably one of the most important extracellular inhibitors of cysteine proteases. • Cystatin 3 has a low molecular weight (~13.3 KD). Due to its small size it is freely filtered by the glomerulus, and is not secreted but is fully reabsorbed and broken down by the renal tubules. This means the primary determinate of blood Cystatin C levels is the rate at which it is filtered at the glomerulus making it an excellent GFR marker.

•

•

• •

17

Serum levels of cystatin C are a more precise test of kidney function than serum creatinine levels. Cystatin C levels are less dependent on age, sex, race and muscle mass compared to creatinine. Cystatin C is an alternative and more sensitive endogenous marker for the estimation of GFR than serum creatinine and serum creatinine based GFR estimations. Cystatin C can be used as a marker of kidney function in the adjustment of medication dosages. Cystatin C can be measured in a random sample of serum using immunoassays such as nephelometry or particle‐enhanced turbidimetry.

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III. Special Function Tests 3. Neutrophil gelatinase‐associated lipocalin (NGAL):

III. Special Function Tests 3. Neutrophil gelatinase‐associated lipocalin (NGAL): Clinical Application: • Creatinine is not useful for early diagnosis. • Urinary NGAL can be used as a marker for the early diagnosis of AKI. • NGAL may be used to detect AKI early in the following cases:

• NGAL is a protein of a small molecular weight (25 kD), belonging to the lipocalin superfamily initially found in activated neutrophils. It is also found in certain epithelia, such as renal tubules, where its expression is dramatically increased in ischemic or nephrotoxic injury. • NGAL is a promising biomarker for early detection of acute kidney injury (AKI). It is specifically released by the damaged kidney and can be detected in both urine and plasma. • Either alone or in combination with other biomarkers they will not only have an impact on medical decisions in future daily clinical routine, but they will also provide the basis for testing novel emergency therapies for a disease that is often recognized too late.

1. Pediatric and adult cardiopulmonary bypass operations. 2. Percutaneous coronary interventions (PCI). 3. Critically ill patients presenting at the emergency department or in the intensive care unit (heart failure, sepsis, multi‐organ failure) 4. Renal transplantation. 5. Patients with chronic kidney disease.

19

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245


Minerals and Electrolytes I Principle Elements (Major Elements= Macrominerals): – These are elements which occur in the body in large amounts and are required in amounts greater than 100 mg / day. – They include Calcium (Ca), Phosphorus (P), Magnesium (Mg), Sodium (Na), Potassium (K), Chloride (Cl) and Sulfur (S). II Trace Elements (Microminerals): – These are elements which occur in the body in small amounts and are required in amounts less than 100 mg / day. – They include Iron (Fe), Copper (Cu), Zinc (Zn), Manganese (Mn), Iodine (I), Fluorine (F), Cobalt (Co), Chromium (Cr), Selenium (Se) and Molybdenum (Mo). – There are other trace elements but not essential for life e.g. Cadium (Cd), Aluminium (Al) and Lithium (Li).


Electrolytes and Minerals 2

Differences between Principle Elements and Trace Elements Item of difference

Principal elements

Trace elements

Occurrence

Large amount

Small amount

Requirement

> 100 mg / day

< 100 mg / day

Plasma level

Expressed in mg / dl (needs to be SI units)

Expressed in µg / dl (needs to be SI units)

Minerals

Electrolytes • Electrolytes are substances whose molecules dissociate into ions when they are dissolved in water. • They include:

Calcium, Phosphorus, Iron, Copper, Zinc, Manganese, Magnesium, Sodium, Iodine, Fluorine, Cobalt, Chromium, Potassium, Chloride, Selenium, Molybdenum, Cadium, Sulfur. Aluminium, Lithium.

3

4

Sodium

Sodium

• Sources: – Table salt (sodium chloride) is the main source of sodium. – Meats contain sodium more than vegetables. • Requirements: – The RDA of sodium is 5.0 g for adults. – Persons with family history of hypertension: 2.0 g. – Hypertensive patients: 0.2 g. • Body sodium: – About ⅓ of total body sodium is present in the skeleton. – About ⅔ of sodium is present in the other tissues and body fluids where sodium is the main extracellular cation.

• Plasma sodium: – The normal plasma sodium level is 135 – 153 mmol/l. – Urine sodium level is 40‐220 mmol/l (24‐hour urine collection)

• Functions of sodium: – Maintenance of osmotic pressure and volume of plasma and extracellular fluid. – Transmission of nerve impulses. – Contraction of muscles. – Regulation of acid‐base balance. 5

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246

Sodium

Sodium

Alteration of plasma sodium: A. Hypernatraemia (excess plasma sodium): – Cushing syndrome: due to excessive glucocorticoids. – Conn’s disease: due to excessive aldosterone. – Diabetes insipidus: due to rapid loss of water. – Drugs: as ACTH or cortisone. B. Hyponatraemia (decreased plasma sodium): – Addison’s disease: due to deficiency of aldosterone. – Renal failure: due to inhibition of sodium reabsorption. – Hypotonic dehydration: due to the treatment of water and sodium loss by water only. – Diuretics: e.g. thiazides that block sodium reabsorption

• Sodium determination: – Ion selective electrode (ISE). – Atomic Emission Spectrophotometry (Flame Photometry). – Atomic Absorption Spectrophotometry. – Chemical (colourimetric) method.

7

8

Potassium

Potassium

• Sources: – Vegetables and fruits (especially citreous fruits). – Fish, meat and organs meat. • Requirements: – The RDA of potassium is 3.0 – 5.0 g. • Body potassium : – About ⅓ of total body potassium is present in the skeleton. – About ⅔ of potassium is present in the other tissues and body fluids where potassium is the main intracellular cation.

• Plasma potassium: – The normal plasma potassium level is 3.5 – 5.0 mmol/l. – Urine potassium level is 25 – 125 mmol/l (24‐hour urine collection).

• Functions of potassium: – Maintenance of osmotic pressure and volume of intracellular fluid. – Transmission of nerve impulses. – Contraction of muscles. – Regulation of acid‐base balance. 9

10

Potassium

Potassium

Alteration of plasma potassium: A. Hyperkalaemia (excess plasma potassium): – Addison’s disease: due to deficiency of aldosterone. – Acidosis due to shift of K+ from intra‐ to extracellular fluid in exchange with H+. – Crush injuries due to leakage of K+ from tissue cells. – Chronic renal failure. – Uncontrolled diabetes mellitus: because the lack of insulin prevents K+ from entering the cells. B. Hypokalaemia (decreased plasma potassium): – Alkalosis. – Treatment of hyperglycaemic coma by insulin without giving potassium as insulin helps K+ to enter the cells. – Excessive vomiting and diarrhoea. – Cushing syndrome: due to excessive glucocorticoids. – Primary and secondary aldosteronism. – Diuretics.

Potassium determination: • Ion selective electrode (ISE). • Atomic Emission Spectrophotometry (Flame Photometry). • Atomic Absorption Spectrophotometry. • Chemical (colourimetric) method.

11

12

Chloride

Chloride

• Sources: – Table salt (sodium chloride) is the main source of chloride.

• Plasma Chloride: – The normal plasma chloride level is 94 – 106 mmol/l. – CSF chloride level is 120 – 132 mmol/l. – Urine chloride level is 110 – 250 mmol/l (24‐hour urine collection).

• Requirements: – The RDA of chloride is 5.0 g for adults.

• Functions of Chloride: – Essential for water balance, osmotic pressure and acid‐base balance. – Essential role in the blood for chloride shift. – Essential for formation of HCl in the stomach. – It activates salivary and pancreatic amylase enzymes.

• Body Chloride: – Chloride is present with sodium in the extra‐cellular fluid compartment. – It has a particularly higher concentration (about 124 mmol/l) in the cerebrospinal fluid (CSF) than in the plasma.

13

14

Chloride

Bicarbonate (Total CO2)

• Alteration of Plasma Chloride:

• Clinical significance:

– Hypo‐ and hyperchloremia is usually associated with hypo‐ and hypernatremia respectively. – Hypochloraemia leads to an increase in plasma bicarbonate as a compensatory mechanism causing alkalosis (hypo‐chloremic alkalosis).

1. Decreased bicarbonate:

A. With elevated pH: • Blood bicarbonate is decreased with elevated blood pH in respiratory alkalosis as seen in the following conditions: – Simple anxiety with increased rate and depth of breathing; the so called “hyperventilation syndrome”. – Salicylates poisoning which stimulate the respiratory center. – Central nervous system lesions such as tumors located in this part of the brain.

• Chloride determination: – Ion selective electrode (ISE). – Chemical (colourimetric) method. 15

16



Clinical significance:

Clinical significance: 2. Increased bicarbonate:

1. Decreased bicarbonate: B. With decreased pH: • Blood bicarbonate is decreased with decreased blood pH in metabolic acidosis as seen in the following conditions: – – – – –

A. With decreased pH: • Blood bicarbonate is increased with decreased blood pH in respiratory acidosis as seen in the following conditions: – Lungs diseases e. g. bronchial asthma and emphysema. – Morphine poisoning.

Diabetic ketoacidosis. Lactic acidosis. Shock Renal failure. Severe diarrhea.

B. With elevated pH: • Blood bicarbonate is increased with increased blood pH in metabolic alkalosis as seen in the following conditions: – Excess intake of sodium bicarbonate as a treatment of gastric hyperacidity e. g. in peptic ulcer. – Severe and prolonged vomiting. 17

18


Blood pH Disorder (Acid‐Base Disturbance)

Bicarbonate determination: • Ion selective electrode (ISE). • Chemical (colourimetric) method. • Calculated from Henderson‐Hasselbalch equation:

• The normal blood pH is 7.4±0.05. • Acid‐base disturbance is the change of blood pH to be either less than 7.35 (acidosis) or more than 7.45 (alkalosis). • Acidosis is the condition in which the blood pH tends to fall below 7.35. • Alkalosis is the condition in which the blood pH tends to rise above 7.45. • Imbalance of blood pH may be:

Where pH and PCO2 (H2CO3) values can be obtained from Blood Gas Analyzer and pKa of carbonic acid (H2CO3) is 6.1 Specimen collection: • Unhaemolyzed fresh serum or heparinized plasma is required for routine electrolyte analysis. • Anaerobic collection in vacutainer tubes is preferred. • Fasting is not necessary although it is recommended.

A. Respiratory: [Changes in H2CO3 (CO2) level]: • This includes respiratory acidosis and alkalosis. B. Metabolic: [Changes in HCO3 level]: – This includes metabolic acidosis and alkalosis. 19

20



Respiratory Acidosis: • It is caused by increased blood H2CO3 due to failure of the lungs to excrete CO2 at the proper rate as in bronchial asthma & morphine poisoning.

Respiratory Alkalosis: • It is caused by increased blood H2CO3 due to failure of the lungs to excrete CO2 at the proper rate as in bronchial asthma & morphine poisoning.

• At first HCO3‐/H2CO3 is decreased below 20:1 (HCO3‐ is normal, H2CO3 is increased and pH is decreased).

• At first HCO3‐ / H2CO3 is increased above 20:1 (HCO3‐ is normal, H2CO3 is decreased and pH is increased).

• This stimulates the kidneys to reabsorb more HCO3‐ increasing blood HCO3‐ till HCO3‐ / H2CO3 becomes 20:1. This is called compensated respiratory acidosis (HCO3‐ is increased, H2CO3 is increased and pH is normal).

• This inhibits reabsorption of HCO3‐ through the kidneys decreasing blood HCO3‐ till HCO3‐ / H2CO3 becomes 20:1. This is called compensated respiratory alkalosis (HCO3‐ is decreased, H2CO3 is decreased and pH is normal). 21

22


Blood pH Disorder Acid‐Base Disturbance

Metabolic Acidosis:

Metabolic Acidosis:

• It is caused by decreased blood HCO3‐ due to increased production and accumulation of acids (β‐hydroxybutyric acid and acetoacetic acid in diabetic ketoacidosis), failure of excretion of acids (renal failure) and increased loss of bases (severe diarrhea). • At first HCO3‐/H2CO3 is decreased below 20:1 (HCO3‐ is decreased, H2CO3 is normal and pH is decreased). • This stimulates the lungs to expirate more CO2 decreasing H2CO3 till HCO3‐/H2CO3 becomes 20:1. This is called compensated metabolic acidosis (HCO3‐ is decreased, H2CO3 is decreased and pH is normal).

•

Plasma anion gap = plasma Na+ – (plasma Cl‐ + plasma HCO3‐)



 

•

Urinary anion gap = (urine Na+ + urine K+) ‐ urine Cl‐  

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249

The anion gap is due to unmeasured anions (e.g. proteins, SO42‐, HPO42‐) that are present in plasma. It is about 12 mmol/l in healthy subjects ranging from 7 ‐ 12 mmol/l. This gap can explain the cause of metabolic acidosis. It is increased in metabolic acidosis due to increased production and accumulation of acids (β‐hydroxybutyric acid and acetoacetic acid in diabetic ketoacidosis), but the gap is normal in metabolic acidosis due to failure of excretion of acids (renal failure) and increased loss of bases (severe diarrhea). This gap can detect the renal cause of metabolic acidosis. If the gap is negative this indicates that there is no renal cause of metabolic acidosis.



Metabolic Alkalosis:

• Acidaemia is uncompensated acidosis where blood pH falls below 7.35. • Alkalaemia is uncompensated alkalosis where blood pH rises above 7.45. • Arterial blood gases (ABG) are investigated by the followings o Arterial blood pH. o PO2. o PCO2. o Bicarbonate (total CO2).

• It is caused by increased blood HCO3‐ due to increased loss of acids (severe vomiting) and increased accumulation of bases (administration of large doses of bicarbonate in the treatment of peptic ulcer). • At first HCO3‐/H2CO3 is increased above 20:1 (HCO3‐ is increased, H2CO3 is normal and pH is increased). • This inhibits expiration of CO2 through the lungs increasing blood H2CO3 till HCO3‐/H2CO3 becomes 20:1. This is called compensated metabolic alkalosis (HCO3‐ is increased, H2CO3 is increased and pH is normal). 25

26

Calcium

Calcium Blood Calcium: • The erythrocytes contain almost no calcium. • Plasma calcium level ranges from 8.5 to 10.5 mg / dl (2.1 to 2.6 mmol / l). • Plasma calcium exists in 2 forms: • Non‐diffusible (45%): This is represented by calcium bound to plasma proteins, mainly albumin. It is physiologically inactive. • Diffusible (55%): This form of calcium may be: • Ionizable (50%): This ionizable calcium is the only physiologically active form. • Non‐ionizable (5%): This is mostly in the form of citrate salt. It is physiologically inactive.

Factors Affecting Plasma Calcium: I Hormonal Factors: The Calcium regulating hormones are: 1. Parathyroid hormone (parathormone = PTH): It is secreted from the four parathyroid glands. It increases the plasma calcium level. 2. Active form of vitamin D3 (1, 25 dihydroxycholecalciferol = calcitriol): It increases the plasma calcium level. 3. Thyrocalcitonin (calcitonin): It is secreted from the para‐follicular‐C cells of thyroid gland. It decreases the plasma calcium level. II Non‐Hormonal Factors: 1. Blood pH: Ionization of calcium occurs at normal blood pH (7.4). Alkalosis decreases ionized calcium. 2. Plasma proteins: In cases of hypoproteinaemia (as in albuminuria), the non‐diffusible calcium decreases. It was found that every one gram loss of albumin in urine leads to a decrease of about 0.8 mg / dl in total calcium level. 3. Plasma phosphate: The solubility product (Ca X P = about 50) must be constant. If plasma phosphate increases (as in renal failure), the plasma calcium decreases to keep Ca/P ratio constant.

27

Calcium

Calcium

Functions of Calcium

Alteration of Plasma Calcium I Hypercalcaemia: It is the increase of plasma calcium level more than 11.0 mg / dl. It is caused by: 1. Hyperparathyroidism, mainly primary (and also secondary and tertiary). 2. Excess intake of vitamin D and/or calcium. 3. Milk‐alkali syndrome where hypercalcaemia is present in patients receiving (for long time) excessive absorbable alkalies and milk for the treatment of peptic ulcer. 4. Malignancy as in leukaemia, multiple myeloma and Paget’s disease. 5. Drugs as thiazides diuretics. 6. Other causes as thyrotoxicosis and Cushing’s syndrome. • N.B.: In hypercalcaemia there is frequent formation of urinary tract stones especially calcium oxalate and triple phosphate stones.

I Unionized calcium: • It enters in the structure of the skeleton. II Ionized calcium: It is important for: • Transmission of nerve impulses. • Contraction of muscles. • Decrease of neuromuscular excitability. So, deficiency of ionized calcium leads to tetany. • Blood and milk clotting. • Maintenance of cell membrane permeability. • Activation of certain enzymes e.g. glycogen phosphorylase and pyruvate kinase. • Mediation of some hormone responses.

29

28

30

250

Calcium

Phosphorus

Alteration of Plasma Calcium: II Hypocalcaemia: It is the decrease of plasma calcium level less than 8.0 mg / dl. It is caused by: 1. Hyporparathyroidism. 2. Alkalosis. 3. Kidney diseases. • N.B.: The decrease in ionized calcium leads to tetany. Deficiency of calcium leads to rickets in children and oesteomalacia in adults.

Sources: • Milk and milk products. • Meat, organs meat and fish. • leafy vegetables and egg yolk. Body Phosphorus: • Total body phosphorus is about 800 g. • About 80% of it is present in the skeleton. • About 20% is present in other tissues (mostly intracellular) and body fluids Requirements: • Adults: 800 mg / day. • Children: 1200 mg / day. • Pregnant and lactating women: 1200 mg / day. 31

32

Phosphorus

Phosphorus

Blood Phosphorus: • Normal plasma inorganic phosphorus is 3.0 – 5.0 mg/dl. • Other forms are present: – In plasma: Phospholipids. – In RBCs: Organic phosphate e.g. ATP, glucose‐6‐phosphate. Factors Affecting Blood Phosphorus: • Parathyroid hormone decreases blood phosphorus. • Active vitamin D3 increases blood phosphorus. • Renal function: In renal failure the plasma inorganic phosphate increases due to failure of its excretion in the urine.

Functions of Phosphorus: • It enters in the structure of the skeleton. • It enters in the formation of blood buffers. • It enters in the structure of the following compounds: 1. Phospholipids: e.g. lecithin, cephalins. 2. Phosphoproteins. 3. Nucleic acids: RNA and DNA. 4. Coenzymes: e.g. NAD and NADP. 5. High‐energy phosphate compounds: e.g. ATP, GTP, creatine phosphate. 6. Cyclic AMP and cyclic GMP. 7. Carbohydrate intermediate e.g. glucose‐6‐phosphate, fructose‐1‐ phosphate. 33

34

Magnesium

Magnesium

Sources: • Green leafy vegetables, legumes, peas and nuts. • Fish, meat and organs meat.

Factors Affecting Plasma Magnesium: • Aldosterone hormone decreases plasma magnesium. • Parathyroid hormone increases plasma magnesium. • Renal function: Renal failure leads to hypermagnesaemia due to failure of excretion of magnesium in the urine. Functions of Magnesium: • It enters in the formation of the skeleton. • It is important for the normal contraction of muscles. • It is important for the normal transmission of nerve impulses. • It decreases the neuromuscular excitability. • It acts as an activator of many enzymes e.g. kinases, phosphatases, phosphorylases Alteration of Plasma Magnesium: • Hypermagnesaemia leads to muscular weakness, paralysis, somnolence and anaesthesia. These effects can be antagonized by calcium. • Hypomagnesaemia may result from parathyroidectomy and leads to tetany which is refractory to calcium therapy.

Requirements: • The recommended daily allowance (RDA) of magnesium is 300 mg for the normal adults. Body Magnesium: • The total body magnesium is about 21 g. • About 70% of it is present in the skeleton. • About 30% is present in the other tissues (mostly intracellular) and body fluids. Blood Magnesium: • Plasma magnesium is 2.0 – 3.0 mg / dl. • Erythrocyte magnesium is 2 – 3 times higher than plasma magnesium 35

36

Iron

Iron Body Iron: • The total body iron is 3 ‐ 5 g. • It is present in the body in two forms:

Sources: • Organs meat (liver, kidney, heart, spleen), meat and fish. • Legumes, vegetables and whole grains.

A. Functional forms (75%): • These are mostly in the form of haemoproteins. They are responsible for cellular respiration. They include:

Requirements: • Adults: 10 mg / day. • Pregnant and lactating women: 30 mg/day.

1. Haemoglobin (67%): This is the main form of iron in the body. It is the haemoprotein present within the red blood cells. 2. Myoglobin (7.5%): This is a haemoprotein found in muscles and heart. 3. Respiratory enzymes (0.5%): These are the haemoproteins which include: 1. Respiratory cytochromes (b, c1, c, a, a3) which are electron carriers in the respiratory chain within the mitochondria. 2. Catalase and peroxidase which are important in the detoxication of hydrogen peroxide (H2O2). 3. Tryptophan oxygenase (Pyrrolase) which is important in tryptophan metabolism. 4. Cytochrome P450 which is found in the mitochondria and microsomes of the liver. It is important in the detoxication of xenobiotic agents (toxic drugs and foreign chemicals). 37

Iron

38

Iron Blood Iron: A. In the red blood cells: • Every gram haemoglobin contains 3 – 4 mg iron. • So, there is about 50 mg of iron per 100 ml blood because there is about 15 gram haemoglobin per 100 ml blood.

B. Non‐Functional forms (25%): These are nonhaeme metalloproteins. They include: 1. Transferrin: the transport form of iron in blood plasma. 2. Ferritin: the storage form of iron in the tissues. It is present in the liver, kidney, spleen, bone marrow & intestinal mucosa. 3. Haemosiderin: found only when the body contains excess iron.

B. In the plasma: • Plasma iron concentration is 60 – 160 μg / dl. • Iron is carried by transferrin which is a glycoprotein synthesized in the liver and runs with β‐globulin in electrophoresis. • Transferrin may carry up to 250 – 400 μg of iron per 100 ml blood plasma. This is known as the total iron binding capacity (TIBC). About 30% of TIBC is saturated. • In iron deficiency anaemia, plasma iron decreases while the TIBC increases. In liver diseases, both plasma iron and the TIBC decreases. • Plasma contains very low concentration of ferritin which is a very good index of iron storage. It decreases in iron deficiency and increases in haemosiderosis. 39

Iron

40

Iron

Alteration of Plasma Iron: A. Iron deficiency anaemia: • Causes: – Deficient intake. – Impaired absorption. – Excessive loss. • Biochemical changes: – Plasma iron is decreased. – Plasma TIBC is increased. – Plasma ferritin is decreased.

B. Iron overload: •Causes: – Repeated blood transfusion. – Intravenous administration of iron. – Hechromatosis (Hemosiderosis; Bronz Diabetes): • Rare hereditary disease characterized by abnormal increase of iron absorption. • Iron is deposited in the form of hemosiderin in: – Liver: causing liver cirrhosis. – Pancreas: causing fibrosis and diabetes mellitus. – Skin: causing bronz discolouration of skin

•Biochemical changes: – Plasma iron is increased. – Plasma TIBC is decreased. – Plasma ferritin is increased. 41

42


Definitions • Enzymes are biological catalysts of protein nature composed of 200 or more amino acids covalently linked in a sequence dictated by cell’s genetic code.


• Isoenzymes are different molecular forms of the same enzyme having the same catalytic activity but differing in physico‐chemical properties.

Clinical Enzymology 2

Plasma Enzymes

1. Functional Plasma Enzymes

• Most enzymes occur at higher concentration intracellularly than in plasma. • Plasma enzymes can be classified into:

• These are enzymes which act on substrates normally present in plasma e.g. coagulation enzymes and pseudocholinesterase.

1. Functional plasma enzymes. 2. Non‐functional plasma enzymes.

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2. Non‐Functional Plasma Enzymes • These are enzymes that are synthesized in the cells where they act.

• Plasma enzymes can also be classified into: 1. Diagnostic enzymes 2. Prognostic enzymes

• They enter the plasma in small amounts as a result of continuous cell aging or due to diffusion during inactivation and catabolism or rarely by excretion into bile or urine. • They include the enzymes of intermediary metabolism e.g. SGOT, SGPT, ALP, LDH, CPK, amylase, etc.

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1. Diagnostic Enzymes

2. Prognostic Enzymes

• These are enzymes by which one can diagnose the disease because of the specificity of the enzyme to the affected organ or tissue e.g.:

• For follow up of certain diseases and not the diagnosis because these enzymes are present in more than one tissue e.g.:

– ALT for liver diseases. – G6PDH for favism. – CPK‐MB for myocardial infarction.

– – – –

AST: for heart , liver and skeletal muscles. LDH : for heart , liver and skeletal muscles. CPK : for heart , brain and skeletal muscles. ALP : for liver and bones.

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When will Plasma Level of Enzymes be Altered? • The normal plasma level of enzymes reflects the balance between:

1. If there is altered synthesis of enzymes within the cells. 2. If there is a change in the amount of enzymes‐ forming tissues (by cell proliferation). 3. If there is a change in cell permeability. 4. If there is alteration in the rate of inactivation and disposal of enzymes. 5. If there is an obstruction to normal pathway of enzyme excretion.

– Release of enzymes during normal cell turnover.

and – Their catabolism and excretion.

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Non‐specific Causes for Raised Plasma Enzyme Levels

Non‐specific Causes for Raised Plasma Enzyme Levels

A. Physiological: • Newborn: e.g. – Creatine phosphokinase (CPK). – Aspartate transaminase (AST).

B. Enzyme induction by drugs: • Diphenylhydantoin & barbiturates therapy increases ALP level. • Alcohol intake increases gamma glutamyl transpeptidase (GGT). C. Artfactual elevation: • Lactate dehydrogenase, acid phosphatase and transaminases are elevated in haemolysed samples. • Amylase may be elevated if mouth pippetting is the rule. • Acid phosphatase is elevated if catheterization or per rectum examination is done within one week before the investigation. • Total CPK is increased after intramuscular injection or after muscular exercise.

• Childhood: e.g. – Alkaline phosphatase (ALP). • Pregnancy: e.g. – ALP (in the last trimester). – CPK & AST (during & immediately after labour).

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AST is a prognosis enzyme increased under the following conditions:

Clinically Important Enzymes

A. Physiological: Newborn .

1. Aspartate Transaminase (AST) or 2. Glutamate Oxaloacetate Transaminase (GOT)

B. Pathological: In the following diseases: • Myocardial infarction. • Liver diseases: – – – – – –

• •

Viral hepatitis. Toxic liver necrosis. Liver cirrhosis. Cholestatic jaundice. Malignant infiltration of the liver. Infectious mononucleosis.

Skeletal muscle diseases: After trauma or surgery. Circulatory failure with shock and hypoxia.

C. Artfactual: Haemolyzed samples. 13

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Clinically Important Enzymes • ALT is a diagnostic enzyme for liver diseases including: – – – – – –

1. Aspartate Transaminase (AST) or 2. Glutamate Oxaloacetate Transaminase (GOT)

Viral hepatitis. Toxic liver necrosis. Liver cirrhosis. Cholestatic jaundice. Liver congestion secondary to cardiac failure. Infectious mononucleosis.

• ALT is also increased in circulatory failure with shock and hypoxia and may be slightly increased in extensive muscle trauma. • In acute liver diseases ALT is increased first followed by an increase in AST. 15

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3. Alkaline Phosphotase (ALP)

B. Pathological: In the following diseases: I. Bone diseases: – – – – – –

• ALP is a prognostic enzyme having five isoenzymes: bone, liver, placenta, intestinal and Regan isoenzymes. • It is increased in the following conditions:

Osteomalacia and rickets. Paget’s disease of bone. Primary hyperparathyroidism with bone involvement. Secondary carcinoma deposits in bone. Extensive osteogenic sarcoma. Healing phase of bone fracture.

II. Liver diseases: – – – –

A. Physiological: 1. Preterm infants. 2. Children. 3. Pregnant women in the last trimester.

Cholestasis. Hepatitis. Liver cirrhosis. Space‐occupying lesions, tumours, infiltrations.

C. Induction by drugs: Barbiturates and diphenylhydantoin.

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4. 5’‐Nucleotidase

5. Gamma Glutamyl Transpeptidase (GGT)

• 5´‐Nucleotidase is a diagnostic enzyme for liver diseases. • Its activity is parallel to that of liver ALP. • It is indicated to exclude the bone diseases as a cause of elevated ALP level where:

• GGT is a diagnostic enzyme for liver diseases increasing in the following conditions: – – – – – –

– In liver diseases: high ALP and high 5´‐Nucleotidase. – In bone diseases: high ALP and normal 5´‐Nucleotidase.

Liver cirrhosis. Metastatic carcinoma. Hepatic infiltration. Cholestasis. Alcoholism. Patients on anti‐convulsant therapy.

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6. Adolase

7. Amylase

• Aldolase is a prognostic enzyme widely distributed mainly in muscles, liver and red blood cells. • Its levels are increased in:

• Amylase is a prognostic enzyme present in both pancreatic juice and saliva. • It is excreted in urine. • It is increased in the following conditions:

– – – –

Myocardial infarction. Extensive muscle trauma. Haemolysis. Generalized malignancy.

– Acute pancreatitis: The enzyme rises temporary within the first 24 hours and returns to normal within 2 or 3 days as the acute attack resolves. – Severe uraemia. – Severe diabetic ketoacidosis. – Perforated peptic ulcer. – Acute cholecystitis. – Ruptured ectopic pregnancy. – Mumps and salivary calculi. 21

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8. Lipase

9. Acid Phosphotase

• It is a diagnostic enzyme for pancreatic diseases especially acute pancreatitis.

A. Prostatic acid phosphatase: • It is a diagnostic enzyme especially for metastatic prostatic carcinoma. • It may also increased in the following conditions:

• It has a similar but slower pattern of rise and fall in pancreatitis than amylase.

1. 2. 3. 4.

• Its assay is of value after 48 hours of the onset where its peak activity is at about 48 hours and the level usually remains high for about a week. 23

Following rectal examination. Acute retention of urine. After urinary catheterization. Chronic constipation.

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10. Creatine Phosphokinase (CPK) Creatine Kinase (CK)

9. Acid Phosphotase

Total CPK is a prognostic enzyme present in cardiac muscle, brain and skeletal muscle. • It is increased in the following conditions: A. Physiological: •

B. Total acid phosphatase •It is a prognostic enzyme present mainly in the prostate and also in other sources including liver, red cells, platelets and bone. •It is increased in the following conditions: 1. 2. 3. 4. 5.

1. 2.

Newborn. After labour for few days.

B. Pathological :

Causes of elevation of prostatic acid phosphatase. Gaucher’s disease. Thrombocytopenia with excessive destruction of platelets. Paget’s disease with high elevation of ALP. Artifactually in haemolyzed samples.

1. 2. 3. 4. 5. 6. 7. 8. 9.

Myocardial infarction. Muscular dystrophies. Muscle injury. After surgery. Severe physical exertion. Hypothyroidism. Alcoholism. Cerebrovascular accident and head injury. Malignant hyperpyrexia.

C. Artfactual: Haemolyzed samples.

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11. Lactate Dehydrogenase (LDH) • LDH is a prognostic enzyme widely distributed in heart, skeletal muscle, liver, kidney, lungs, brain, erythrocytes and malignant tissues. • It is increased in the following conditions:

• CPK is a dimmer enzyme composed of 2 subunits of M & B types the combination of which results in the formation of 3 isoenzymes:

A. Pathological: 1. Myocardial infarction. 2. Haematological disorders as pernicious anaemia, leukaemia and haemolytic anaemia. 3. Circulatory failure with shock and hypoxia. 4. Viral hepatitis. 5. Pulmonary embolism. 6. Infectious mononucleosis. 7. Occasionally in cerebral and renal infarction.

1. CK‐MM: Specific for skeletal muscle disorders. 2. CK‐MB: Specific for heart (Myocardial infarction). 3. CK‐BB: Specific for brain.

B. Artfactual: Haemolyzed samples.

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Timing of Cardiac Markers in Relation to Myocardial Infarction

• LDH is a tetramer composed of 4 subunits of H & M types the combination of whish results in the formation of 5 isoenzymes: 1. 2.

LDH1 = H4 (HHHH) LDH2 = H3 M (HHHM) • They are predominant in cardiac muscle (LDH1 > LDH2), red blood cells and malignant tissues (LDH2 > LDH1). • In myocardial infarction, they are increased with LDH1/LDH2 > 1. • These isoenzymes can be substituted by hydroxybutyryl dehydrogenase (HBDH) which is also called cardiac LDH . • In haemolytic and pernicious anaemias, these isoenzymes are increased with LDH1/LDH2 < 1. 3. LDH3 = H2 M2 (HHMM) • It is predominant in lungs and kidneys. • It is increased with LDH2 in acute leukaemia. • It is increased in pulmonary and renal infarction. 4. LDH4 = H M3 (HMMM) 5. LDH5 = M4 (MMMM) • They are predominant in skeletal muscle and liver. • They are increased in muscle diseases and acute hepatitis. 29

Cardiac Marker

Onset of Rising (Hour)

Peak of Rising (Hour)

Duration of Rising (Days)

CPK‐MB

3 – 5

8 – 12

1 – 2

Total CPK

4 – 6

12 – 24

1.5 – 3

AST (GOT)

8 – 12

24 – 36

3–6

LDH

12 – 24

48 – 72

6 – 12

Myoglobin

About one

4–8

0.5 – 1

Cardiac troponin I & T

3–5

24 - 48

7 - 10 30

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12. Pseudocholinesterase Two enzymes of cholinesterase are present in the body: A. True cholinesterase (Acetyl cholinesterase): • It is found in the nervous tissues and skeletal muscles with lower concentration in red blood cells. • It hydrolyzes the excess acetylcholine to prevent continuous discharge of the nerve impulse or continuous contraction of the muscle after completion of impulse. B. Pseudocholinesterase (Plasma cholinesterase) • It is widely distributed in the body including the liver (site of its synthesis) and plasma. • It has no effect on acetylcholine present in the nerve endings but it destroys any acetylcholine which might escape the action of acetyl cholinesterase and reach the blood. • It destroys other choline and non‐choline esters e.g. succinylcholine used as a muscle relaxant during general anaesthesia. • So, it is advised to measure the enzyme activity before general anaesthesia to avoid the danger of prolonged periods of apnea after major operations in susceptible persons.

• Pseudocholinesterase activity is decreased in:

•

3.

Enzyme activity is markedly decreased before the toxic effect of organophosphorus compounds on the central nervous system. So, estimation of the enzyme activity is used as a monitor for

patients of

organophosphorus poisoning and for workers in insecticides factories.

3.

Inherited abnormal cholinesterase variants with low biological activity.

• Its activity is increased in: 1.

Recovery from liver damage.

2.

Nephrotic syndrome.

3.

Obesity, thyrotoxicosis, hypertension and alcoholism.

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13. Glucose‐6‐Phosphate Dehydrogenase (G6PDH) • G6PDH is the main enzyme of hexose monophosphate (HMP) shunt pathway.

Enzyme activity is markedly decreased before the toxic effect of organophosphorus compounds on the central nervous system. So, estimation of the enzyme activity is used as a monitor for patients of organophosphorus poisoning and for workers in insecticides factories.

NADPH+H+

Pentose Phosphate Pathway

Inherited abnormal cholinesterase variants with low biological activity.

Glucose-6-phosphate Dehydrogenase

NADP

G-S-S-G

FAD

Glutathione Reductase

2 G-SH

2 H2O

Se

Glutathione Peroxidase

H 2O 2

• It is important for the integrity of red blood cells through the production of reduced coenzyme II (NADPH + H+). • Its deficiency may cause haemolytic anaemia called Favism which is precipitated by administration of certain oxidizing agents such as Fava beans, anti‐malarial drugs, sulpha drugs, phenylbutazone and vitamin K analogues. • Its synthesis is induced in these patients by the age of 9 – 11 years.

• Its activity is increased in: 1. 2. 3.

Organophosphorus poisoning:

•

Hepatic parenchymal diseases (hepatitis, cirrhosis, congestion). Organophosphorus poisoning: •

Hepatic parenchymal diseases (hepatitis, cirrhosis, congestion).

2.

•

• Pseudocholinesterase activity is decreased in: 1. 2.

1.

Recovery from liver damage. Nephrotic syndrome. Obesity, thyrotoxicosis, hypertension and alcoholism.

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Role of Liver in Metabolism The liver plays an important regulatory role in: I. Carbohydrate Metabolism. II. Lipid Metabolism. III. Protein Metabolism. IV. Metabolism of Foreign Organic Substances. V. Vitamin Metabolism. VI. Mineral Metabolism.


Liver Metabolism 2

Role of Liver in Carbohydrate Metabolism

Role of Liver in Lipids Metabolism

The liver is the “blood glucostat”.

Major site for fatty acid oxidation. Synthesis, mobilization and oxidation of triglycerides. Site for fatty acids biosynthesis from excess glucose. Site for synthesis of cholesterol from active acetate. Main site for ketogenesis. Major site for phospholipids & lipoproteins synthesis. Site for degradation of cholesterol, phospholipids and lipoproteins. • Only site for synthesis of bile salts. • Site of storage of fat‐soluble vitamins. • Only site for detoxication of steroid hormones & drugs. • • • • • • •

A. After carbohydrate meal: • The liver prevents excessive hyperglycaemia by increasing the uptake and utilization of glucose by oxidation (glycolysis and Kreb’s cycle), glycogenesis and lipogenesis. • It converts fructose and galactose into glucose preventing their excretion in the urine. B. During fasting: • The main source of blood glucose is the liver where glycogenolysis and gluconeogenesis occur. 3

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Role of Liver in Metabolism of Foreign Organic Substances

Role of Liver in Protein Metabolism • Deamination of amino acids. • Formation of urea from the ammonia resulting from the deamination of amino acids. • Gluconeogenesis from the carbon skeleton of the deaminated amino acids. • Biosynthesis of the non‐essential amino acids. • Biosynthesis and degradation of its own proteins as well as of most of the plasma proteins. • Biosynthesis of creatine, purines and pyrimidines. • Catabolism of purines, pyrimidines and haemoglobin.

• Numerous drugs are detoxicated by the liver and some are excreted in bile. • Benzoic acid is detoxicated by conjugation with glycine to form hippuric acid which is excreted in urine.

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Role of Liver in Vitamin Metabolism

Role of Liver in Mineral Metabolism

• Liver has storage function for fat‐soluble vitamins. • It helps absorption of the fat‐soluble vitamins through the formation of the bile salts. • It converts carotenes to vitamin A, and vitamin D3 to 25‐ hydroxy vitamin D3. • It converts tryptophan to niacin. • It utilizes vitamin K for the synthesis of prothrombin and blood clotting factors VII, IX and X.

• The liver is an important storage site for iron and copper. • It is the main route of excretion of copper.

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Bilirubin Metabolism


• The average life span of the red blood cells is 120 days. • Every day, about 250 mg of bile pigments are produced by the catabolism of about 6.25 g of haemoglobin. • The formation of the bile pigments can be divided into 3 stages: 1. In the Reticuloendothelial Cells. 2. In the Liver. 3. In the Intestines.

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Van den Bergh Reaction • This is a reaction between bilirubin and Ehrlich diazo (diazotized sulfanilic acid) reagent giving violet colour. • Conjugated bilirubin reacts directly with the reagent. So, it is called direct bilirubin. • Unconjugated bilirubin does not react with the reagent directly except after addition of methyl alcohol. So, it is called indirect bilirubin.

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Differences Between Unconjugated and Conjugated Bilirubin Unconjugated Bilirubin

Conjugated Bilirubin

1. Present normally in plasma.

1. Present normally in bile.

2. Attached to albumin.

2. Conjugated to glucuronic acid.

3. Insoluble in water.

3. Soluble in water.

4. Cannot be filtered through the kidney.

4. Can be filtered through the kidney.

5. Can cross blood-brain barrier.

5. Cannot cross blood-brain barrier.

6. Gives indirect Van den Bergh reaction.

6. Gives direct Van den Bergh reaction.

Jaundice • Jaundice is the yellowish discoloration of the skin, mucous membranes and sclera due to increased plasma bilirubin level more than 2.0 mg/dl.

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Jaundice

Haemolytic Jaundice (Pre‐Hepatic Jaundice)

• Normally: Plasma total bilirubin is 0.2 – 1.0 mg/dl, direct bilirubin is 0.00 – 0.25 mg/dl and indirect bilirubin is 0.20 – 0.80 mg/dl. • Hyperbilirubinaemia results when plasma bilirubin level exceeds 1.0 mg/dl. • Values between 1.0 and 2.0 mg/dl are considered latent jaundice. • Hyperbilirubinaemia may be due to increased conjugated and/or unconjugated bilirubin. • Jaundice can be classified into: 1. Haemolytic (Pre‐hepatic) jaundice. 2. Hepatocellular or Toxic (Hepatic) Jaundice. 3. Obstructive or cholestatic (Post‐hepatic) Jaundice.

• This is due to excessive haemolysis leading to increase in the amount of plasma unconjugated bilirubin more than the conjugating capacity of the liver. • Biochemical changes are: 1. Unconjugated hyperbilirubinaemia. 2. Increased urobilinogen in urine and stools. 3. No bilirubin appears in urine.

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Hepatocellular Jaundice (Toxic Jaundice) (Hepatic Jaundice)

Obstructive Jaundice (Cholestatic Jaundice) (Post‐Hepatic Jaundice)

• It is due to liver damage by cirrhosis, hepatitis and toxins. • There is an associated obstruction of some biliary canaliculi. • Biochemical changes are:

• Cholestasis may be due to obstruction of biliary tree by gall stone in common bile duct, cancer head pancreas or carcinoma of biliary tree. • Biochemical changes are: 1. Conjugated hyperbilirubinaemia. 2. Urobilinogen is absent in urine and stools. 3. Bilirubin appears in urine.

1. Unconjugated & conjugated hyperbilirubinaemia. 2. Trace amount of urobilinogen in urine & stools. 3. Bilirubin appears in urine.

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Biochemical Changes in Normals and the 3 Types of Jaundice

Causes of Jaundice

Plasma Condition

Urine

T. Bilirubin (mg/dl)

D. Bilirubin (mg/dl)

Bilirubin

Urobilinogen (mg/day)

Foecal Stercobilinogen (mg/day)

1. Normals

0.2 – 1.0

0.0 – 0.25

Negative

0 – 3

30 – 300

2. Haemolytic jaundice

Increased

Normal

Negative

Increased

Increased

3. Hepatic jaundice

Increased

Increased

Positive

Decreased – Normal

Decreased – Normal

4.Obstructive jaundice

Increased

Increased

Positive Decreased

Decreased

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Physiological Neonatal Jaundice

Congenital Hyperbilirubinaemia

• This is a transient condition occurring in some newborn infants especially if they are premature due to immaturity of UDP‐ glucuronyltansferase enzyme and accelerated haemolysis of RBCs.

Gilbert’s Disease: • Asymptomatic unconjugated hyperbilirubinaemia due to a defect in the uptake of bilirubin by the liver cells and a mild deficiency of UDP‐ glucuronyltransferase enzyme. Crigler‐Najjar Syndrome: • Severe unconjugated hyperbilirubinaemia due to marked reduction of UDP‐ glucuronyltransferase. Dubin‐Johnson syndrome: • Conjugated hyperbilirubinaemia due to a defect in the hepatic secretion of direct bilirubin into the bile. Rotor’s syndrome: • Conjugated hyperbilirubinaemia with normal liver histology. • Its cause has not been identified, but it may be due to a defect in transport by hepatocytes for organic anions, including bilirubin.

• This leads to unconjugated hyperbilirubinaemia and jaundice which lasts 2 – 3 days in full‐term infants and 1 – 2 weeks in premature infants. • If plasma indirect bilirubin exceeds the capacity of plasma albumin (20 – 25 mg/dl), free bilirubin can cross blood‐brain barrier causing kernicterus (toxic encephalopathy) which can lead to mental retardation. • Neonatal jaundice is treated by Phenobarbital and by exposure of the infant to visible light (phototherapy). 21

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Bile Acids and Bile Salts II. Secondary Bile Acids: • They are synthesized in the intestine from primary bile acids. • They include: 1. Deoxycholic acid. 2. Lithocholic acid.

• Greater proportions of these bile acids are reabsorbed from the intestine to the liver again and then are re‐excreted in the bile forming what is called “enterohepatic circulation of bile acids”. • The non‐absorbed bile acids are excreted in foeces.

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Gallstones

Liver Function Tests I. Synthetic Function Tests: 1) Plasma total protein:

• Gallstones are solid concretions that are most commonly formed in the gallbladder and occasionally in the bile ducts. • There are three major types of gallstones: 1. Cholesterol gallstones: –

•

2) Plasma albumin: • •

They can be examined and detected by the Liebermann‐Burchard reaction.

(1 – 2/1).

Plasma albumin A/G = ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ Plasma total protein – (Plasma albumin + Fibrinogen)

The pigment is mainly bilirubin which is present as calcium bilirubinate. They can be examined and detected by using Ehrlich diazo reagent.

Serum albumin A/G = ‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ Serum total protein – Serum albumin

3. Mixed gallstones: –

(3.8 – 5.0 g/dl).

Albumin is synthesized in the liver. Hypoalbuminaemia occurs in acute and chronic liver diseases.

3) Albumin / globulin ratio (A/G):

2. Pigmented gallstones: – –

(6.5 – 8.0 g/dl).

All plasma proteins are synthesized in the liver except γ‐globulin.

They may contain a mixture of cholesterol, bilirubin, calcium phosphate, calcium carbonate and mucoproteins.

4) Prothrombin time: (Concentration: 70 – 120%). •

Measurement of Prothrombin concentration and its response to vitamin K is a useful test of liver function. 26

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Liver Function Tests

Liver Function Tests

II. Excretory Function Tests: 1. Plasma total bilirubin: (0.2 – 1.0 mg/dl). – It is increased in all types of jaundice. 2. Plasma direct bilirubin: (0.0 – 0.25 mg/dl). – It is increased in obstructive and hepatocellular jaundice. 3. Alkaline phosphatase: – It is normally increased in children, and pregnant women in last months. – It is increased in obstructive jaundice and osteolytic bone diseases.

III. Tests Depending on Integrity of Liver cells (Liver Enzymes): • The following enzymes are increased in acute hepatocellular damage as in viral hepatitis: 1. Plasma alanine transaminase (ALT) = Plasma glutamate pyruvate transaminase (GPT) 2. Plasma aspartate transaminase (AST) = Plasma glutamate oxaloacetate transaminase (GOT) 3. Plasma gamma glutamyl transpeptidase (GGT) It is induced by alcoholic intake and by hypnotics. 4. Other plasma enzymes: –

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These include 5`‐nucleotidase, aldolase and lactate dehydrogenase (LDH) especially LDH5.

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HEART Training Program for Health Institute Graduates Laboratory Technician

Cardiac Biomarkers 2

The Heart

Sources of Energy for the Heart

• The heart is an efficient and durable pump. • It is a muscular organ responsible for moving blood through blood vessel to all parts of the body. • Work of the heart is generated by the alternating contraction and relaxation of heart muscle fibres. • These muscle fibres are composed of cardiac‐specific contractile proteins (actin and myosin) and regulatory proteins (troponin and tropomyocin). • They also contain proteins (such as myoglobin) and enzymes (such as creatine kinase, lactate dehydrogenase and aspartate transaminase) that are vital for energy use.

• Energy is liberated from various substances that are used in the heart as fuels by several pathways: 1. 2. 3.

Embden‐Meyerhof glycolytic pathway. Fatty acid oxidation. Kreb’s citric acid cycle.

• The heart uses free fatty acids as its predominant fuel. It also consumes significant quantities of glucose and lactate, as well as lesser amounts of pyruvate, ketone bodies and amino acids. • Most of the energy for cardiac function is obtained from the breakdown of metabolites through the citric acid cycle and the oxidative phosphorylation

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Storage of Energy in Cardiac Muscle

Encountered Medical Terms

• The heart requires an effective storage method to maintain a reserve of ATP. • This is achieved through the synthesis of creatine phosphate, which acts as an available store for rapid regeneration of ATP on need.

• • • • • • •

Mg2+

Atherosclerosis. Acute coronary syndrome. Acute myocardial infarction. Deep venous thrombosis. Pulmonary circulation. Pulmonary embolism. Heart failure.

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Artherosclerosis

Coronary Atherosclerosis

• Atherosclerosis is the deposition of plaques containing cholesterol and lipids on the intima (innermost layer of the walls) of large‐ and medium‐sized arteries.

• Coronary atherosclerosis is an inflammatory disease characterized by the accumulation of white blood cells, cell debris, fatty substances (cholesterol and fatty acids), calcium, and fibrous tissue (plaque or atheromas) on the walls of the coronary arteries that supply the heart muscle.

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Coronary Stenosis

Coronary Stenosis

• As plaque slowly increases in size over many years, the artery narrows in places (stenosis), and blood flow to the heart is reduced.

• Cholesterol‐containing plaques are highly dangerous even without narrowing of the vessel because the fibrous cap can be softened and rupture suddenly during acute heavy exercise or activity. This can cause bleeding from the blood vessel wall, resulting in blood clot formation that may obstruct the vessel.

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Myocardial Ischaemia

Acute Coronary Syndrome

• The stenosis of coronary arteries may become so significant that the blood supply is inadequate to meet the needs of the heart (myocardial ischaemia), and the affected part of the heart muscle no longer functions normally. • Myocardial ischaemia typically results in chest pain (angina pectoris), but may also cause no symptoms (silent ischaemia). Total blockage of a coronary artery results in a heart attack (myocardial infarction).

• Acute coronary syndrome is a spectrum of conditions involving chest discomfort or other symptoms caused by lack of oxygen to the heart muscle (the myocardium) due to sudden, reduced blood flow to the heart (insufficient blood supply to the heart muscle that results from coronary artery disease; CHD). • Acute coronary syndrome is treatable if diagnosed quickly.

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• Acute myocardial infarction is the myocardial tissue death (necrosis) due to prolonged ischaemia that is usually caused by arteriosclerosis with narrowing of the coronary arteries, leading to thrombosis (clot formation).

Diagram of a myocardial infarction • LCA: Left coronary artery • RCA: Right coronary artery 1. 2.

Branch of the left coronary artery Tip of the anterior wall of the heart (an apical infarct).

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Deep Venous Thrombosis

Pulmonary Circulation

• Deep venous thrombosis (DVT) is clotting of blood in a deep vein of an extremity (usually calf or thigh) or the pelvis. • It is a form of thrombophlebitis (inflammation of a vein with clot formation). • DVT results from conditions that impair venous return, lead to endothelial injury or dysfunction, or cause hypercoagulability). • DVT is the primary cause of pulmonary embolism.

• Deep venous thrombosis (DVT) is clotting of blood in a deep vein of an extremity (usually calf or thigh) or the pelvis. • It is a form of thrombophlebitis (inflammation of a vein with clot formation). • DVT results from conditions that impair venous return, lead to endothelial injury or dysfunction, or cause hypercoagulability). • DVT is the primary cause of pulmonary embolism. 15

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Pulmonary Circulation

Pulmonary Embolism

• Pulmonary circulation is the portion of the cardiovascular system which carries venous non‐oxygenated blood from the right ventricle of the heart, to the lungs, and then returns oxygenated blood back to the left atrium of the heart.

• Pulmonary embolism is the obstruction of the pulmonary artery or a branch of it leading to the lungs by a blood clot, usually from the leg, or foreign material causing sudden closure of the vessel.

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Heart Failure

Cardiac Markers

• Heart failure is a clinical syndrome characterized by systemic perfusion inadequacy to meet the body's metabolic demands as a result of impaired cardiac pump function. • This may be further subdivided into systolic or diastolic heart failure. • In systolic heart failure, there is reduced cardiac contractility, whereas in diastolic heart failure there is impaired cardiac relaxation and abnormal ventricular filling. • Heart failure may be due to failure of the right or left or both ventricles.

• These markers help in the early diagnosis of acute myocardial infarction (AMI) resulting from coronary heart disease (CHD) in addition to the clinical picture of the patient and electrocardiography (ECG). • When myocytes become necrotic, they loose their membrane integrity, & intracellular macromolecules diffuse into the cardiac interstitial space & ultimately into the cardiac microvasculature and lymphatics, then these macromolecules are detectable in the peripheral circulation. 19

Cardiac Markers

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Ideal Cardiac Marker • Diagnostically it has: 1. High sensitivity (Detection of AMI positive cases). 2. High specificity (Absent in non‐myocardial injury). 3. Rapidly release at a detectable concentration. 4. Correlates efficiently with extent of the infarction. 5. Persists in blood for valuable time (Prolonged ½ life) • Analytically it has: 1. High sensitivity (Low detectable limit). 2. High specificity (Less interferences). 3. Easy, inexpensive and tested rapidly (Short TAT). 21

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Importance of Cardiac Markers

Classification of Cardiac Biomarkers

1. To confirm the diagnosis of AMI when diagnosis by ECG is unclear (no ST‐segment elevation). 2. To distinguish patients with unstable angina from those with non‐Q wave AMI. 3. To provide prognostic information: as a non‐ invasive assessment of the likehood that the patient has undergone successful reperfusion when thrombolytic therapy is administered.

I. Traditional cardiac markers: A. Cardiac enzymes: 1. 2. 3. 4.

Creatine kinase (CK) = Creatine phosphokinase (CPK). Aspartate transaminase (AST). Lactate dehydrogenase (LDH). Hydroxy butyrate dehydrogenase (HBDH).

B. Cardiac proteins: 1. 2.

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Myoglobin. Cardiac Tropinin I & T

24



II. Recent cardiac markers: A. Markers for AMI:

III. Future cardiac markers:

1. 2. 3.

1. 2. 3. 4.

Cardiac Tropinin I & T. CK‐MB mass. Myoglobin.

Ischaemia Modified Albumin (IMA). Heart type‐Fatty Acid Binding Protein (H‐FABP). Glycogen Phosphorylase‐BB (GPBB). Copeptin.

B. Marker for heart failure: 1.

B‐natriuretic peptides (B‐NP & Pro B‐NP).

C. Marker for pulmonary embolism: 1.

D‐dimer. 25

26

Creatine Kinase (CK) Creatine Phosphokinase (CPK)

Creatine Kinase (CK)

• It is also called total CK where it is found in most tissues, but in high concentration in skeletal muscle, cardiac muscle and brain. So, it is a prognostic but not diagnostic enzyme. • It plays an important role in production of energy for muscle on need. Creatine Kinase Creatine + ATP Creatine Phosphate + ADP Mg2+

Its level is increased in: 1. Muscular diseases e.g. muscular dystrophy &polymyositis, and muscle fatigue as after severe muscular exercise. 2. AMI where the enzyme starts to increase 4 – 6 hours after the chest pain, then reaches its peak 12 – 24 hours after the attack and lastly returns to its normal level 36 – 72 hours after the onset of infarction. 3. Cerebrovascular accidents and severe neurogenic shock.

• Its level in males is more than that in females because of the difference in muscle bulk. 27

28



CK is a dimmer composed of two subunits: M (muscle) and/or B (brain). The combination of these subunits results in the formation of 3 isoenzymes:

• For CK assay in patients with AMI, the blood sample should not be aspirated within the first 3 hours or after 3 days of the attack.

1. CK‐MM: Specific for skeletal muscle. 2. CK‐BB: In brain, bladder, placenta, prostate, lungs and thyroid glands. 3. CK‐MB: About 45% in cardiac muscle and < 2% in skeletal muscle. Its level starts to increase 3 – 5 hours after AMI, then reaches its peak 8 – 12 hours after chest pain and lastly returns to its normal level 24 – 48 hours after the attack.

• During the follow up of these patients by total CK assay, the patients must not take analgesics or sedatives by intra‐muscular route to avoid any increase in CK‐MM that leads to misdiagnosis of superimposed infarction. 29

30

268

CK‐MBmass

CK‐MBmass • The mass assay for CK‐MB measures the amount of CK‐MB present in human serum or plasma for confirmation of acute myocardial infarction. The mass CK‐MB assay gives more specific patient results than traditional measurement of enzyme activity assays. • CK‐MBmass relative index (%RI) is calculated as follows:

• For CK assay in patients with AMI, the blood sample should not be aspirated within the first 3 hours or after 3 days of the attack. • During the follow up of these patients by total CK assay, the patients must not take analgesics or sedatives by intra‐muscular route to avoid any increase in CK‐MM that leads to misdiagnosis of superimposed infarction.

•

%RI = [CK‐Mbmass (μg/L) ÷ Total CK activity (U/L)] X 100

• Increased RI suggests myocardial origin. • RI > 3 ‐ 6 % with increased total CK activity suggests myocardial necrosis. • RI helps to rule‐out patients with skeletal muscle injury. 32

31

Aspartate Transaminase (AST)

Lactate Dehydrogenase (LDH)

• It is also called glutamate oxaloacetate transaminase (GOT).

• LDH is a reversible hydrogen transfer enzyme that catalyzes the reduction of lactate to pyruvate and vice versa using NAD as a hydrogen carrier. LDH Lactate + NAD Pyruvate + NADH+H+

• It is also a liver enzyme. So, it is a prognostic enzyme, but not a diagnostic one.

• It is also called total LDH where it is found in skeletal muscle, liver, heart, kidney and red blood cells.

• Its level starts to increase 8 – 12 hours after infarction, then reaches its peak 24 – 36 hours after onset of chest pain and lastly returns to its normal level 3 – 6 days after the infarction. 33

34



It is a tetramer composed of 4 subunits which are of two types; H (heart) and M (muscle). The combination of these subunits results in the formation of five isoenzymes: 1) LDH1 (HHHH) 2) LDH2 (HHHM) 3) LDH3 (HHMM) 4) LDH4 (HMMM) 5) LDH5 (MMMM) 35

269



3) LDH3 (HHMM):

• Blood sample for assay should be in plain tube because plasma samples may contain platelets that are rich in LDH. • During LDH assay, the blood sample must be free from haemolysis because red blood cells contain high concentration of this enzyme. • EDTA, oxalate and borates are interfering substances in its assay.

• It is predominant in lungs and kidneys. • It is increased with LDH2 in leukaemia. • It is increased in pulmonary and renal infarction. 4) LDH4 (HMMM): 5) LDH5 (MMMM):

• LDH4 and LDH5 are predominant in skeletal muscle and liver. • They are increased in muscle diseases and acute hepatitis. 37

38

Myoglobin

Myoglobin

A.

• Unlike haemoglobin, myoglobin does not exhibit cooperative binding of oxygen. Instead, the binding of oxygen by myoglobin is unaffected by the oxygen tension in the surrounding tissue. • Its low molecular weight and cytoplasmic location account for its early appearance in the circulation following muscle injury. • Serum myoglobin is increased after trauma to either skeletal or cardiac muscle as in crush injuries or AMI respectively.

B.

Myoglobin is an oxygen‐binding haemoprotein of cardiac and skeletal muscle with a molecular weight of 17,800 daltons. Myoglobin consists of a single protein chain with 153 amino acids and one heme group that stores oxygen in the muscle cells.

39

40

Myoglobin

Myoglobin

• The methods of serum myoglobin determination are unable to distinguish the tissue of origin. So, even minor skeletal muscle injury may result in elevated serum myoglobin concentration which may lead to misdiagnosis of myocardial infarction. • Myoglobin is the most sensitive marker in the early phase of a heart attack, when other cardiac markers may often still be normal. An acute myocardiac infarction (AMI) can be ruled out if the myoglobin level does not rise within 4 ‐ 6 hours after the onset of the symptoms. • Myoglobin is most useful when combined with an ECG. Elevated myoglobin and ST segment changes on an ECG are very indicative of an AMI.

• Myoglobin is an early marker of myocardial necrosis. It is often used as a negative marker for acute myocardial infarction (AMI) since two consecutive results below the cutoff, along with other clinical information, could be used to rule out a diagnosis of AMI. • Its level is increased as early as one to two hours after AMI with a peak at 4 – 8 hours and then is cleared and returns to its normal level 12 – 24 hours after infarction. 41

42

270

Cardiac Tropinin I & T

Cardiac Tropinin I & T

• Troponin is one of the minor protein components of the muscle. • It is a regulatory protein of contractile proteins of the myofibril. • It is a complex of 3 protein subunits: tropinin C (calcium‐binding component) troponin I (inhibitory component) and troponin T (trobomyosin‐binding component).

• It is found in both skeletal and heart muscles. However, cardiac‐specific troponin‐I (cTnI) and cardiac‐specific troponin‐T (cTnT) have been identified. • In contrast to cTnI & cTnT, troponin‐C is identical in both cardial and skeletal muscle. So, troponin C is not useful as a cardiac marker. • Due to their absolute coronary specificity and high sensitivity, cTnI & cTnT are the preferred biomarker for diagnosing cardiac muscle damage. • They serve for confirmation, even if the acute event is about 2 weeks in the past. 43

44

Timing of Cardiac Markers in Relation to Myocardial Infarction

Cardiac Tropinin I & T • Cardiac specificity of cTnI & cTnT eliminates a false diagnosis of AMI in patients with increased CK‐MB after skeletal muscle injury. • cTnI or cTnT has significant prognostic usefulness in unstable angina patients. Patients presenting with unstable angina who had concentrations above the normal level of this troponin had 5 ‐ 10 times greater risk of MI or death than patients with normal troponin levels. • The levels of cardiac cTnI & cTnT start to rise 3 – 5 hours after chest pain with a peak at 24 – 48 hours. The levels can remain elevated up to 7 – 10 days for cTnI or up to 10 – 15 days for cTnT after AMI.

Onset of rising (Hours) 3 – 5

Peak of rising (Hours) 8 – 12

Duration of rising (Days) 1 – 2

Total CPK

4 – 6

12 – 24

1.5 – 3

AST (GOT)

8 – 12

24 – 36

3 – 6

LDH

12 – 24

48 – 72

5 – 10 0.5 – 1

Cardiac marker CPK‐MB

MYOGLOBIN

1 ‐ 2

4 – 8

Cardiac troponin I

3 – 5

24 ‐ 48

7 ‐ 10

Cardiac troponin T

3 – 5

24 ‐ 48

10‐ 15

45

46

Natriuretic Peptides

Natriuretic Peptides

• Natriuretic peptide is one of the peptides that causes natriuresis. • The natriuretic peptides are produced by the heart and vasculature: – A‐type natriuretic peptide is secreted largely by the atrial myocardium in response to dilatation. – B‐type natriuretic peptide is manufactured mainly by the ventricular myocardium. – C‐type natriuretic peptide is produced by endothelial cells that line the blood vessels. • The natriuretic peptides serve to maintain intravascular homeostasis through their diuretic, natriuretic & vasodilator properties.

47

48

271

B‐Natriuretic Peptides


• B‐natriuretic peptide is a small peptide (32 amino acids) secreted by heart myocytes for regulation of blood pressure and fluid balance. • This peptide is synthesized by ventricular cells and stored as ProBNP (108 amino acids). • Proteolysis of ProBNP results in: 1. Active BNP (half‐life 20 minutes) containing 32 amino acids (77 – 108). It is the biologically active part. 2. N‐terminal fragment designated NT‐ProBNP (half‐life 120 minutes) that contains 76 amino acids (1 – 76). It is biologically inactive.

Release of BNP and NT‐proBNP 49

50



• B‐natriuretic peptide is useful in the diagnosis of heart failure. The finding of a low level of this peptide tends to exclude heart failure. • There is excellent clinical and statistical correlation between assays for BNP and NT‐proBNP. • Both BNP and NT‐pro‐BNP are sensitive, diagnostic markers for heart failure. • Plasma concentrations of both BNP and NT‐proBNP are significantly increased in patients with asymptomatic and symptomatic left ventricular dysfunction.

• Due to the longer half‐life in the circulation, NT‐proBNP levels in blood plasma are generally 6‐10 times higher than BNP. The better stability and wider dynamic range for NT‐proBNP may provide an advantage. • However, in the clinical setting, their overall diagnostic and prognostic abilities are comparable and both BNP and NT‐ proBNP have been shown to be extremely helpful in the diagnosis and management of patients with heart failure. • Changes in NT‐proBNP concentration can be used to evaluate the success of treatment in patients with left ventriculardysfunction. 51

52

D‐Dimer

D‐Dimer • The D‐dimer concentration is an indicator for the fibrinolytic activity of plasmin in the vascular system. • A higher concentration of D‐dimer indicates increased coagulation and fibrinolysis activity. • D‐dimer is a specific marker of degradation of fibrin clot and an indirect marker of clot formation • D‐Dimer is elevated in several clinical conditions including deep venous thrombosis (DVT), pulmonary embolism (PE) and disseminated intravascular coagulation (DIC ). • Acute deep veinous thrombosis and pulmonary embolism can be ruled out within shortest time and high accuracy with the D‐dimer assay.

• D‐dimers are specific degradation products of cross‐linked fibrin that are released when the endogenous fibrinolytic system attacks the fibrin matrix of fresh venous thromboemboli. • It is so named because it contains two crosslinked D fragments of the fibrinogen protein

53

272

Ischaemic Modified Albumin (IMA)


• IMA is a novel marker of ischaemia. • It is produced when circulating plasma albumin comes in contact with ischaemic heart tissues. • During ischaemia, N‐terminus of albumin is altered, probably through a series of chemical reactions involving free radical damage‐altered albumin, forming Ischaemic Modified Albumin • IMA is unable to bind metals, such as cobalt, at the N‐ terminus. • It is produced continually during ischaemia leading to rapid increase of its blood concentration that remains elevated during the ischaemic event

• Ischaemic patients have proportionately more IMA than non‐ ischaemic ones. • IMA has twice the sensitivity of cardiac troponin for diagnosing patient with AMI. It has value as rule out AMI. • Negative IMA can be used to predict subsequent negative troponin. • Combination of negative IMA and troponin and non‐ diagnostic ECG yields a negative predictive value of 99%. • IMA has specificity of 45 – 65%.

55

56


Heart type‐Fatty Acid Binding Protein (H‐FABP)

• IMA level starts to increase 6 – 10 minutes after the ischaemic cardiac event, then reaches its peak about 6 hours after the attack and lastly returns to its baseline about 12 hours after the event. • IMA may increase falsely in:

• Fatty acid binding proteins (FABPs) are members of cytosolic protein family. • FABP is relatively tissue specific. They are most abundantly found in heart (H‐FABP), liver (L‐FABP) and intestine (I‐FABP). • H‐FABP is a new cardiac marker. • It is released into the circulation shortly after the onset of ischaemia where it achieves its diagnostic level before 3 hours and returns to the normal level 12 ‐24 hours of the attack.

1. 2. 3. 4. 5.

Some cancers. Acute infections. End stage renal failure. Liver cirrhosis. Brain ischaemia.

57

58

Heart type‐Fatty Acid Binding Protein (H‐FABP)

Glycogen Phosphorylase‐BB (GPBB)

• H‐FABP can be considered as a diagnostic marker of acute coronary syndrome due to:

• Glycogen phosphorylase is the main enzyme in the glycogenolysis process (second source of blood glucose; the fuel of cardiac muscle). • GPBB is one of glycogen phosphorylase isoenzymes. • This isoform exists in heart and brain. • Other isoenzymes are found in liver (GPLL) and muscle (GPMM). • Due to an intact blood‐brain barrier, this marker can be regarded as a cardiac specific.

High myocardial content (more than 10 folds of skeletal muscle content) Present mainly in the cytosol Low molecular weight ( about 15 K‐dalton) Relative specificity Early appearance in plasma and urine after AMI onset

59

60

273

Glycogen Phosphorylase‐BB (GPBB)

Copeptin

• GPBB is a sensitive marker for the AMI diagnosis. • It has also been shown that GPBB is increased in a considerable proportion of AMI patients within 3 – 4 hours from onset of chest pain. • Level of GPBB is increased early in patients with unstable angina.

• Copeptin is glycopeptide formed of the C‐terminal fragment (39 amino acids) of provasopressin; the precursor of vasopressin hormone. • As a marker of endogenous stress, Copeptin is increased immediately after the onset of acute myocardial infarction and then steadily decreases. • Copeptin can be used in conjunction with cardiac troponin to improve the speed of diagnosing or ruling out myocardial infarction. • Copeptin predicts prognosis in patients with heart failure. • Moreover, copeptin has been found to be a prognostically relevant biomarker in a variety of illnesses such as sepsis, shock, pneumonia and acute exacerbation of COPD.

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62

Relation of Infarct Size to Cardiac Markers and ECG

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274


Sources of Lipids Dietary lipids • Triglycerides: Chief dietary lipids. • Cholesterol & phospholipids: Small amounts. • Fat‐soluble vitamins & carotenoids: Trace amounts.


Lipids 2

Digestion and Absorption


• The end products of triacylglycerols digestion are:

•

– 2‐monoacylglycerol + two fatty acids (72%) – glycerol + three fatty acids (22%) – 1‐monoacylglycerol + two fatty acids (6%)

•

• The end products of phospholipids digestion are: – – – –

•

Glycerol. Two fatty acids. Phosphoric acid. Nitrogenous bases (choline, serine, ethanolamine).

• •

• Cholesterol itself undergoes no digestion and is absorbed as such. • Cholesterol esters are hydrolyzed by pancreatic cholesterol esterase into cholesterol and fatty acids.

•

With the aid of bile salts, fatty acids and monoglycerides are dispersed into smaller particles known as micelles which enter the intestinal mucosal cells where fat digestion may be completed by intestinal lipase liberating glycerol and fatty acids. Short‐chain fatty acids and glycerol are absorbed through portal circulation to the liver. Long‐chain fatty acids are utilized for resynthesis of triglycerides within mucosal cells. The droplets of resynthesized triglycerides are then coated with a layer of protein and phospholipids, forming chylomicrons. Chylomicrons are then carried by lymphatics to the thoracic duct which transports them to general circulation. Free cholesterol is esterified within the mucosal cells forming cholesterol esters which are carried by lymphatics to enter the thoracic duct to the general circulation.

3

4


Fate of Absorbed Lipids

Steatorrhea • Steatorrhea is a disease due to defects in digestion and/or absorption of lipids. • It is the appearance of excessive amounts of lipids (<5 gm/day) in stools. • Normal fat content of stools is >5 gm/day. • Steatorrhea may be caused by one or more of the following: – Deficiency of pancreatic lipase. – Deficiency of bile salts. – Deficiency of healthy intestinal mucosa.

• Uptake by tissues. • Utilization by tissues. – A. Oxidation of fatty acids. There are three pathways: 1. 2. 3.

β‐Oxidation (Knoob’s theory). Alpha oxidation. Omega oxidation.

– B. Conversion to glucose. – C. Formation of tissue fat. • Storage. • Secretion. 5

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275

β‐Oxidation

Alpha Oxidation

• The major pathway for oxidation of fatty acids. • Carnitine is necessary for β‐Oxidation to be started • Importance of β‐Oxidation:

• • • •

1. Source of energy. 2. Production of acetyl‐CoA. 3. Ketone bodies formation.

A minor pathway for fatty acid oxidation. In α‐position of β‐methyl FA e.g. phytanic acid. Mainly in the brain & also in liver tissue. No energy production.

Refsum’s disease: 1. Failure of α–oxidation. 2. Accumulation of large amounts of phytanic acid in the brain, liver and blood. 3. Polyneuropathy, deafness and blindness occur at young age. 7

8

Omega Oxidation

Differences between Tissue Fat and Depot Fat

• At ω‐carbon (terminal CH3‐) of Fatty acid. • Formation of dicarboxylic acids. • Necessary for the structure of cell membrane of the brain cells.

Item of difference

Tissue fat

Depot fat

Site

Every cell e.g. cell membranes e.g. myelin sheath.

Fat cells of adipose tissue

Composition

Cholesterol, phospho‐lipids, glycollipids with little triglycerides

Mainly triglycerides with small amounts of cholesterol and phospholipids

Stability

Not affected by the nutritional state (constant element)

Affected by nutritional state (variable element)

Source of energy

No

Yes

Functions

Membrane permeability Respiratory enzymes Insulator to nerve impulse

Insulator to heat Protection Support

9

10

Plasma Lipids

Plasma Lipids

Cholesterol: • • • • • • • • • • • •

Hypercholesterolaemia: • It is the increase of blood cholesterol level. • It is due to: – Dietary e.g. diet rich in fat and carbohydrates. – Hypothyroidism. – Diabetes mellitus. – Nephrosis. – Obstructive jaundice. – Familial hyperlipoproteinaemia

Cholesterol is an animal esterol. All tissues synthesize their own cholesterol. Acetyl‐CoA is the source of all carbon atoms in cholesterol. HMG‐CoA reductase is the key enzyme in the biosynthesis of cholesterol. Plasma cholesterol is of both intrinsic and extrinsic origin. Liver is the only source of blood cholesterol. Plasma total cholesterol concentration is 150 – 250 mg/dl. In plasma: free cholesterol 30% ‐ cholesterol ester 70%. In plasma: LDL‐cholesterol 60% ‐ LDL‐cholesterol 40%. Functions of cholesterol: Formation of lipoproteins and cell membranes. Precursor of: 1. Bile salts. 2. Steroid hormones. 3. Vitamin D3.

11

12

276

Plasma Lipids

Plasma Lipids

Hypocholesterolaemia: • It is the decrease of blood cholesterol level • It is due to: 1. Dietary: 2. Starvation. • Low dietary cholesterol and carbohydrates. • Liver diseases: Hepatocellular damage. • Severe anaemia. • Hyperthyroidism. • Infection e.g. tuberculosis.

Triacylglycerols (triglycerides): • Plasma triglycerides are of two origins: 1. Intrinsic origin: • Synthesized in the liver. • Secreted into the plasma as very low density lipoproteins (VLDL) which represent endogenous triglycerides. 2. Extrinsic origin: • Absorbed from dietary sources. • Transported into the intestinal lymph and then into the plasma in the form of chylomicrons that represent exogenous or dietary triglycerides.

•

In the postabsorptive state (12 – 14 hours): – – –

Source of plasma triglycerides is the endogenous one. Exogenous triglycerides of chylomicrons are cleared from the circulation by the clearing factor (lipoprotein lipase enzyme). Plasma triglycerides concentration is 50 – 150 mg/dl.

13

14

Plasma Lipids

Plasma Lipids

Phospholipids: • The major phospholipids in plasma are lecithin and sphingomyelin. • Phospholipids are mainly synthesized in the mucosa of the small intestine and in the liver. • They circulate in the plasma as a component of HDL. • They are used as important constituents of all cells particularly those of the nervous system. • The normal plasma phospholipids level is 150 – 250 mg/dl.

Free fatty acids (FFA): • They are also called non‐esterified fatty acids (NEFA). • FFA are carried mainly bound to plasma albumin. • The normal plasma FFA level is10 – 30 mg/dl. Steroid hormones, fat‐soluble vitamins and carotenoids: • They are present in minute amounts. • They are called derived lipids because they are associated with lipids in nature and related to them in properties and metabolism. • They are transported in plasma carried on specific carrier protein for each.

15

16

Lipoproteins

Lipoproteins

• Lipids are relatively insoluble in water but they are carried in the body fluids as soluble protein complexes known as lipoproteins. • The lipoprotein particles contain the polar (water soluble) coat of protein and phospholipids at their surface, whereas the non‐polar (water insoluble) molecules such as cholesterol esters and triglycerides are present at the core of the lipoprotein molecules. • This structure makes it possible for these complex particles to transport water‐insoluble lipids in plasma.

•

The protein components of lipoproteins are known as apolipoproteins that are divided into 5 groups known as apolipoproteins: Apo‐A (apo A‐I, apo A‐II and apo A‐IV), Apo‐B (apo B‐48 and apo B‐100), Apo‐C (apo C‐I, apo C‐II and Apo C‐III), Apo‐D (apo D), and E (apo E2, apo E3 and apo E4).

•

Apolipoprotein‐a (apo‐a) is the protein moiety of the plasma human lipoprotein‐ a (Lp‐a), whose concentration is highly correlated with coronary artery disease. The protein moiety of this lipoprotein consists of apo‐a and apo B‐100, linked by one or more disulfide bonds.

•

They are transported in plasma in the form of lipoproteins that can be separated by ultracentrifugation or electrophoresis into chylomicron, VLDL (pre‐ β‐lipoprotein), LDL (β‐lipoprotein), HDL (α‐lipoprotein), and FFA & albumin.

17

18

Lipoproteins

Lipoproteins

1. Chylomicrons: • They transport mainly the absorbed (exogenous) triglycerides with small amounts of cholesterol and phospholipids. • The lowest density of lipoproteins, and contain very little protein. • They are synthesized in the intestinal mucosa and reach the systemic circulation via thoracic duct. • Their apolipoproteins are C, B and E.

3. Low‐density lipoproteins (LDL) = Beta‐lipoproteins: • Beta (β) lipoproteins on separation by electrophoresis. • They are formed in the liver from VLDL through the formation of intermediate density lipoproteins (IDL) by the removal of more triglycerides and apolipoproteins, before being secreted into the plasma. • About 60% of total cholesterol with smaller amounts of phospholipids and triglycerides are carried on them. • Transport cholesterol from the liver to the peripheral tissues. • Their apolipoproteins are mostly apo B (96%), with only small amounts of apo C (2%) and apo A (1%). • LDL is considered as a positive risk factor for atherosclerosis and ischaemic heart diseases, i.e. if LDL is increased the risk will be increased. • N.B.: Intermediate density lipoproteins (IDL) are usually transient Intermediates in the metabolism of VLDL to LDL.

2. • • •

Very low‐density lipoproteins (VLDL): They are also called pre‐beta (pre‐β) lipoproteins on separation by electrophoresis. They are mainly formed in the liver and to a lesser extent by the intestinal mucosa. They are responsible mainly for the transport of endogenous triglycerides with a small quantity of cholesterol from the liver to the cells. • Their apolipoproteins are C (55% = apo C‐I 5%, apo C‐II 20%, apo C‐III 30%), B (35%) and E (10%).

19

20

Lipoproteins

Hypolipoproteinaemias

4. High‐density lipoproteins (HDL): • Alpha (α) lipoproteins on separation by electrophoresis. • The smallest lipoprotein particles but are the densest because they contain a large amount of proteins. • Their lipids are chiefly phospholipids with smaller amounts of cholesterol (40% of total cholesterol) and triglycerides. • They transport cholesterol from peripheral tissues to the liver to be metabolized and excreted. • Their apolipoproteins are mostly apo A‐I (70%) and apo A‐II (20%), with only small amounts of apo C (10%). • HDL is considered as a negative risk factor, i.e. if HDL is increased it will protect against atherosclerosis and ischaemic heart diseases

• •

Decreased plasma lipoproteins. The following familial diseases are known: – Tangier disease (α‐lipoprotein deficiency): • It is due to very low plasma apo A‐I which results from increased rate of catabolism of such apolipoprotein. • Only traces of HDL are present in plasma. • Cholesterol esters accumulate in the tissue.

– A betalipoproteinaemia: • There is complete absence of apo‐B. • VLDL and LDL are absent from the plasma. • Plasma cholesterol and triglycerides are very low.

– Hypobetalipoproteinaemia: • Synthesis of apo‐B is decreased but VLDL and LDL, although reduced, are present in plasma. • Plasma cholesterol is decreased but not as markedly as in abetalipoproteinaemia.

5. Free fatty acids and albumin: • Free fatty acids are transported in plasma bound to albumin. 21

22

Hyperlipoproteinaemias

Hyperlipoproteinaemias – B. Type‐II hyperlipoproteinaemia: 1) Type‐IIa hyperlipoproteinaemia (hyper‐β lipoproteinaemia):

• They are disorders of metabolism in which one or more of plasma lipoproteins are increased. • They may be primary (inherited) or secondary (to diseases such as diabetes mellitus, obesity, nephrotic syndrome and hypothyroidism). • Hyperlipoproteinaemias can be classified as follows:



It is characterized by increased LDL, plasma is clear and plasma cholesterol is elevated.

2) Type‐IIb hyperlipoproteinaemia (hyper‐β and hyper‐pre‐β lipoproteinaemia): 

– A. Type‐I hyperlipoproteinaemia: • This is also known as hyperchylomicronaemia or exogenous hypertriglyceridaemia. • It is due to deficiency of lipoprotein lipase enzyme leading to accumulation of chylomicrons. • There is no increased risk of atherosclerosis. • The principle of treatment is to reduce the level of circulating chylomicrons by reducing dietary fats. 23

Increase in both LDL and VLDL, plasma may be turbid, and both cholesterol and triglycerides are elevated.



Very high level of plasma cholesterol (500 – 1000 mg/dl).



Defective LDL receptors in peripheral tissues.



Xanthelasma (deposits of cholesterol around the eyes) and xanthomas (deposits of cholesterol in skin and tendons).



Severe atherosclerosis and death from coronary artery disease are common.



Dietary therapy is important by decreasing cholesterol and saturated fat‐rich diet, and increasing its polyunsaturated fat.

24

278



– C. Type‐III hyperlipoproteinaemia: • Broad‐β or floating‐β hyperlipoproteinaemia. • It is similar to type‐IIb hyperlipoproteinaemia. • It is due to defect in IDL catabolism.

– E. Type‐V hyperlipoproteinaemia: • Hyperchylomicronaemia with hyperprebetalipoproteinaemia. • Elevation of chylomicron & VLDL causing Hypertriglyceridaemia and hypercholesterolaemia. • Frequent xanthomas but incidence of atherosclerosis is not risky. • Increased incidence of glucose intolerance and hyperuricemia.

– D. Type‐IV hyperlipoproteinaemia: • Hyper‐pre‐β lipoproteinaemia or endogenous hypertriglyceridaemia. • The most frequent one of hyperlipoproteinaemias. • Increase in VLDL without other lipoprotein abnormalities. • Hypertriglyceridaemia with hypercholesterolaemia and glucose intolerance are common.

25

26

Cholesterol and Atherosclerosis

Lipogenesis Synthesis of triacylglycerols from carbohydrates.

• Atherosclerosis is the deposition of cholesteryl ester and other lipids in the connective tissue of the arterial wall. • LDL/HDL cholesterol ratio helps in predicting atherosclerosis where:

• Site: – Liver, adipose tissue and lactating mammary gland. • Steps: 1. Biosynthesis of active glycerol:

– Increased LDL/HDL ratio predisposes to atherosclerosis. – Decreased LDL/HDL ratio gives protection against atherosclerosis.

• Glycerol is synthesized from intermediates of glucose oxidation through glycolysis.

• Atherosclerosis may lead to coronary heart disease and myocardial infarction.

2.

Biosynthesis of fatty acids and their activation: • Extramitochondrial system (De novo synthesis of fatty acid). • Microsomal system. • Mitochondrial system.

3.

Combination of active glycerol and three fatty acids to form triacylglycerols.

27

28

Lipolysis

Ketone Bodies Ketone bodies are substances which are normally formed in small amounts including: • Acetone. • Acetoacetic acid • β‐Hydroxybutyric acid Normally, the plasma concentration of ketone bodies does not exceed 2 mg/dl and its urinary excretion is less than 10 mg/day.

• Hydrolysis of stored triacylglycerols in adipose tissue into glycerol & free fatty acids.

Ketogenesis: • Formation ketone bodies from active acetate of fat origin in the mitochondria of the liver. Ketolysis: • Complete oxidation of ketone bodies to CO2 and H2O in the mitochondria of extrahepatic tissues. 29

30

279

Ketone Bodies

Bile Acids and Salts

Ketosis: • Increased ketone bodies in the blood (ketonaemia) and in the urine (ketonuria). • It occurs in states of decreased carbohydrates utilization e.g. starvation, low carbohydrates‐high fat diet and severe uncontrolled diabetes mellitus. • If ketosis is not treated, the buffer system (mainly bicarbonate) is depleted leading to acidosis i.e. ketoacidosis (decreased blood pH) which may lead to coma & death in severe & advanced cases.

Bile acids are the excretory products of cholesterol. • I. Primary Bile Acids: – They are synthesized in the liver from cholesterol. 1. 2.

Cholic acid. Chenodeoxycholic acid.

– They are conjugated with glycine (75%) or taurine (25%) to form conjugated bile acids which are secreted in bile as their sodium or potassium salts. – Bile salts are: 1. 2. 3. 4.

• •

Na (K) glycocholate. Na (K) taurocholate. Na (K) glycochenodeoxycholate. Na (K) taurochenodeoxycholate.

Bile salts are important for the digestion &absorption of fats because of their ability to lower the surface tension leading to emulsification of fat. In obstructive jaundice, bile salts are increased in blood leading to itching and bradycardia.

31

32

Bile Acids and Salts

Role of Liver in Lipid Metabolism

• II. Secondary Bile Acids:

• Fatty acid oxidation when the physiological conditions of the body need lipid oxidation. • Synthesis, mobilization and oxidation of triglycerides. • Fatty acids biosynthesis from excess glucose. • Synthesis of cholesterol from active acetate. • Ketogenesis. • Phospholipids and lipoproteins synthesis. • Degradation of cholesterol, phospholipids and lipoproteins. • Synthesis of bile salts facilitating the digestion and absorption of fats. • Storage of most fat‐soluble vitamins. • Detoxication of steroid hormones and drugs by conjugating them with sulfate and/or glucuronic acid.

– They are synthesized in the intestine from primary bile acids by deconjugation and 7α‐dehydroxylation. – They include: • Deoxycholic acid. • Lithocholic acid.

– Greater proportions of these bile acids are reabsorbed from the intestine to the liver again and then are re‐excreted in the bile forming enterohepatic circulation of bile acids. – The non‐reabsorbed bile acids are excreted in foeces.

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34

Fatty Liver

Lipotropic Factors

• Fatty liver is the accumulation of excessive amounts of lipids (mainly triglycerides)in the liver. • The lipid content of the normal liver is about 4% of which only about ¼ are triglycerides. • In severe cases of fatty liver, the lipid content may reach up to 40% of the liver weight. • In prolonged conditions, liver cells die and become fibrosed leading to liver cirrhosis and fibrosis with impaired liver function. • Causes:

• Lipotropic factors are the factors which help the mobilization of triacylglycerols from the liver. • Deficiency of these factors leads to undermobilization of fat from the liver to the blood with the development of fatty liver. • Lipotropic factors include:

1. 2. 3.

– A. Substances important for the biosynthesis of phospholipids: • • • • •

Overmobilization of fat from extrahepatic tissue to the liver. During high carbohydrate diet. Undermobilization of fat from the liver to the blood.

Essential fatty acids. Inositol. Choline Amino acids including methionine and serine. Vitamins including vitamin B12 and folic acid.

– B. Substances important for the biosynthesis of proteins: • Proteins of high biological value providing essential amino acids.

35

36


Lecture Outline • •


Special Tests in Clinical Chemistry

Protein Electrophoresis • Special Proteins – Human Immunoglobulins (IgG, IgA and IgM) – Apolipoproteins – Haptoglobulin – α1‐Acid Glycoprotein – Cystatin C – Urinary Microalbumin – β2‐Microglobulin – α1‐Microglobulin – α2‐Macroglobulin – Prealbumin

Non‐routine investigations in Clinical Chemistry: – Glucose‐6‐Phosphate Dehydrogenase (G6PDH) – Urinary 17‐Ketosteroids – Urinary 5‐Hydroxy Indole Acetic Acid (5‐HIAA) – Urinary Vanilyl Mandelic Acid (VM) – Urinary Porphyrins and Porphobilinogens – Neutrophil gelatinase‐associated lipocalin (NGAL) – Nephrolithiasis (Urinary Calculi) – Gallstone 2

Protein Electrophoresis

Protein Electrophoresis (continued)

• Electrophoresis is the migration of charged particles in a liquid medium under the influence of an electric field. • A charged particle placed in an electrical field migrates towards the anode (+) or cathode (‐), depending on the net charge carried by the particle. The rate of migration in a porous medium varies with its net charge and the strength of the electrical field.

Using Protein Electrophoresis, 5 main groups of proteins can be distinguished: • Albumin. • Alfa‐1 globulin. – Alfa‐1‐antitrypsin – Alfa‐1‐glycoprotein – Alfa‐lipoproteins

• Alfa‐2 globulin. – Alfa‐2‐macroglobulin – Haptoglobulin – Ceruloplasmin

• Beta globulin. – Beta‐lipoproteins – Transferrin – Several components of the complement system.

• Gamma globulin.

3

Special Proteins I. Human Immunoglobulins (IgG, IgA and IgM) II. Apolipoproteins III. Haptoglobulin IV: α1‐Acid Glycoprotein V: Cystatin C VI: Urinary Microalbumin VII: β2‐Microglobulin VIII: α1‐Microglobulin IX: α2‐Macroglobulin X: Prealbumin 5

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4

I: Human Immunoglobulins (IgG, IgA and IgM) Human IgG Subclasses 1 – 4

I: Human Immunoglobulins (IgG, IgA and IgM): • Immunoglobulins are formed by plasma cells as a humoral immune response to contact of the immune system with antigens. • The primary reaction after the initial contact is the formation of antibodies of the IgM class followed later by IgG and also IgA antibodies. • Quantitative determination of the immunoglobulins can provide important information on the humoral immune status. • Decreased serum immunoglobulin concentrations occur in :

•

Diminished IgG2 concentrations occurs in :

•

Diminished IgG1 and IgG2 concentrations occurs in:

•

Diminished IgG3 concentrations occurs in :

•

Diminished IgG4 concentrations occurs in:

•

Changes in the concentrations of IgG subclasses have also been reported in patients with autoimmune diseases, neurological syndromes and HIV infection.

•

– General immunodeficiency

– Primary immunodeficiency conditions – Secondary immune insufficiencies e.g. in advanced malignant tumours, lymphatic leukaemia, multiple myeloma and Waldenstrom’s disease.

– Infections of the upper airways and in bronchopulmonary infections.

• Increased serum immunoglobulinconcentrations occur in : – Polyclonal or oligoclonal Ig proliferation, e.g. in hepatic diseases (hepatitis, liver cirrhosis), acute and chronic infections, autoimmune diseases as well as in the cord blood of neonates with intra‐uterine and perinatal infections. – Monoclonal immunoglobulin proliferations in the serum are found e.g. in plasmacytomas, Waldenstrom’s disease and heavy‐chain disease. – Local immune reactions result in elevated immunoglobulin levels, particularly IgG, in the cerebrospinal fluid. – Elevated urinary concentrations of IgG are found in patients with non‐ selective glomerular proteinuria.

•

The human IgG antibodies are composed of the four subclasses IgG‐1, IgG‐2, IgG‐ 3, IgG‐4. The differences between the IgG subclasses are reflected in different, biologically important functions such as antigen recognition, complement activation and cell surface receptor binding. Diminished IgG1 concentrations occurs in :

•

– Nephrotic syndrome, but particularly in minimal change nephritis. – Virus‐related infections of the urinary tract. – Patients with chronic bronchopulmonary diseases and bronchiectases

7

8

II: Apolipoproteins

II: Apolipoproteins

Apolipoprotein A‐II: • Apolipoprotein A‐II is a structural protein of HDL which has phospholipid‐binding properties. • Apo A‐II consists of two identical protein chains linked by a disulfide bridge, each chain containing 77 amino acids. • The dimeric molecule has a molecular weight of 17 440 daltons. Genetically‐related structural variants of Apo A‐II have not been reported. • The human plasma contains different isoforms of Apo AII with similar molecular weights and immunological properties but with different isoelectric points. • The synthesis site of Apo A‐II is the liver. • The percentage concentrations of Apo A‐II in the serum lipoproteins are: chylomicrons (traces), VLDL (traces), LDL (traces), HDL2 (10 %), HDL3 (23%). • The half life of Apo A‐II in the blood amounts to 4 ‐ 5 days. • Studies performed in patients with coronary heart disease or myocardial infarctions were unable to establish a clear correlation between the plasma concentrations of Apo A‐II and coronary risk. • In some centres Apo A‐II is measured in order to obtain information on the HDL2 and HDL3 fractions from the ratio of Apo A‐I to Apo A‐II. • Diminished Apo A‐II concentrations have been found in Tangier disease (a very rare Apo A‐I defect), in patients with a high consumption of cigarettes and in hepatic insufficiency. • Elevated Apo A‐II concentrations have been found in cases of high alcohol consumption.

Apolipoprotein A‐I: • Apolipoprotein A‐I is the main protein component of high density lipoprotein (HDL) and accounts for approximately 65% of the total protein content of HDL. • Apo A‐I activates lecithin cholesterol acyltransferase which catalyses the esterification of cholesterol. • The resulting esterified cholesterol can then be transported to the liver, metabolized and excreted. • Persons with atherosclerotic vascular changes frequently exhibit decreased levels of Apo A‐I. • Decreased concentrations of Apo A‐I also occur in dyslipoproteinaemias, acute hepatitis, hepatic cirrhosis and in insulin‐treated diabetics. 9

II: Apolipoproteins

II: Apolipoproteins

Apolipoprotein B: • Apolipoprotein B is the main protein component of low density lipoprotein (LDL) and accounts for approximately 95% of the total protein content of LDL. • Apo B is necessary for the reaction with LDL receptors in the liver and on cell walls and is thus involved in transporting cholesterol from the liver to the vessel cell. • Elevated levels of Apo B are frequently found in patients with atherosclerotic vascular changes and are a risk factor for atherosclerosis.

Apolipoprotein E • Apolipoprotein E has a molecular weight of 34 000 daltons (299 amino acids) and is synthesized in the liver. • Approx. 10 ‐ 20 % of the total protein in VLDL consists of Apo E. • Elevated Apo E concentrations indicate raised levels of the highly atherogenic degradation products of chylomicrons and VLDL ("remnants"). 11

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10

III: Haptoglobin

IV: α1‐Acid Glycoprotein •

• Haptoglobin binds hemoglobin which is released during erythrocyte lysis. The haptoglobin/hemoglobin complex is rapidly eliminated from the bloodstream. • Increased release of hemoglobin due to intravascular hemolysis results in a reduction in the haptoglobin concentrations, and during severe hemolysis, to complete consumption of the haptoglobin. • In children haptoglobin has lower physiological serum concentrations and therefore is not suited for hemolysis testing. • Haptoglobin is an acute‐phase protein which can develop very high serum levels during inflammatory conditions.

•

•

• •

•

13

V: Cystatin C • •

•

•

• • •

14

VI: Urinary Microalbumin

Cystatin C or cystatin 3 is a cysteine proteinase inhibitor mainly used as a biomarker of kidney function. In humans, all cells with a nucleus produce cystatin C as a chain of 120 amino acids. It is found in virtually all tissues and bodily fluids. It is a potent inhibitor of lysosomal proteinases and probably one of the most important extracellular inhibitors of cysteine proteases. Cystatin 3 has a low molecular weight (~13.3 KD). –

α 1‐acid glycoprotein is a plasma glycoprotein with a carbohydrate content of approx. 40 %. In healthy persons differences in its serum levels can be found within the reference interval. In particular, compared to men, lower concentrations occur in women of childbearing age, on oral contraceptives and during pregnancy. As an acute‐phase protein, the serum levels of α 1‐acid glycoprotein are elevated during infections as well as acute and chronic inflammatory processes (e.g. Crohn's disease). In these cases a highly sensitive assessment of the condition of the patient can be obtained by preparing a prognostic index of α 1‐acid glycoprotein and other parameters such as CRP. Patients with injuries, burns or tumours exhibit elevated serum concentrations. Patients with chronic renal failure are found to have high serum concentrations of α 1‐acid glycoprotein, with no major difference reported between dialyzed and non‐dialyzed patients. Diminished serum concentrations due to restricted production of α 1‐acid glycoprotein are found in patients with chronic liver diseases. Low serum concentrations due to increased excretion of the protein are associated with nephrotic syndrome.

• • •

Due to its small size it is freely filtered by the glomerulus, and is not secreted but is fully reabsorbed and broken down by the renal tubules. This means the primary determinate of blood Cystatin C levels is the rate at which it is filtered at the glomerulus making it an excellent GFR marker.

•

Serum levels of cystatin C are a more precise test of kidney function than serum creatinine levels. Cystatin C levels are less dependent on age, sex, race and muscle mass compared to creatinine. Cystatin C is an alternative and more sensitive endogenous marker for the estimation of GFR than serum creatinine and serum creatinine based GFR estimations. Cystatin C can be used as a marker of kidney function in the adjustment of medication dosages. Cystatin C can be measured in a random sample of serum using immunoassays such as nephelometry or particle‐enhanced turbidimetry.

•

Normally, albumin is not present in urine because it is filtered from the bloodstream by the kidneys. Microalbuminuria occurs when there is an abnormally high permeability for albumin in the renal glomerulus. Microalbuminuria cannot be detected by urine dipstick methods but there is specific Microalbumin urine test to determine the presence of the albumin in urine. Microalbuminuria is diagnosed from elevated concentrations (30 to 300 mg/L) on at least two occasions. An albumin level above these values is called "macroalbuminuria", or just albuminuria. To compensate for variations in urine concentration in spot‐check samples, the albumin/creatinine ratio (ACR) is calculated. –

•

Microalbuminuria is defined as ACR ≥2.8 mg/mmol (male) or ≥2.0 mg/mmol(female).

The significance of microalbuminuria test is: • An indicator of subclinical cardiovascular disease. • Marker of vascular endothelial dysfunction. • An important prognostic marker for kidney disease – In diabetes mellitus. – In hypertension. • Increasing microalbuminuria during the first 48 hours after admission to an intensive care unit predicts elevated risk for acute respiratory failure, multiple organ failure, and overall mortality.

15

VII: β2‐Microglobulin

VIII: α1‐Microglobulin

• β2‐microglobulin has a molecular weight of 11,800 daltons and occurs on all nucleated cells as a component of the HLA complex. • It is constantly released into the blood in small quantities. As it is freely filtered and reabsorbed in the kidney the serum levels found in healthy persons remain at a consistently low level whereas the urine is found to contain almost no β2‐microglobulin. • An increase in the release of β2‐microglobulin (due to increased activity of the immune system, cell death) or diminished elimination (due to glomerular renal damage) leads to a rise in the serum concentration. • The serum concentration of β2‐microglobulin is thus a sensitive marker for the glomerular filtration capacity of the kidney. • Chronic inflammations and autoimmune diseases such as SLE, rheumatoid arthritis and Sjögren’s syndrome are also associated with elevated levels.

• The clinical relevance of the α1‐microglobulin assay relates to the identification of tubular proteinurias. • α1‐microglobulin, also referred to as protein HC, is a low‐molecular weight glycoprotein (33 KD), the free proportion of which is quantitatively filtered through the glomeruli. As with other plasma proteins which are filtered through the glomeruli, reabsorption and catabolism take place in the proximal tubules. • Detection of elevated urinary concentrations of α1‐ microglobulin can be indicative of tubular damage as may occur in the context of nephritides, advanced diabetic nephropathy, after exposure to heavy metals or after administration of nephrotoxic medicaments. • Detection of elevated urinary concentrations of α1‐microglobulin patients with urinary tract infections is indicative of renal involvement.

17

16

18

283

IX: α2‐Macroglobulin • • • • • • • • •

X: Prealbumin

α2‐macroglobulin is a proteinase inhibitor which exerts a particular effect on endopeptidases. It transports hormones and enzymes, exhibits effector and inhibitor functions in the development of the lymphatic system and inhibits components of the complement system and haemostasis system. The reference values are slightly higher in women than in men. In hyperfibrinolytic states, after major surgery, in septicaemia and severe hepatic insufficiency the measured levels of α2‐macroglobulin are often low. Patients with acute pancreatitis exhibit low serum concentrations which correlate with the severity of the disease. Acute myocardial infarction patients with low α2‐macroglobulin concentrations have a significantly better prognosis. The assay of α2‐macroglobulin is of major significance for the differential diagnosis of nephrotic syndrome. Here, an elevated α2‐macroglobulin/albumin ratio is indicative of post‐renal haematuria. In patients with liver cirrhosis and diabetes the levels of α2‐macroglobulin are found to be elevated.

• Prealbumin acts as a binding protein for thyroxin, and RBP are the transport protein for retinol (vitamin A). • Prealbumin and retinol‐binding prptein (RBP) are synthesized in the liver. • The serum concentrations of both proteins reflect the synthesis capacity of the liver and are markedly diminished in malnutrition and other conditions. • Due to their short half lives of approx. two days and twelve hours respectively prealbumin and RBP may be suitable for monitoring the nutritional status and efficacy of parenteral nutrition. 19

20

Glucose‐6‐Phosphate Dehydrogenase (G6PDH) (continued)

Glucose‐6‐Phosphate Dehydrogenase (G6PDH)

• Important for the integrity of red blood cells through the production of reduced coenzyme II (NADPH + H+). Most of the interest of G6P‐DH focuses on its role in the erythrocyte. Here, it functions to maintain NADPH in its reduced form. An adequate concentration of NADPH is required to regenerate sulfhydryl‐containing proteins such as glutathione form the oxidized to the reduced state. Glutathione in the reduced form, in turn, protects hemoglobin from oxidation by agents that may be present in the cell.

• G6PDH is the main enzyme of hexose monophosphate (HMP) shunt pathway.

22

21

Glucose‐6‐Phosphate Dehydrogenase (G6PDH) (continued)

Urinary 17‐Ketosteroids

G6PDH Deficiency •

•

•

•

•

May cause haemolytic anaemia called Favism – precipitated by administration of certain oxidizing agents such as Fava beans, anti‐malarial drugs, sulpha drugs, phenylbutazone and vitamin K analogues. Deficiency results in an inadequate supply of NADPH – Inability to maintain reduced glutathione levels. – When erythrocytes are exposed to oxidizing agents, hemolysis occurs because of oxidation of hemoglobin and to damage of the cell membrane. Deficiency is an inherited sex‐linked trait. – Full expression of trait occurs in hemizygous males where the single X chromosome carries the mutant gene and in homozygous females where both sex chromosomes (XX) carry a mutant gene. – Intermediate expression is found in heterozygous females where expression is variable. • Female heterozygotes have two populations of red cells: one with normal and the other with deficient enzyme activity. The proportion of the two populations in different heterozygotes results in G6PDH activities which may vary from almost normal to those found for hemizygotes – Synthesis is induced at the age of 9 – 11 years. The disorder can result in several different clinical manifestations, eg drug‐induced hemolytic anemia. When exposed to an oxidant drug such as primaquine, an antimalarial drug, affected individuals experience a hemolytic episode. Research suggests that the deficiency confers some protection against Falciparum malaria. – May lessen the severity of malarial infections in young children and infants.

• They are breakdown products of adrenal androgens in both males and females. • These steroids include androstenedione, androsterone, estrone, and dehydroepiandrosterone. • The reference range of urinary 17‐ketosteroids is 8‐20 mg/day in males and 6‐12 mg/day in females. • Increases in levels of 24‐hour urinary 17‐ketosteroids are associated with adrenal tumours, cushing syndrome, ovarian cancer, testicular cancer and polycystic ovarian syndrome. • Decreased levels are associated with Addison disease, hypopituitarism, myxoedema, nephrosis. 23

24

Urinary 5‐Hydroxy Indole Acetic Acid (5‐HIAA)

Urinary Vanilyl Mandelic Acid (VM)

• 5‐HIAA is the excretory metabolite of serotonin. • It is most frequently performed for the diagnosis of carcinoid tumors of the enterochromaffin cells of the small intestine, which release large amounts of serotonin. • Values more than 25 mg/day are strong evidence for carcinoid. The normal range is 2 to 6 mg/day.

• • • • •

Vanilyl Mandelic acid (VMA) is the metabolite of catecholamine measured in urine of patients suffering from pheochromocytoma; a benign adrenal tumor. Measurement of VMA is important as because even small adrenal tumor secrete large amount of catecholamine. Catecholamine secretion is usually paroxysmal, so the measurement in 24‐hour urine sample is more useful than single plasma measurement for screening. No particular restriction in the diet preceding the urine collection is required, as the blank sample subtracts dietary vanillin, which may give falsely high values. Some drugs influence the VMA urinary excretion: – Increased values result from administration of insulin, reserpine, epinephrine, norepinephrine – Decreased values result from administration of morphine, pentobarbital, chloropromazine, iproniazid.

• •

The VMA/creatinine ratio in urine allows performing the test on a single micturition and may give quite precise results, within certain limits, for screening purposes. However, 24 hour urine VMA is preferred and it is required when the VMA/creatinine ratio is close or lightly beyond normal values limit.

25

26

Urinary Porphyrins and Porphobilinogens

Neutrophil gelatinase‐associated lipocalin (NGAL)

• These are intermediates in haemoglobin synthesis. They are excreted in patient’s urine of porphyrias which are group of diseases, resulting from deficiency of one or more of the enzymes involved in heme synthesis. • Urine porphyrins are useful for the evaluation of cutaneous photosensitivity to exclude porphyria cutanea tarda. • Urine porphobilinogen is useful for the evaluation of neurologic and/or psychiatric symptoms to exclude acute porphyrias such as acute intermittent porphyria.

• NGAL is a protein of a small molecular weight (25 kD), belonging to the lipocalin superfamily initially found in activated neutrophils. It is also found in certain epithelia, such as renal tubules, where its expression is dramatically increased in ischemic or nephrotoxic injury. • NGAL is a promising biomarker for early detection of acute kidney injury (AKI). It is specifically released by the damaged kidney and can be detected in both urine and plasma. • Either alone or in combination with other biomarkers they will not only have an impact on medical decisions in future daily clinical routine, but they will also provide the basis for testing novel emergency therapies for a disease that is often recognized too late. 27

28

Neutrophil gelatinase‐associated lipocalin (NGAL) – Clinical Application

Nephrolithiasis (Urinary Calculi)

• Creatinine is not useful for early diagnosis. • Urinary NGAL can be used as a marker for the early diagnosis of AKI. • NGAL may be used to detect AKI early in the following cases: – Pediatric and adult cardiopulmonary bypass operations. – Percutaneous coronary interventions (PCI). – Critically ill patients presenting at the emergency department or in the intensive care unit (heart failure, sepsis, multi‐organ failure) – Renal transplantation. – Patients with chronic kidney disease.

• Condition characterized by the presence of renal calculi (stones). • Due to nutritional, environmental or genetic factors. • Urinary calculi occur in kidneys, the ureters or the bladder. They may be: – Single stones which may be composed of any of: calcium oxalate, uric acid, calcium carbonate, calcium phosphate or magnesium ammonium phosphate (triple phosphate). – Mixed stones which may be composed of two or more of the mentioned constituents. – Cystine or xanthine stones which are rare and found in the inherited metabolic abnormalities: cystinuria or xanthinuria respectively. • Both qualitative and quantitative analyses of the chemical constituents of kidney stones may be useful in establishing the aetiology and in planning adequate therapy. • Radiological examinations are required to explore the degree of intrarenal calcification and papillary damage.

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Gallstone

Summary •

Gallstones are solid concretions that are most commonly formed in the gallbladder and occasionally in the bile ducts.

•

There are three major types of gallstones: 1. Cholesterol gallstones: •

2.

They can be examined and detected by the Liebermann‐Burchard reaction.

Pigmented gallstones: • •

3.

The pigment is mainly bilirubin which is present as calcium bilirubinate. They can be examined and detected by using Ehrlich diazo reagent.

•

Mixed gallstones: •

They may contain a mixture of cholesterol, bilirubin, calcium phosphate, calcium carbonate and mucoproteins.

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286

Electrophoresis is the migration of charged particles in a liquid medium under the influence of an electric field. Using Protein Electrophoresis, 5 main groups of proteins can be distinguished. Special Proteins – VI: Urinary – I. Human Immunoglobulins (IgG, IgA and IgM) Microalbumin – II. Apolipoproteins – VII: β2‐Microglobulin – III. Haptoglobulin – VIII: α1‐Microglobulin – IV: α1‐Acid Glycoprotein – IX: α2‐Macroglobulin – V: Cystatin C – X: Prealbumin Non‐routine investigations are done in Clinical Chemistry, that include: – Glucose‐6‐Phosphate Dehydrogenase (G6PDH) – Urinary 17‐Ketosteroids – Urinary 5‐Hydroxy Indole Acetic Acid (5‐HIAA) – Urinary Vanilyl Mandelic Acid (VM) – Urinary Porphyrins and Porphobilinogens – Neutrophil gelatinase‐associated lipocalin (NGAL) – Nephrolithiasis (Urinary Calculi) – Gallstone

32


Body Fluids Body fluids include the following: 1. Blood. 2. Urine. 3. Cerebrospinal fluid (CSF). 4. Milk. 5. Seminal fluid. 6. Pleural Fluid. 7. Pericardial. 8. Peritoneal (Ascetic) fluid. 9. Synovial fluid. 10. Amniotic fluid.


Body Fluids 2

Body Fluids

Urine

Biochemically, the following body fluids are discussed in this lecture:

• Urine is a typically sterile fluid secreted and then excreted through a process called micturition (urination) by the urinary system which is the main excretory system in the human body especially for excreting water‐ soluble chemicals from the body. • These chemicals can be detected and analyzed by urinalysis. • Certain diseases can result in pathogen‐contaminated urine.

1. 2. 3. 4. 5. 6. 7.

Urine. Cerebrospinal fluid (CSF). Pleural Fluid. Pericardial. Peritoneal (Ascetic) fluid. Synovial fluid. Amniotic fluid.

Clinical significance of urine examination: 1. Diagnosis and management of renal or urinary tract diseases. 2. Detection of metabolic or systemic diseases not directly related to the kidney.

3

4

Urine

Urine

Collection of urine samples: • Single specimens of urine (random or morning samples) are used for ward examinations and qualitative tests. • Double‐voided specimen is the urine excreted during a time period after complete emptying of the bladder by 15 ‐ 30 min. • 24 hours‐urine collections are preferred for quantitative tests due to the diurnal variation in the excretion of some substances. • The 24 hours‐collection is as follows:

Storage of urine samples: • It is satisfactory in most cases to use specimens collected in cool, clean containers. • Urine sample can be stored for about one week in the refrigerator at 2 – 8°C. • Urine samples can be stored for many months at ‐20°C without any addition. • Concentrated hydrochloric acid (HCl), thymol or chloroform can be used for urine storage. • Acid should not be used for storage of proteins, creatinine and steroids determination.

– At a suitable time (e.g. 8 :00am), the patient empties his bladder and the urine is discarded. – All urine passed during the following 24 hours is saved in specific container. – At the same time of the next morning, the patient empties his bladder and the urine is added to the collected one.

•

N.B.: Creatinine level of the urine sample can be used as a rough check on the reliability of the collection to exclude adulteration of the urine sample..

5

6

287

Physicochemical Examination by Urine Strips • • •

Physicochemical Examination by Urine Strips

Urine strips are dry reagent strips which are plastic supports containing one or more chemically impregnated test sites on an absorbent pad. When the strips come in contact with urine, the reagents are activated and a chemical reaction occurs. The chemical reaction is a specific colour change, which may be:

Urine strips contain one or more of the following tests: • pH: 5 ‐7 • Specific Gravity: 1015 ‐ 1025 • Ascorbic Acid: Negative • Nitrites: Negative • White blood cells: Negative • Blood (Intact red blood cells and haemoglobin): Negative • Protein/Albumin: Negative • Glucose: Negative • Ketones/Acetone: Negative • Bilirubin: Negative • Urobilinogen: Trace

– Observed visually and compared with a special colour chart. Or – Measured electronically (usually by reflectance) by an instrument designed to be used with the reagent strips.

• •

The intensity of the colour formed is proportional to the amount of substance present in the specimen when observed at a specific time. Some test areas are used as: – Screening tests which are reported as positive or negative. Or – Semiquantitative estimation of the amount of certain substance present in the specimen. These are reported in a plus system or in international values (e.g. mg/dl, g/dl, mmol/l).

7

8

Urine Chemical Investigations


1.

4.

Proteins: o o

2.

Albumin (Microalbumin): o o o

3.

Creatinine clearance: o It is the practical and most convenient method of obtaining an acceptable accurate estimation of glomerular filtration rate (GFR). o It ranges from 90 to 130 ml/min in males and from 80 to 120 ml/min in females. 5. Minerals and electrolytes: o They include calcium, phosphorus, sodium, potassium, chloride and bicarbonate. 6. Osmolality: o It indicates the number of particles of solute per unit of solution and reflects the relative degree of concentration or dilution of a urine specimen. o The normal adult with a normal fluid intake will produce a urine of about 300‐900 mOsm/kg water.

Normally, protein in urine is up to 150 mg/day. Abnormal gamma globulin (Bence‐Joones protein) can be detected in the urine of multiple myeloma patients. It is diagnosed from elevated concentrations (30 to 300 mg/L) on at least two occasions. An albumin level above these values is called "macroalbuminuria", or just albuminuria. Values below 30 mg/L indicate negative microalbuminuria.

Non‐protein nitrogenous (NPN) compounds: o

These compounds include urea (20 – 40 g/day), uric acid (About 1.0 g/day), creatine (0.0 – 0.2 g/day), creatinine (About 1.5 g/day), ammonia (About 0.7 g/day) and amino acids (150 – 200 mg/day).

9

10



7.

10. 17‐Ketosteroids: o They are breakdown products of adrenal androgens in both males and females. o These steroids include androstenedione, androsterone, estrone, and dehydroepiandrosterone. o The reference range of urinary 17‐ketosteroids is 8‐20 mg/day in males and 6‐12 mg/day in females. o Increases in levels of 24‐hour urinary 17‐ketosteroids are associated with adrenal tumours, cushing syndrome, ovarian cancer, testicular cancer and polycystic ovarian syndrome. o Decreased levels are associated with Addison disease, hypopituitarism, myxoedema, nephrosis.

Neutrophil gelatinase‐associated lipocalin (NGAL): o It is a promising biomarker for early detection of acute kidney injury. o It is specifically released by the damaged kidney and can be detected in both urine and plasma. 8. Vanilyl Mandelic Acid (VMA): o VMA is one of catecholamines metabolites. o Urinary excretion of VMA is a monitor of adrenal medullary function, and is increased in pheochromocytoma (benign medullary tumour). 9. 5‐Hydroxy Indole Acetic Acid (5‐HIAA): o 5‐HIAA is the excretory metabolite of serotonin. o It is most frequently performed for the diagnosis of carcinoid tumors of the enterochromaffin cells of the small intestine, which release large amounts of serotonin. o Values more than 25 mg/day are strong evidence for carcinoid. The normal range is 2 to 6 mg/day. 11

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288


Cerebrospinal Fluid (CSF)

11. Porphyrins and Porphobilinogens:

•

o

o o

These are intermediates in haemoglobin synthesis. They are excreted in patient’s urine of porphyrias which are group of diseases, resulting from deficiency of one or more of the enzymes involved in heme synthesis. Urine porphyrins are useful for the evaluation of cutaneous photosensitivity to exclude porphyria cutanea tarda. Urine porphobilinogen is useful for the evaluation of neurologic and/or psychiatric symptoms to exclude acute porphyrias such as acute intermittent porphyria.

• •

•

It provides a fluid cushion to protect the brain and spinal card. It carries nutrients and removes waste products. It maintains the pressure inside the head and around the spinal cord. It is the pathway whereby hypothalamus releasing factors are transported to the cells of the median eminence. – It maintains central nervous system ionic homeostasis. – – – –

12. Drugs and Toxicological Tests: o o

Cerebrospinal fluid (CSF) is a fluid contained within the membranous coverings of the spinal cord and brain within the space known as the subarachnoid space. Most CSF in formed by the choroid plexuses of the brain ventricles. It is produced by ultrafiltration and active secretion of the blood plasma. CSF is constantly produced (250 – 750 ml / day), but it is also constantly reabsorbed from the subarachnoid space into the blood stream, hence the volume remains constraint. Functions:

These tests are performed to monitor the therapeutic level of the drugs and to detect any toxicological doses. There are also urinary screening tests for certain addictive drugs

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14

Cerebrospinal Fluid

Cerebrospinal Fluid

Characteristics of CSF: – Volume: 50 – 150 ml. – pH: 7.4 – Aspect: Clear – Color: Colourless

Chemical Composition:

CSF supernatant color Pink Yellow Orange Yellow‐green Brown

– – – – –

Associated diseases/disorders RBC lysis/hemoglobin breakdown products RBC lysis/hemoglobin breakdown products Hyperbilirubinemia CSF protein > 150 mg/dL (1.5 g/L) RBC lysis/hemoglobin breakdown products Hypervitaminosis A (carotenoids) Hyperbilirubinemia (biliverdin) Meningeal metastatic melanoma

Glucose : 50 ‐ 70 mg/dl. Proteins: 15 ‐ 45 mg/dl. Lipids: Cholesrerol (about 0.4 mg/dl). NPN: as urea (20 ‐40 mg/dl). Minerals: Na+, K++, Mg++, and Phosphorus.

15

16

Specimen Collection • •

•

• • • •

CSF Chemical Investigations

Cerebrospinal fluid may be obtained by lumbar, cisternal or lateral cervical puncture or through ventricular cannulas or shunts. Up to 20 ml of CSF can be safely removed from an adult although this amount is not usually required. Clinician should provide clinical history to the laboratory. The puncture site (i.e., lumbar, cisternal, etc.) should be noted since cytologic and chemical parameters vary at different sites. 3 – 4 ml of CSF are allowed to drip into 3 plain tubes; the first tube should be used for chemical tests, the second for microbiological tests, and the third for microscopic and cytological examination. Highly bloody sample for cell count should be collected in EDTA tube to prevent formation of clot. Glass tubes should be avoided since cell adhesion to glass affects the cell count. Specimen should be delivered to the laboratory and processed immediately to minimize cellular degradation, which begins within 1 hour of collection. Refrigeration is contraindicated for culture specimens because organisms like Haemophilus influenzae and Neisseria meningitidis will not survive.

1. Proteins (total and specific): • Over 80% of CSF Protein content is derived from the plasma, in concentrations less than 1% of their blood level. • An increased CSF protein serves as a useful but nonspecific indicator of disease. Increased CSF protein (more than 45 mg/dl) may be caused by: – – – –

•

Low lumber CSF total protein levels (less than 15 mg/dl) occur in: – – – – –

•

17

Increased permeability of the blood‐brain barrier. Decreased reorption at the arachnoid villi. Mechanical obstruction of CSF flow due to spinal block above the site of puncture. Increase in intrathecal immunoglobulin (Ig‐G) synthesis as in multiple sclerosis and in conditions associated with lymphocytic and plasmacytic infiltrate of the central nervous system (CNS). Normally in children between 6 months and 2 years. Following removal of large volume of CSF. CSF Leakage by trauma or lumber puncture. Increased intracranial pressure. Hyperthyroidism.

Protein electrophoresis of concentrated normal CSF reveals two distinct differences from serum: prominent transthyretin band & extra transferrin band 18

289



2. Glucose : • CSF glucose results should be compared with plasma levels for clinical interpretation. So, the necessity for simultaneous plasma glucose should also be considered. This is best obtained 2‐4 hours before lumbar puncture because of the delay in plasma‐CSF equilibrium. The normal CSF /plasma glucose ratio varies from 0.3‐0.9. • CSF values below 40 mg/dl (2.2 mmol/L) or ratios below 0.3 are considered to be abnormal. Low glucose level is a characteristic finding of:

3. • • •

– – –

• •

Lactate: CSF lactate concentration is largely independent of blood glucose levels. The primary source of CSF lactate is CNS anaerobic metabolism. Any condition associated with tissue hypoxia of the CNS may cause increase in CSF lactate as in: – – – – – – – – –

Bacterial, tuberculous and fungal meningitis. Some cases of viral meningoencephalitis also have low glucose levels, but generally not to the degree seen in bacterial meningitis. Meningeal involvement by a malignant tumor, sarcoidosis, cysticercosis, trichinosis, amoeba (Naegleria), and acute syphilitic meningitis, intrathecal administration of radioiodinated serum albumin, subarachnoid hemorrhage, symptomatic hypoglycemia and rheumatoid meningitis may also produce low CSF glucose levels.

CSF glucose levels normalize before protein levels and cell counts during recovery from meningitis, making it a useful parameter in assessing response to treatment. Increased CSF glucose is of no clinical significance. It reflects increased blood glucose level.

Traumatic brain injury. Cerebral oedema. Intracranial haemorrhage. Cerebral infarct. Hydrocephalus. Brain abscess. Cerebral ischaemia. Metastatic neoplasm. Meningitis.

19

20



4.

6. Enzymes:

Chloride : – CSF chloride (Cl) reflects blood electrolyte only, but in tuberculous meningitis, a decrease of 25% may exceed the serum Cl decrease. – It is not useful in diagnosis of tuberculous meningitis. –

5.

– Lactate dehydrogenase (LDH): • LDH is useful in differentiating a traumatic tap from intracranial hemorrhage since a current traumatic tap with intact RBC’s does not significantly elevate the LDH level. • LDH activity is also significantly higher in:

Ammonia and Glutamine: – – – –

Ammonia levels in CSF are about one third that of arterial blood ammonia. There is relation between increased CSF ammonia and hepatic encephalopathy. Elevated ammonia levels lead to high CSF glutamine levels. Accordingly, increased CSF glutamine reflects increased brain ammonia. Elevated CSF glutamine occurs in hepatic encephalopathy, septic encephalopathy, & encephalopathy secondary to respiratory failure.

– –

Bacterial meningitis. Patients with CNS leukemia, lymphoma, metastatic carcinoma, bacterial meningitis, and subarachnoid hemorrhage.

– Creatine kinase (CK): • Assay of total CK activity in CSF has only limited value in clinical diagnosis because any changes obtained are irregular & often nonspecific. • CSF elevations can be found in: – –

Epileptic patients. Patients with brain tumour, cerebral infarcts cerebral haemorrhage or any cerebral damage.

• CSF CK‐BB increases after certain types of neurological injury. 21

22



6. Enzymes (continued):

Adult Lumbar CSF Reference Values

– Aspartate transaminase (AST) :

Analyte Protein Glucose Lactate Chloride Ammonia Glutamine LDH CK AST Cholinesterase

• Changes in AST are irregular and generally of limited diagnostic value. • AST elevations occur in : – Cases of large brain infarct during the first 10 days. – About 40% of CNS tumour. – Cerebrovascular accidents.

– Cholinesterase : • Cholinesterase activity is very low in CSF. • Its activity is increased in : – – – –

Brain tumours and brain abscess. Hydrocephalus. Meningitis. Multiple sclerosis.

23

Reference Interval 15 – 45 mg/dl 50 – 70 mg/dl 10 – 22 mg/dl 115 – 130 mmol/L 10 – 35 μg/dl 6 – 15 mg/dl ~10% of serum value 0 – 5 U/L 7 – 49 U/L 13 – 21 U/L 24

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Pleural Fluid

Pleural Fluid

• The pleural cavity is a potential space lined by mesothelium of the visceral and parietal pleura. • The pleural cavity normally contains a small amount of fluid that facilitates movement of the two membranes against each other. • Pleural fluid may be transudate or exudate. • Turbid pleural effusion may be due to septic or nonseptic inflammation, tuberculosis, rheumatoid disease or rheumatic fever. • Hemorrhagic effusions suggest malignancy, pulmonary infarct and trauma. • Chylous (milky) fluid is due to trauma or obstruction to the thoracic duct.

Specimen collection: • Thoracocentesis is indicated for any undiagnosed pleural effusion or for therapeutic purposes in patients with massive symptomatic effusions. • This collection/handling and often under testing or inappropriate testing is more common than with other body fluids. • Specimen is usually divided into three serially collected sterile tubes: tube 1 for chemical investigations; tube 2 for microbiologic examination; and tube 3 for total and differential cell count. • Highly bloody sample, for cell count, is better to be collected in EDTA tube to prevent clot formation.

25

26

Chemical Investigations on Pleural Fluid


1.

4.

Protein: o o

2.

o

Lactate Dehydrogenase (LDH) : o

5.

Glucose: o

3.

Proteins are of value in differential diagnosis of transudates (protein < 3 g/dl) or exudates (protein > 3 g/dl). Pleural fluid to serum proteins ratio of < 5 is with transudates while if > 5 is with exudates.

Lipids : o

Pleural fluid to serum glucose ratio under 0.5 may be found in tuberculosis, SLE, empyema and rheumatoid pleurisy. Low pleural fluid glucose may also be present in malignancy, tuberculosis, nonpurulent bacterial infections, lupus pleuritis, and esophageal rupture.

o

Lactate: o o o

Pleural fluid levels of lactate can be useful in the rapid diagnosis of infectious pleuritis. Levels are significantly higher in bacterial and tuberculous pleural infections than other pleural effusions. Values more than 90 mg/dl have a positive predictive value for infectious pleuritis of 94% and negative predictive value of 100%.

o

6.

High LDH level is associated with pneumonic effusion, rheumatoid pleurisy and some malignant effusions. Some serous effusions appear to be chylous (i.e., a milky appearance) but are not (pseudochylous) Lipid measurements are also helpful in identifying chylous effusions. Thus, pleural fluid triglyceride levels above 110 mg/dl indicate a chylous effusion. Nonchylous and pseudochylous effusions generally have triglyceride levels below 50 mg/dl. A total cholesterol value < 55 mg/dl are found in transudates, and > 55 mg/dl in exudates.

Tumour Markers : o

Combined CEA and CA‐125 have a sensitivity of 75 – 100% and specificity of 95% for detection of malignant effusions due to carcinoma of lung, heart, gastrointestinal tract and ovary.

27

28


Pericardial Fluid •

Laboratory Criteria for Pleural Fluid Exudate Pleural fluid/serum protein ratio Pleural fluid/serum LDH ratio

≥ 0.60

Pleural fluid LDH

≥ ⅔ upper limit of normal serum LDH

Pleural fluid cholesterol

> 45 mg/dl

Pleural fluid/serum cholesterol ratio Serum–pleural fluid albumin gradient Pleural fluid/serum bilirubin ratio

•

≥ 0.50

• •

•

≥ 0.30 •

≤ 1.2 g/dl ≥ 0.60

• 29

10 ‐ 50 ml of fluid is normally present in the pericardial space. Normal pericardial fluid is pale yellow and clear. Increased fluid in the pericardial cavity may be caused by inflammation, tumor or hemorrhage. Pericardial effusions are most often caused by viral infection. They may also develop as a result of bacterial, tuberculous or fungal infections, HIV infection, autoimmune disorders, renal failure, myocardial infarction, mediastinal injury, tumor, hemorrhage, due to the effects of various drugs or it may be idiopathic. Large effusions (>350 ml) are most often caused by malignancy or uremia or are idiopathic. Infection or malignancy typically produces turbid effusions, whereas effusions due to uremia are usually clear and straw colored. These and several other disorders may produce hemorrhagic effusions. A milky appearance suggests the presence of a chylous or pseudochylous effusion. 30

291

Pericardial Fluid

Clinical Investigations on Pericardial Fluid

Specimen Collection

1.

Protein: o Pericardial effusions are either transudates (protein < 3 g/dl) or exudates (protein > 3 g/dl). 2. Glucose: o It is decreased to less than 40 mg/dl in effusions due to bacterial or tuberculous infections, rheumatoid disease and malignancy. 3. Lactate Dehydrogenase (LDH): o A pericardial fluid LDH level more than 300 U/L is of significance to differentiate pericardial exudates from transudates. 4. Lipids: o Separation of true chylous from pseudochylous effusions may be facilitated by triglyceride and cholesterol measurements as well as lipoprotein electrophoresis for chylomicrons.

• Fluid is obtained either by pericardiotomy following limited thoracotomy, or by pericardiocentesis. • Specimen is usually divided into three serially collected sterile plain tubes: the first one is for chemical investigations; the second for microbiologic examination; and the third one for total and differential cell count. • Blood‐like fluid obtained by pericardiocentesis might represent a hemorrhagic effusion or inadvertent aspiration of blood from the heart. • For cell count, fluid is better to be collected in EDTA tube to prevent clot formation.

31

32

Peritoneal Fluid • • •

Chemical Investigations on Peritoneal Fluid

Up to 50 ml of fluid is normally present in this mesothelial lined potential space that represents the peritoneal cavity. Ascites is the pathologic accumulation of excess fluid in the peritoneal cavity. Abnormal fluid may be collected in the peritoneal cavity in certain pathological conditions.

1. Protein : o Peritoneal effusions are either transudates (protein < 3 g/dl) or exudates (protein > 3 g/dl). o Transudates occur in cases of congestive heart failure, hepatic cirrhosis and hypoproteinemia as in nephritic syndrome. o Exudates are frequent in infections due to tuberculosis, primary bacterial peritonitis, and secondary peritonitis e.g. in appendicitis and mesothelioma, trauma, pancreatitis and bile peritonitis e.g. ruptured gall bladder. o The serum–ascites albumin gradient, defined as the serum albumin concentration minus the ascitic fluid albumin concentration, is widely considered as the most reliable method to differentiate peritoneal transudates from exudates. o Ascites caused by portal hypertension has a gradient of at least 1.1 g/dl (transudate) whereas ascites produced by other causes has a gradient less than 1.1 g/dl (exudate).

Specimen collection: • Diagnostic paracentesis in performed in most patients with new ascitis, or if there is a change in the clinical picture of a patient with ascitis. A minimum of 30 ml is needed for complete evaluation. • Diagnostic peritoneal lavage (DPL) is no longer used routinely in the evaluation of abdominal trauma. • Specimen is usually divided into three serially collected sterile tubes: tube 1 for chemical investigations; tube 2 for microbiologic examination; and tube 3 (EDTA tube) for total and differential cell count. 33

34

Chemical Investigations on Peritoneal Fluid

Chemical Investigations on Peritoneal Fluid 6.

2. Glucose :

Lactate Dehydrogenase (LDH): o

o An ascetic fluid to serum glucose ratio < 1.0 may occur in patient with bacterial peritonitis.

3. Lactate:

7.

o Ascitic fluid lactate has been used with pH measurements to differentiate bacterial from uncomplicated ascites. o Sensitivity and specificity are approximately 90% using a cutoff value of about 62%. Malignant and tuberculous ascites are associated with elevated lactate.

Creatinine and Urea: o o

4. Amylase:

8.

o Enzyme activity is elevated in patients with acute pancreatitis, pancreatic trauma or pancreatic pseudocyst. The ratio of ascitic fluid to serum amylase is over 2.0.

9.

o 35

292

Ascitic fluid/serum bilirubin ratio over 1.0 suggests a ruptured gallbladder.

Cholesterol: o

o This measurement may be helpful in the differentiation of primary bacterial peritonitis from secondary bacterial peritonitis due to bowel perforation.

Measurement of creatinine and urea nitrogen is useful to differentiate between peritoneal fluid and urine. Elevated peritoneal fluid urea nitrogen and creatinine, in association with elevated serum urea but normal serum creatinine suggest urinary bladder rupture.

Bilirubin: o

5. Alkaline Phosphatase:

Enzyme activity is elevated in patients with spontaneous bacterial peritonitis and malignant effusions.

The ascitic fluid cholesterol has been used for differential diagnosis of uncomplicated ascites versus ascites caused by malignancy. A cutoff value of above 1.2 mmol/l provides a good sensitivity, specificity, positive and negative predictive value, and overall diagnostic accuracy for differentiating malignant from nonmalignant.

36

Synovial Fluid

Synovial Fluid

• Synovium refers to the tissue lining synovial tendon sheaths, bursae, and joints. • It is composed of one to three cell layers that form a discontinuous surface overlying fatty, fibrous or periosteal joint tissue. • Synovial fluid is ultrafiltrate of blood plasma combined with hyaluronic acid produced by the synovial cells. Small ions and molecules (e.g., Na+, K+, glucose, urea, etc.) readily pass into the joint space and are therefore similar in concentration to plasma while large molecules are absent or present in trace amounts. • Resorption of synovial molecules is by the lymphatics and is not size dependent. • Synovial fluid acts as a lubricant and provides nutrients for the avascular articular cartilage.

Specimen collection: • Joint fluid aspiration (arthrocentesis) should be performed by an experienced operator using good sterile technique. • The specimen should ideally be separated into three parts: 3‐ 10 ml into a sterile heparinized tube or syringe for microbiological studies, 2–5 ml in an anticoagulant tube (sodium heparin) for microscopic examination; and about 5 ml in a plain tube for chemical analysis.

37

38

Clinical Investigations on Synovial Fluid 1.

Chemical Investigations on Synovial Fluid 4.

Protein: o o

5.

2.

Glucose: o

o

3.

o o o o

LDH is elevated in rheumatoisd arthritis, gout, failed arthroplasties, and infectious arthritis.

The mean normal protein concentration is about is 1.0 ‐ 3.0 g/dl. With increasing inflammation, larger proteins (e.g., fibrinogen) enter the synovial space. Spontaneous clot formation may be detected in non‐anticoagulated specimen tubes.

Lipids: o

Proper interpretation of synovial fluid glucose values requires comparison with serum levels. The serum–synovia difference is less than 10 mg/dl in normal and many noninflammatory conditions. In septic arthritis, this difference ranges from 20 ‐ 60 mg/dl.

Lactate Dehydrogenase (LDH): o

Uric Acid: o o

The mean normal protein concentration is about is 1.0 ‐ 3.0 g/dl. With increasing inflammation, larger proteins (e.g., fibrinogen) enter the synovial space. Spontaneous clot formation may be detected in non‐anticoagulated specimen tubes.

In contrast to plasma, normal synovial fluid contains extremely low concentrations of lipids. Synovial fluid lipid abnormalities include: Rare cholesterol‐rich pseudochylous effusions associated with chronic rheumatoid arthritis. Lipid droplets associated with trauma. Rare chylous effusions seen in association with rheumatoid arthritis, systemic lupus erythematosus, filariasis, pancreatitis and trauma.

N.B.: In case of sticky sample, dilution of 1:1 should be done with normal saline and the result will be multiplied by 2. 39

40

Chemical Investigations on Synovial Fluid

Amniotic Fluid • • •

Reference Intervals for Synovial Fluid Constituents Constituent Total protein Hyaluronic acid Glucose Uric acid Lactate

Albumin Gamma‐globulin

Synovial fluid

Plasma

1 – 3 g/dl 55 – 70% 10 – 14% 0.3 – 0.4 g/dl

6 – 8 g/dl 50 – 65% 12 – 22%

70 – 110 mg/dl

70 – 110 mg/dl

2 – 8 mg/dl

2 – 8 mg/dl

9 – 29 mg/dl

9 – 29 mg/dl

• • •

• •

41

Amniotic fluid is the protective liquid contained by the amniotic sac of a pregnant female. It is clear pale yellow fluid with pH of about 7.2 and a specific gravity of 1.006 – 1.008. At very early stages the amniotic fluid is secreted by the amniotic cells, later most of it is derived from the maternal tissue fluid by diffusion across the amniochorionic membrane and from the placenta. The composition of the amniotic fluid changes with gestation age. In early pregnancy it is similar to maternal and fetal serum. 98‐99% of the amniotic fluid is water. A large number of dissolved substances such as creatinine, urea, bile pigments, renin, glucose, fructose, proteins (albumin and globulin), lipids, hormones (estrogen and progestrone), enzymes, minerals (Na+, K+, Cl‐). Some undissolved materials (such as some fetal epithelial cells) are suspended in it. During the second half of gestation its osmolarity decreases and is close to dilute fetal urine with added phospholipids and other substances from fetal lung and other metabolites.

42

293

Amniotic Fluid

Chemical Investigations on Amniotic Fluid

Specimen collection: • Amniotic fluid is collected by amniocentesis which is the aspiration of a small amount of amniotic fluid from the sac around the baby. • This is usually performed at 16 weeks in pregnancy. • A fine needle is inserted under ultrasound guidance through the mothers' abdomen into a pool of amniotic fluid.

1.

Lecithine/sphingomyelin ratio: o o o

2.

Bilirubin : o

3.

Alpha‐fetoprotein (AFP): o o o

o

5.

High levels of AFP in the amniotic fluid indicate the presence of a severe neural tube defect whereas low levels of alpha‐fetoproteins may indicate chromosomal abnormalities. It is more accurate than that in maternal serum screening. It has been measured as early as 8 weeks. It cannot be measured around 14 weeks because amniotic fluid AFP at that time may lead misdiagnosis of neural tube defects at that gestational age. Increased amniotic fluid AFP should be tested for foetal haemoglobin which is a sensitive marker of foetal blood contamination.

Chromosomal analysis of the cells: o o

It is a useful adjunct in the diagnosis of neural tube defects. This enzyme is relatively specific for neural tissue.

44

Chemical Investigations on Amniotic Fluid 4.

It indicates the degree of fetal red blood cell destruction, where abnormally high levels could indicate serious cases such as mother fetal blood incompatibility.

Acetylcholinesterase (AChE): o

43

This test predicts foetal lung maturity more reliably than it predicts immaturity. As the lung matures, the concentration of phospholipids (especially lecithin) increases since lecithin is the major lung surfactant. This test is done to assess the maturation of the fetal lungs, a ratio 3/1 indicates mature lungs and a ratio less than 3/1 indicates immature lungs.

This is usually performed during the second trimester, after increasing the number of foetal cells by culture. This analysis is done to achieve early diagnosis of both numerical and structural disorders.

45

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Blood Bank

295

Kingdom of Saudi Arabia – Ministry of Health General Directorate of Training and Scholarship Training Program for Health Institute Graduates Blood Bank

Donor Recruitment & Retention - Slide 39 – Importance of regular voluntary non remunerate blood donation With the rapid increase in the number of people with transfusion-transmissible infections, and in particular the human immunodeficiency virus (HIV) Whatever your job, your role in this process is extremely important and you need to develop the knowledge and skills to ensure that blood donation is safe, both for donors themselves and for the recipients of their blood. - Slide 58 and 59 – Donor Complaints Give some examples of donor complaints and explain how the quality system should deal with them. Emphasise the quality system aspect NOT the complaint The BTS must ensure that there are standard procedures for dealing with donor complaints no matter how trivial. All staff need to be trained to the standard procedures. The procedures should ensure that all complaints are investigated and where necessary, corrective and preventive action is taken. Hear, empathise, apologise, take responsibility for your actions - Slide 61 – Positive Outcomes Discuss how donors can contribute to the quality cycle Donors can often have insight into how they would like to be treated. Listen to any suggestions. When introducing new campaigns, awards and practices, one way of soliciting donor suggestions is to ask them to fill in a short questionnaire about the BTS activities.

Blood Grouping Discrepancies - Slide 33 – 1. Mixed-field Agglutination Mixed cell populations resulting from massive transfusion of another blood group such as an B individual receiving "O" red blood cell donor units since the transfusion center did not have enough B donor units. Bone marrow transplant patients may have both some of their original type of cells and the type of the bone marrow transplant. Weak subgroups of A3 traditionally give a mixed field reaction. Rarely the condition called chimerism due to intrauterine exchange of erythrocyte precursors between twins or 2 fertilized eggs fuse into one individual. You should try to find cause of mixed field agglutination before setting up blood to transfuse so be sure to check the patient's transfusion records and clinical history. If it appears to be a weak subgroup performed the tests discussed under Unexpected Anti-A - Slide 38 – Acquired B Galactosamine results from the deacetylating reaction, resembling D-galactose (found in Group B individuals). This sugar cross-reacts with the reagent anti-B, giving a weak reaction (but still technically it is “extra”). Patients should receive Group A units. Acquired B usually goes away when the condition resolves. Antibody Identification - Slide 31 – Guidelines DTR – delayed transfusion reaction (donor cells are sensitized with patient’s antibody)

Transfusion Transmitted Diseases - Slide 5 – Types of Blood Borne Pathogens Human Immunodeficiency Virus (HIV): viruses or bacteria that are carried in blood and cause disease in people. - Slide 8 – Hepatitis A (HAV) HAV- May not have symptom


-

-

-

-

Slide 15 – Hepatitis C (HCV) These persons are at risk for developing cirrhosis and liver cancer. Slide 16 – Hepatitis C Virus – Geographic Distribution HCV was discovered in 1989 and was soon recognized as the primary cause of post-transfusion non-A, non-B hepatitis. Slide 17 – Hepatitis C Virus – Mode of Transmission HCV is transmitted primarily through large or repeated percutaneous (i.e., passage through the skin) exposures to infectious blood, such as Injection drug use (currently the most common means of HCV transmission in the United States); Receipt of donated blood, blood products, and organs (once a common means of transmission but now rare in the United States since blood screening became available in 1992); Needlestick injuries in healthcare settings; Birth to an HCV-infected mother HCV can also be spread infrequently through sex with an HCV-infected person (an inefficient means of transmission); Sharing personal items contaminated with infectious blood, such as razors or toothbrushes (also inefficient vectors of transmission); Other healthcare procedures that involve invasive procedures, such as injections (usually recognized in the context of outbreaks) Slide 24 – Human Immuno-deficiency Virus (HIV) Drugs are available to reduce the viral load of the HIV virus and can prevent cross infection with other people. Slide 26 - 27 – Transmission of HIV and Incubation Period of HIV HIV was discovered in 1989 and was soon recognized as the primary cause of post-transfusion non-A, non-B hepatitis. Slide 50 – Compliance Control Methods All human blood and certain human bodily fluids are treated as known to be infectious for HIV, HBV, and other BBPs. Change PPE between patients and wash hands each time after removal of glove. Slide 51 – Compliance Control Methods Change gloves between tasks and procedures on the same patient and after contact with material that may contain a high concentration of germs. Change gloves if they become damaged or torn. Wash hands or use alcohol gel immediately after removing gloves. Always change gloves between patients. Slide 53-54 – Compliance Control Methods Don’t take food and drink in work areas. Take care to minimize splashing of all materials. Cover any open cuts, scrapes, rashes and broken skin. Don’t touch anything that’s contaminated, such as sharps or body fluids.

Screening & Confirmatory - Slide 35 – Thick blood film The blood smear must be thick enough to just be able to make out the print on a newspaper (or similar) when the slide is placed on top of that piece of paper.

NAT Types - Slide 13 – Window Period Window Period: Infection to Detection This information comes from Model Testing based on Sero Conversion Panels. Note the significant differences in NAT versus Ab or Ag for HIV and HCV HBV takes longer to replicate and therefore the difference is not quite as dramatic, yet it is significant.

297


-

Slide 19 - Published Yield Cases - Demonstrated NAT Yield in Developing Countries (Ekiaby et al. 2010) Kehua – Manual extraction, single-lex using ABI 7300 Slide 27 – Mechanisms for OBI occurrence Need to define OBI for students

298


Introduction • The proper donor selection is the first line of defense against the transfusion transmitted diseases. • The primary responsibility of a blood bank is to attract blood donors and to ensure their return for continuous donation. • Blood substitutes are meant to be used on a short‐term basis and are not meant to be replacement for blood. • Volunteer blood donors are the only precious source for providing this gift of life. • Care should be given to ensure safety of both blood donor and recipient.


Criteria of Donor Selection and Deferral 2

Steps of Donor Selection

Donor Registration

There are certain critical steps to establish blood donor suitability for collection of a safe unit of blood or hemapheresis procedure • Donor Registration. • Physical examination. • Medical History. • Deferral Policy

Donor registration makes it possible to trace a unit from the beginning of the donation process to component preparation and testing to the distribution of the unit to its final destination.

3

4

Donor Registration

Physical Examination

1. 2. 3. 4. 5. 6.

• The purpose of the limited physical examination is to determine if the donor appears to be in good health. If there is evidence that donor is under the influence of drugs or alcohol, or if the donor shows obvious signs of a cold or of excessive nervousness, then the donor should be deferred. • Should be done by qualified blood bank physician, or qualified well trained nurse or interviewer under the supervision of the blood bank physician.

Date of donation. Name: first, middle, last. Age or date of birth Nationality Sex Type of donation: Voluntary, Directed, Autologous, Replacement, Apheresis or other. 7. Address: residence or business 8. Telephone No. 9. Donor ID No.: Type, Date of issue & place of issue. 5

6

299



Age: 18 ‐65 years Weight: >50kg. Temperature: 37.5 C (99,5 F) Blood Pressure: Systolic: 100 – 180 mm Hg Diastolic: 60 – 100mm Hg • Pulse: 50 – 100 beat/minute, Regularity, Force, Deficits.

Hemoglobin or Hematocrit: Method: Accepted levels:

• • • •





Routine and Apheresis Hb:12.5 – 18 gm/dl & Hct: 38 – 50% Autologous: Hb: 11 gm/dl. Hct: 33%

• Arm Inspection • Apheresis Donors: Plat. Count: 200 x 109/L for double product. 150 x 109/L for single product.  WBC’s: <129 /L  Total Protein:> 60 g/L  IgG:> 6.8 g/L  IgA:> 0.5 g/L  IgM:> 0.3 g/L 

7

8

Medical History

Medical History

• Information provided to the donor: Educational information about blood function, blood donation, and transfusion transmitted disease and specially HIV.

• The donor should feel comfortable during the interview process as well as during the donation process. • The prospective donor may be asked the questions orally, or may complete his own questionnaire. If the donor completes a questionnaire, he must be reviewed with a qualified interviewer before his acceptance as a donor.

• It is essential that the donor understand the information presented and be able to make an informed decision to donate.

9

10

Medical History

Medical History Questionnaire

• Ensure the privacy during the blood donor interview.

1.

In the past 8 weeks have you donated blood or its components? 

• Verbal privacy allows the prospective donor and interviewer to discuss confidential medical history which may affect the donor's suitability .

Frequency of donation: routine, automated 2 unit PRBC’s, autologous, apheresis:  Whole blood: every 8 weeks, not more than 5 time/year  Automated 2 units PRBC’s: every 16 weeks  Apheresis donation: every 48 hours, not more than 2 times/week, or 24 times/year.  Autologous donation: every week, no sooner than 3 days before surgery.

11

2.

Have you ever been rejected as a blood donor? Why?

3.

Are you feeling well and healthy today?

12

300



4. Have you ever had contact with AIDS patients?

7. In the past one week have you had any dental surgery?

5. Have you been outside the Kingdom for any time in the past twelve months?

8. In the past 12 months have you had any surgery or severe illness?

6. Do you know that, if you have AIDS virus, you can transmit it even with negative AIDS test?

9. Have you ever been I.V. drug user, or used intranasal cocaine? 10. Have you had growth hormone, or ever injected with beef insulin since 1980? 13

14



11. Have you or one of your family members ever had Mad Cow disease?

14. In the past 12 months: 1. 2. 3. 4. 5.

12. Have you ever had brain surgery for dura‐mater transplant or reside in U.K for 6 months?

6.

13. For female donors: during the past 6 week, have you been pregnant or delivered a baby?

Have you or your spouse received blood or organ transplant? Have you been given rabies shots? Have you been dialysis unit nurse, rape victim, in a prison or a patient in mental hospital? Have you had a tattoo, acupuncture, ear piercing or needle sticks? Have you had contact with hepatitis B patient, or received hepatitis B immunoglobulin. Have you had sex with someone who has hemophilia A or B or has taken money or drugs for sex?

15. During the past 4 weeks have you had: vaccination, Acutane, Proscar, Propecia, or Prozac medications. 16. Have you had: Asprin, Soriatane or other Medications. 15

16



17. Do you suffer or have you suffered from: • Syphilis or Gonorrhea • Hepatitis or Jaundice • Diabetes/Insulin • Aids symptoms:

• Malaria: o Treatment. o Travel to endemic area o Citizen of endemic area

• SARS:

o Prolonged fever or diarrhea o Enlarged lymph nodes o Unexplained weight loss

• • • •

o Patient o Travel to endemic area during last month o In contact with a patient during last month

T.B., Asthma allergy or any severe lung disease Epilepsy, cancer, bleeding abnormalities Brucellosis, Babesiosis, Leshmanasis or shagas disease Stroke

18. For Autologous donation: • Medication, illness, cardiovascular fitness and bacteremia • Not necessary to ask about TTD 17

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Deferral Policy

Deferral Policy (continued) 8. 9. 10. 11. 12. 13. 14. 15. 16.

• Donors should be deferred permanently if they suffer from or if they have, had: 1. 2. 3. 4. 5. 6. 7.

Bleeding abnormalities/Blood clots Cancer Chaga's disease Diabetes/Insulin Epilepsy Heart disease/chest pain Hepatitis

Severe Kidney disease Visceral Leishmaniasis Severe Lung disease SARS Positive HIV, serology (AIDS Patients) T.B Family member with Creutzfeldt‐Jacob’s disease I.V. drug users or used intranasal cocaine Stroke

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Deferral Policy (continued)

Deferral Policy

18. Dura‐matter transplant or reside in UK for 6 months. 19. Symptoms of AIDS

Donors should be deferred for 3 years if they have: o Been from countries with endemic malaria. o Had been diagnosed and treated from Malaria. o Soriatane medication, because of its long acting teratogenic effect. o Had been diagnosed and treated from Brucellosis.

• • • • • •

Prolonged fever or diarrhea. Enlarged lymph nodes Unexplained weight loss (more than 5kg) Night sweats Persistent cough White spots in mouth 2 1

22

Deferral Policy

Deferral Policy

Donors should be deferred for 1 year (12 months) if they suffer from or they have had: o Himself or his spouse received blood or organ transplant. o Rabies shots o Been a nurse for kidney dialysis unit. o Been incarcerated in a prison more than 72 hours

• • • • • • • • • • •

23

Been a patient in a mental hospital Has a tattoo Acupuncture Ear or nose piercing Needle stick Stab wound In contact with AIDS patient Body fluid splash to mucous membrane Gonorrhea, after treatment Syphilis, after treatment In contact with hepatitis patient, or receive Anti HB immune globulins 24

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Deferral Policy

Deferral Policy

• Been treated with anti‐malarial treatment as prophylaxis • Traveled to Malaria endemic area without symptoms • Animal bite • If they have had any surgery or severe illness • Have had sex with someone who has hemophilia A or B

• Female donors should be deferred for 6 weeks if they have been pregnant or delivered a baby. • Donors should be deferred for 4 weeks if they have had: – – – – – – – –

Low hemoglobin (less than 12.5g/dl) High pulse rate (more than 100 beat/minute) Low pulse rate (less than 50 beat / minute) High blood pressure (more than 180mm Hg for systole and or more than 100mm Hg for diastole) Vaccination, travel to endemic area or in contact with SARS patient. Accutane Medication for Acne. Proscar mediation for prostate. Propecia or prozac medications.

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Deferral Policy

Deferral Policy

Donors should be deferred for one week if they have had:

• Donors should be deferred for 72 hours if they have had:

o o o o o

Mild Fever Flu or common cold Sore throat Dental extraction Antibiotics

o Aspirin or any aspirin containing medication, if we intend to separate platelet concentrate.

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Donation Consent

Confidential Unit Exclusion (CUE)

I read, understood & answered accurately all the above questions to the best of my knowledge. I hereby grant permission to the blood bank to draw one unit of whole blood or to perform apheresis procedure & use it the way it may deem desirable.

• All donors must be given the opportunity to indicate confidentially whether their blood is safe for transfusion, or not. • The CUE may be accomplished during or after the donation process, allowing the donor another chance to indicate whether the unit is suitable for transfusion.

Signature:

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Criteria for Autologous Donor

Criteria for Platelet Pheresis Donor

• Written consent from the patient/donor • In case of minors, parents/guardians can give consent • Age: no bar any person who can bear the physio‐ dynamic changes can be accepted • Weight: No weight restriction can collect 8 ml/Kg. Body weight • Hb.: Donor should not be less than 11 gm.%

• Donor should meet all the acceptable criteria for routine whole blood donation however : • Age of the donor‐18 to 50 years. • Weight of the donor‐> than 55‐60kg. • TTI Results ‐ non‐reactive • The pre‐procedure platelet count should be more than 150,000 per cubic mm.

3 2

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Criteria for Platelet pheresis Donor

Donation Interval

• Donor should not have taken aspirin or any other platelet inhibitor in last 72 hours. • The donor should not be fasting prior to the procedure, however should refrain oily/spicy food, also citrus fruits or juices. • Donor should have a prominent and easily accessible central anticubital vein in at least one of the arm.

• The minimum time gap between two blood donations should be 12 weeks/3 months • Whole blood donation must be deferred for at least 72 hours after plateletpheresis • In case of re‐infusion failure, donor should not donate whole blood for 12 weeks

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Approval of Donor Suitability

References

• Approval must be in writing by blood bank physician before donation • Accordingly the donor will be sent for phlebotomy.

1. American Association of blood bank – Technical Manual, 14th edition, Bestheda, MD, 2002 2. Standards for Blood Transfusion services, AABB, 22nd edition, Bestheda, MD, 2003

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Principle • Characteristics of blood container: – Sterile – Pyrogen‐free – approved by FDA or CE


• The venipuncture site must be aseptic. • Blood and anticoagulant must be mixed continually during the procedure of blood collection, using calibrated blood mixer. • After the donation is completed, the donor recovery will be observed and monitored. • Refreshments (Juice & Biscuit) will be given. • The donor will receive the post phlebotomy care instructions prior to being allowed to leave the donation room.


Phlebotomy (Collection of Blood)

2

Procedure

Procedure

1. All phlebotomy staff must don disposable gloves and gowns prior to touching the patient / donor. 2. Prior to phlebotomy write unit number and donor name on the main collection bag and all transfer bags, donor medical history form, three red top tubes, and thick film for malaria.

5.

Prepare donor arm: –

Scrub 4 cm area in all directions from intended site with 2% PVP iodine solution for 30 seconds (if a donors sensitive to iodine, use alcohol swab.)

–

Apply 10% PVP iodine swab stick, start at the center with concentric spiral outward for 30 seconds.

–

Cover area with sterile 4x4 gauze, and do not touch the skin.

3. Place the blood collection set on the blood mixer scale, and thread donor tubing on the blood mixer scale. 4. Inspect arm for suitable vein (usually in the antecubital fossa.) 5. Apply tourniquet, identify suitable vein, and release it. 3

4

Procedure

Procedure

6.

Apply tourniquet again

7.

Remove the needle cover. (16‐gauge needle), and perform phlebotomy, by inserting the needle with it’s bevel upward in straight steady motion in the vein.

11. When 585 gm (469 ml ) of blood has been collected, the device will automatically stop the blood flow, and alarm will sound (complete draw within 10‐15 minutes; to separate all blood components collection time must not exceed 10 minutes, if it reach 15 minutes we can separate only PRBCs and discard platelet rich plasma, and if it exceed 15 minutes we stop donation discard blood due to slow bleed)

8. Tape needle in place on arm with adhesive strips. 9. Release tourniquet 10. Switch on blood mixer.

12. Apply hemostat (this needs to be explained as to what it is ? A clamp) to tubing near venipuncture and make a tight knot from previously prepared loose knot just distal to inline needle and hemostat.

11. Have donor open and close fist (squeezing foam ball every 10‐12 seconds.) 5

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Procedure

Procedure

13. Cut tubing by scissor between the tight knot and hemostat and separate the blood collection set.

17. Record time started and finished and unit weight.

14. Obtain blood samples by unclamp the hemostat, fill the tubes with blood and reclamp again.

18. Seal tubing next to the knot, strip donor tubing three times and heat seal into segments with clear readable numbers up to the main bag.

15. Release the tourniquet, (shouldn’t the tourniquet have been released at the beginning????) withdraw the needle, and apply pressure with a gauze pad, and have donor raise arm.

19. Remove the first segment and label it with unit number and place it into daily segment tray. 20. Put all blood collection set with rubber band and send to component preparation.

16. Discard needle assembly into a “sharps” container. 7

8

Procedure

Reporting Results

21. Place red top tubes in rack for sending to donor processing and infectious disease screening.

• •

22. Complete the donor medical history form.

Record adverse reactions Record any exception – – – – – – –

23. Assess donation site. If satisfactory, apply Band‐Aid. 24. Allow donor to sit up and stay with him. 25. Provide donor with juice and cookies and observe him.

Double phlebotomy (explain this further) Incomplete bleed Slow bleed (more than 10 minutes) Arterial puncture Contamination Overweight unit (>522 g) +wt of set Low volume unit (<316g)

26. Allow donor to leave after 10 minutes rest, and in absence of any adverse reaction. 9

10

Procedure Notes • Units take more than 10 minutes not suitable for preparation of components. only WB or PRBC’S ) • In case of cardiac arrest call emergency room team in the hospital. • In mobile site cardiac arrest cases call ambulance, or Police or Fire • Contamination of venipuncture site, repeat entire procedure. • Double phlebotomy, use different site and set. • Units from donors ingested aspirin within last 3 days, not suitable for platelet preparation.

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Introduction • Blood donors normally tolerate the donation very well, but occasionally adverse reactions of variable severity may occur before, during or at the end of the collection.


• The adverse reactions that occur in donors can be divided into local reactions and systemic reactions.

Donor Adverse Reactions 2

Adverse Reactions

Psychological Factors

• Definition: side‐effect; an undesirable or allergic

• Seeing of his/her own blood

response that happens during the donation process. • Anxiety over a donor’s condition

• Causes:

• First time donating

– Psychological factors. – Physical reasons.

• Fear • Witnessing of another donor’s reaction • Group/individual excitement 3

4

Physical Reactions • Hyperventilation

• First time donors > Repeated donors

• Shift of body fluids

• Female donors > Male donors

• Lack of sleep • Younger donors (under 20) > Older donors

• Decreased fluid intake (dehydration) • Physical activity

• Low body weight > High body weight

• Lack of nutrition

• No difference in Race 5

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1. Arm Injury – i. Vessel Injury

Types of Donor Reactions

A. Haematoma (bruise) 1. Arm injury i. Vessel injury ii. Nerve injury iii. Local allergy

2. Vasovagal event i. ii.

Vasovagal reaction Vasovagal syncope

3. Hyperventilation 4. Epileptic crisis 5. Cardiovascular event

6. Allergic reaction i. ii.

Mild allergic reaction Anaphylactic reaction

B. Arterial puncture

7. Haemolytic reaction 8. Air embolism 9. Citrate toxicity 10.Chills and /or rigors 11.Hypotension

C. Arteriovenous fistula D. Thrombophlebitis (superficial)

i. Angina ii. Myocardial infarction iii. Stroke

E. Deep venous thrombosis 7

8

A. Hematoma (bruise)

A. Hematoma (bruise)

• Definition: An abnormal, localized collection of blood under the skin.

• How to take care : – Remove tourniquet & pull the line. Apologize & Reassure everything is appropriate. – Apply pressure to site & elevate arm. – Apply ice X 15 min. & periodically throughout the day. – If pain continues, may take Tylenol.

• Signs and symptoms: – Colour change in the skin (bruise, if no other signs) – Swelling – Pain or tenderness at the venipuncture site

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B. Arterial Puncture • Definition: Accidental puncture of an artery.

• How to take care : – – – –

• Signs and symptoms: – High blood flow rate (blood unit <4 minutes). – Bright red colour of the collected blood. – Pulsating needle.

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Pull line immediately & add pressure for 10 minutes. Apologize to the donor. Wrap with flexible bandage. Do NOT Restick Donor!

C. Arteriovenous Fistula

D. Thrombophlebitis (superficial)

• Definition: Formation of a channel between a vein & an artery following laceration of the vessels by the penetrating needle.

• Definition: Formation of a blood clot in the punctured superficial vein associated with an inflammatory reaction of the vein. • Signs and symptoms:

• Signs and symptoms:

– Tenderness and hardness of the vein. – Redness of the overlying skin.

– Bruise, haematoma, stiffness, swelling & pain in the arm. – Distal veins dilated and pulsate. – Pulsating mass with a continuous murmur and palpable thrill.

• How to take care : – Require medical evaluation.

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E. Deep Venous Thrombosis

1. Arm Injury – ii. Nerve Injury

• Definition: Formation of a blood clot in a deep vein with very little reaction in the vein wall.

• Definition: Injury of a nerve by the needle or by a haematoma.



– – – – –

May be asymptomatic. Swelling & Discoloration of the arm . Antecubital tenderness. Increasing arm pain. Venous distention of the arm.

– Sensory changes (numbness, tingling). – Excessive/burning/ radiating pain in the arm. – Loss of arm or hand strength.

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16

1. Arm Injury – iii. Local Injury

2. Vasovagal Event – i. Vasovagal Reaction

• Definition: Allergic reaction to an adhesive tape or skin preparation solution

• Definition: A feeling of discomfort just before, during, or shortly after blood donation. • Signs and symptoms: – – – – – – – – –

• Signs and symptoms: – Erythema, pruritus

• How to take care: – Treatment of symptoms is with antihistamines and the counter topical steroids.

17

Pallor Weakness Dizziness Sweating Anxiety Nausea Vomiting Hypotension Bradycardia

18

Vasovagal Event – ii. Vasovagal Syncope

Vasovagal Event

• Definition: Donor unconscious for a short period of time. Can’t remember all what happened. A syncope may occur after the donor left the collection site

• How to take care : – – – – –

• Signs and symptoms: – Symptoms of a vasovagal reaction. – Loss of consciousness. – Incontinence & Convulsions.

Remove needle immediately. Reassure the donor. Loosen clothing, monitor & record vital signs. Cold pack behind the neck, bend knees & elevate feet. Administer aromatic spirits of ammonia by inhalation.

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3. Hyperventilation

4. Epileptic Crisis

• Definition:

• Definition: Sudden attack of loss of consciousness or awareness associated with abnormal movements or convulsion

– Faster and deeper breathing resulting in exhaling excessive amounts of carbon dioxide. – This causes a decreased blood carbon dioxide level and increased pH level. – Both can cause cerebrovascular constriction.

• Signs and symptoms: – – – –

• Signs and symptoms: – Paresthesias/tingling, twitching – Anxiety, sensation of suffocation

Sudden onset. Loss of consciousness. Tonic‐convulsive movements. Upturning eyes


• How to take care : – Ask the donor to breathe into paper bag.

Turn the donor on his side to protect the airway. Use a padded tongue blade to prevent injury to the tongue. Prevent falls and injury to the donor Instruct the donor to avoid further donations

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22

5. Cardiovascular Event

6. Mild Allergic Reaction

i. Angina ii. Myocardial infarction iii. Stroke

• Definition: An allergic reaction to a substance that is transfused to the donor during an aphaeresis procedure.

• How to take care:


– – – –

– Pruritus, rash, urticaria.

Very rare incident D.D. between vasovagal reaction & cardiac shock Call hospital emergency Start CPR in cardiac arrest

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310

6. Anaphylactic Reaction

7. Haemolytic Reaction

• Definition: Immediate severe hypersensitivity reaction.

• Definition: Return of haemolysed blood

(mechanical) or haemolytic reaction in the donor following accidental infusion of a hypotonic solution during an aphaeresis procedure


– Erythema, urticaria, laryngeal, pharyngeal & facial oedema. – Bronchospasm, respiratory distress. – Hypotension, shock.



Stop procedure. IV Anti‐allergic. IV Decadron. IV Calcium.

– Haemoglobinuria, haemolysed plasma – Renal dysfunction – Hypotension, DIC and fever

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8. Air Embolism

8. Air Embolism

• Definition: Fast infusion of a large air bubble into a donor during an aphaeresis procedure.

• How to take care : – Turn on Left side – Lower head & elevate legs. – Call for emergency department.

• Signs and symptoms: – – – – – –

Abrupt onset Cough Dyspnoea Cyanosis Hypotension Cardiac arrhythmia 27

28

9. Citrate Toxicity

10. Chills and/or Rigor

• Definition: Citrate infusion during an aphaeresis procedure causing reduced free calcium and associated symptoms. Subsides with reduction of the blood flow.

• Definition: Return of cold blood during an aphaeresis procedure may cause a cold feeling.



– Paresthesia/tingling – Nausea, vomiting – Arrhythmia

– Chills, rigor.


• How to take care :

Warm blankets. Aphaeresis staff to decrease Inlet ‐ check Citrate Monitor Monitor and reassure donor Oral/IV calcium

– Warm blankets, – Monitor donor – Reassure donor 29

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11. Hypotension

Steps for Prevention

• Definition: Decrease blood pressure.

• Although the number of donors who developed disturbances before, during or at the end of blood donations was very low, it is nevertheless desirable to reduce risks to a minimum.

• Signs and symptoms: – – – –

Light headedness Increase pulse rate Shallow respiration Perspiration

• A set of advices is provided for preventing problems.

• How to take care : – Lower head – Raise feet – Give fluids bolus

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32



• Shorten the waiting times.

• Avoid traumatic needle insertion with invasive and painful maneuvers.

• Allow the donor a light breakfast, excluding sugar, milk & milk products.

• Identify the best venous access, by inspecting both forearms.

• Ensure a comfortable room temperature and humidity.

• If the first venipuncture is failed, allow the donor to rest and reassure him or her, before attempting a new one.

• Engage particularly anxious donors in conversation, in order to distract their attention from what is happening. 33

34



• Invite donors to wear comfortable clothes, avoiding tight ties and belts.

• React swiftly to the initial symptom of pallor, by putting the subject in the Trendelenburg position.

• Do not let the donors drink very hot or very cold drinks during the recovery phase.

• Do not let the donor leave the donor site too quickly.

• Do not allow donors to eat solid foods during the donation.

• Continuous adequately training for the blood donation staff.

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312

Who is responsible for ensuring an adequate blood supply?


A. Government


B. Ministry of Health


C. The Donors D. Blood Banks E. Community

Donor Recruitment and Retained Strategies

2

Who is Best Qualified to Perform the Function of Donor Recruitment?

Problems in Donor Recruitment

A. Doctors

1.

Absence of professional donor recruiters.

2.

Limiting recruitment to national level.

3.

Misconceptions regarding blood donation.

4.

Lack of understanding of donor motivation.

B. Nurses C. Blood bank technical staff D. Public officials E. Marketing professionals 3

4

Problems in Donor Recruitment

The Challenge

5.

Poor goal setting and wasting of resources.

6.

Lack of sustained educational and recruitment efforts.

“Motivating potential donors to donate and organize them to provide adequate sustainable and safe blood supply all year around.”

7.

Poor donor treatment.

8.

Donor confusion when recruited by competing blood center. 5

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313

Guiding Principles

Guiding Principles

• Donor shall be volunteers and shall not be paid.

• Regular donors are safer than new donors.

• It is easier to introduce young people to blood donation than their parents.

• Donor recruitment must be a steady year round activity.

• Group leadership is the best way to recruit new donors.

• Donation by public leaders can set a valuable example. 7

8

Donor Recruitment

Donor Recruitment

I.

Field study

VII.

Blood Donation Plan

II.

Assessing needs and setting goals

VIII.

Counseling and Donor care

III.

Team work IX.

Retaining donors

IV.

Motivating donors

V.

Educational Program

X.

Record maintenance

VI.

Selecting safe donors

XI.

Monitoring and evaluation

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10

Field Study • Assess and identify the strengths and weaknesses of the Blood Bank in terms of the availability of trained manpower and expertise , public relations and the absorptive capacity and financial resources.

I. Field Study

• Identify opportunities and obstacles in the surrounding environment and the study of culture and lifestyle behaviors among a random sample of donors and non ‐ blood donors. 11

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Field Study • Determine the list of hospitals located in the vicinity of the blood bank, and determine it’s need to blood.

II. Setting Goals

• Alert strengths and work to improve the weaknesses and exploit the chances of success and try to avoid obstacles or foreign cope with it.

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Reasons of Increasing The Target Than The Actual Requirement

Setting Goals

• Some donors are refused either temporarily or permanently.

• One of the most important and most difficult steps in effective donor recruitment.

• Some donors don’t complete the donation. • Some units of blood had positive serology results.

• Goals must be set at higher level than the apparent need for blood.

• Blood units that are exposed to damage or contamination. • Units of blood , which expire without use . • As a strategic stock for emergency response. 15

16

Approach to Setting Collection Goals 1. Total Population : 2% 2. Acute Hospital Beds: o o o

III. Team Work

Primary Units: 5 to 7 / Bed. Specialized Hospitals: 25 to 30 / Bed. Surgery : 10 ‐ 12 / bed / year.

3. Medical Facilities in the area. 4. Annual Blood Usage: Past, Present & Future identified with an annual increase of about 10%.

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315

Team Work

The Team

• Team work is the concept of the people working together cooperatively as a team in order to accomplish the same goal. • The good understanding of team work and team building a critical for every work success.

• Choose the members of the team (doctors, administrators, nurses, motivators and coordinators campaigns, workers) depends on the size of the program and the nature of the work to be carried out. • Put the job description with identifying tasks and responsibilities for each of them and the issuance of ways of working standard and train to become a high degree of knowledge and skill. 19

20

Team Relationship

What are Characteristics of Effective Teams

• There must be a mechanism for cooperation and communication between the different departments of the Center and the supervisor on the recruitment program.

• • • • •

• Work relationships seem to have rules: – – – –

• • • •

Accept fair share of work. Cooperate in sharing resources. Help each other when asked. Mutual trust

Members have a clear goal. The focus is on achieving results. There is a plan for achieving the goal. Members have effective interpersonal skills. They know each other well and have good relationships. The team has the support of management. The team is open to new ideas. There is periodic self-assessment. There are sufficient resources to support the team work.

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22

IV. Motivating Donors

1. People don’t give blood unless they are asked to do that . 2. People are not naturally motivated to donate their blood. 3. There are more than enough potential blood donors. 4. There many misconception about blood donation in public.

IV. Motivating Donors

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Stages of Motivation

Stages of Motivation

1. Awareness: Essential but not sufficient to cause people actually to give blood.

3. Desire : Usually come through personal or public crisis . This a normal human reaction but not sufficient for sustained blood supply.

2. Interest: Develops over time through discussion and reconsideration.

4. Action: This is the target of the motivation reconsideration process. 25

26

Oral Communication • Most effective method of recruiting donors • Talk on need of blood, shortage of blood, ease of donation and myth about blood donation, possibly illustrated by films is very effective

V. Educational Program

• The Speakers/Recruiters must have the persuasive power to appeal to the humanitarian feelings of the audience. • Time should be available in the end of talk for the audience to ask questions and to give precise answers 28

27

Printed Communication

Educational Institutions

• Brochures, Posters and informative leaflets are various forms of communication

• Education among the young is useful to remove superstition and myth connected with blood donation.

• Material must catch the eye and be easy to understand • Publicity materials eg Posters, television, advertisements, cartoons etc ,should be prepared by professionals

• It is important to introduce the subject of blood donation into Schools as part of Science and civic studies.

• Greeting Cards on Birthday, Marriage Anniversaries, New Year’s day or other auspicious days carrying motivational slogans for voluntary donation are also effective

• The young are potential Donors. 29

30

Role of Media

Role of Media

• Participation of media is the most effective way to mobilise voluntary blood donation.

• Journalists are always looking for good stories which doesn’t involve any cost, for egg: interview of person with his family sharing his experience how blood saves his life and personally thanking the donors for the same.

• Ability to reach large numbers of people is most important advantage. • Media seems to be expensive?

• Involvement of media gives a emotional touch to public towards blood donation.

• Although newspapers, radio and television may charge for advertisements or regular public service broadcasts. 31

32

Role of Media

Role of Media

• We can encourage internet service providers and search engines to promote blood donation on their home pages on World Blood Donor Day and other special events.

• Request mobile telephone companies to send SMS/text messages to all subscribers urging them to become blood donors. • Utilising entertainment industry to add story‐lines on blood donation in their television programmes, plays and movies.

• Or an FM radio station to broadcast date and location of blood donation camp.

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Blood Donors • Voluntary Non Remunerated Blood Donors are the low risk donors for: • Safe blood. • Sustainable blood Supply.

VI. Selecting Safe Donors

• Replacement Donors‐not a preferred system: • Risks of professional Donors in garb of replacement donors. • Risk of hidden information. 36

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318

Regular voluntary non‐remunerated blood donor

Characteristics of regular voluntary non‐remunerated blood donors

Is defined as:

• Donate for altruistic reasons. • Entrust their blood donation to be used according to the need. • Are not under pressure to give blood. • Are committed to give blood when required. • Provide the foundation of a safe and sustainable blood supply.

"A voluntary non remunerated blood donor who donates at least once per year ‐or more according to national standards‐ for two consecutive years “.

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38

Importance of regular voluntary non remunerated blood donation

Characteristics of paid & family replacement donors

• Blood safety.

• This occurs in countries where: – blood supplies are scarce.

• Sustainability and availability of blood.

– there is no culture of voluntary blood donation. – There is no trust in blood banking facilities.

• Avoid exploitation and commercialization of the

• Where family replacement donors are forced into donating blood, they are less likely to be truthful about their health status or high‐risk behavior.

human body, the poor & vulnerable. • Altruism and social solidarity.

• This results in a higher incidence and prevalence of TTIs compared to a system based on voluntary blood donation 39

40

Incentives & Risk to Blood Safety • The use of incentives differ from country to country. • Depending on each person’s hierarchy of needs e.g. cultural beliefs, personal values, economic factors. • The use of incentives should never:

VII. Blood Donation Plan

– Compromise the safety of the blood supply. – Be conditional on the person actually giving blood.

41

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319

Blood Donation Plan

Plan of Action

• Recruitment process should be based on scientific studies not assumptions.

• Train some of our young active employee to work as recruiters. • Increase the number of blood drives. • Form information data base for arrange the process of donation and donors recall. • Develop the Appropriate Screening Questions Form. • Target Low Risk Groups.

• Local culture & religious beliefs must be understood to motivate people appropriately. 43

44

Convert replacement donors to voluntary blood donors

Recall Infrequent or Temporary Deferred Donors

• Act as a important target population despite the risks associated with them. • They understand the importance of the timely availability of blood because of their direct experience of the urgent need of donors. • Shifting family/replacement donation system to voluntary blood donation system requires a change in practice by hospitals itself. • Encourage and educate hospital and health centre staff to discuss voluntary blood donation with them.

• They also constitute an important target donor population. • These donors are already awarded – About donation process – Need of blood – And already motivated.

• Reactivating former donors can be more easy than recruiting new donors. • They simply need a reminder and encouragement to return. • Donor database or donor register is a vital tool for recalling previously active donors. 45

46

Pre‐donation Counselling • Provide a counselling session before every blood donation

VIII. Counseling & Donor Care

• Pre‐donation interview/session should be: – Private – Confidential – Not interrupted – 2 – way communication 47

48

Objectives of Pre‐Donation Counselling

Objectives of Pre‐Donation Counselling (cont)

•

To make a preliminary assessment of donor state of health.

•

To encourage self‐deferral of people who may have a history of risk behaviour

•

Explain in advance every step involved in blood donation.

•

To discourage people from using the blood service as a centre for voluntary counselling and testing

•

Obtain informed consent for blood donation and testing.

•

Emphasise the need to maintain a healthy lifestyle

•

•

Dispel myths and misconceptions

To increase awareness about testing for TTIs and the possible consequences of positive results. 49

50

Post‐ Donation Counselling

General rules of donor care

• Post‐ Donation Counselling is offered to a person who has donated blood and has learnt the results of; HIV, Hepatitis B &C, Syphilis and has been told diagnosis of these diseases by the counselor.

• Convenient location, operating hours and accessibility. • Well advertised with high visibility. • Staff should be always smart, clean in appearance and maintain a high standard of personal hygiene. 51

52

During blood donation •

• • • •

During blood donation (cont)

Staff should be well trained and qualified on venepuncture. Donor bed comfortable & clean sheets. Comfortable position. Personal attention. Explanation of procedures & technology.

• Ensure quality and safety standards. • Maximising an education opportunity. • Observing and listening to donor. • Manage uneventful donations. • Provide post donation advice.

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321

Post donation

Post donation (cont)

• Appropriate resting time.

• Manage reactions professionally

• Provide complimentary refreshments.

• Encourage donor feedback

• Appropriately trained staff /or volunteers

• Thank the donor, remind him of next donation and invite him to bring a friend

• Good observation skills for donors‘ reactions.

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56

Donor feedback

Donor Complaints

• Encouraged, welcomed and acknowledged e.g.

• To ensure donor satisfaction and retention any donor complaints must be taken seriously:

suggestion box, donor surveys, donor complaint

– complaints may be about anything that they have experienced in their contact with the BTS.

form.

– complaints may range from trivial to serious.

• Positive and negative feedback is dealt with and recorded. • Respond to all donor feedback. 57

58

Donor Complaints

Dealing with Complaints

• Standard procedures for handling complaints.

• Complaints should be dealt with professionally. • Donors must feel that their complaint has been listened to and understood, and will be investigated. • Complaints must be investigated. • Feedback should be given to the donor

• Trained staff for dealing with complaints. • Measures to ensure that errors that are identified are corrected and prevented from recurring.

– immediate if appropriate – later in writing

• All complaints must be logged and the investigations and outcomes recorded 59

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Positive Outcomes

Donor Recognition

• Donor complaints may be useful in identifying areas of poor practice ‐ poor quality • The donors are the best people to assess how they are actually treated at a donor clinic • The BTS must always be open to criticism and be willing to learn from it • Positive responses to complaints may make it easier to retain the donor

• Forms of Donor Recognition – – – – – – – –

Letter of appreciation Greetings cards / Thank you letters Certificate Badge Memento Annual felicitation by way of convocation Donors card Special award for 10/25/50/100 time donation

• Recognition and awards act as an incentive to existing donors and motivate potential donors to become regular voluntary donors. 61

62

Recognize Donors’ Contribution

Key Points

• Recognition plays a major role in donor recruitment strategies. • Recognition of voluntary blood donor means honour without any monetary value. • Recognition of donors helps as:

• Voluntary donors are priceless. • Quality donor care is a basic donor right. • Mobile donor clinics are more convenient to donor population.

 Automatic incentive.  Social prestige .  Motivation to panel donors .  Spurs donors to greater participation.  Feeling appreciated and honoured.  Forms stronger commitment.  Help in retention of donors .

• Donor must be well‐informed, safe & appreciated. • Emphasize the importance of donor satisfaction as the first step in donor retention. • A satisfied and committed donor is the best ambassador of recruiting other donors. 63

64

Why Donor Retention?

 It is difficult to recruit new donors.

IX. Retaining Donors

 It costs money to recruit new donors.  It costs more to take one donation only.  It is safer to use regular donor blood.

66

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How to do it standards 

How to do it standards (cont.)

The first important message to the blood collection team is to consider the voluntary donor as a partner in the supply of blood for the community



Donors are the owners of the service and not only customers.



Identify donor’s expectations.



Identify donor’s needs.



Views valued, respected and acted upon.



Warm welcome.



Ensure donor is appreciated, thanked and encouraged to return.

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68

What to do? 

Always address the donor by his name.



Always make eye contact.



Explain the donation process.



Explain what happens next.



Ensure privacy and discretion.



Answer questions/queries honestly and tactfully.



Talk up the system‐be its advocate.



Be positive during the donation process.

What to do? (cont.) 

Talk to the donor during blood collection



Chat to the donor if they want.



Ask donors regularly if they are well.



Assess according to eye contact.



Escort donor to the refreshment area.



Make them feel welcome and valued.

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70

What not to do 

Abandon the donor‐ even for a short period.



Talk jargon when explaining.



Talk over with other colleagues.



Challenge donor’s beliefs .



Give information you are not sure about.



Disregard donor’s observations.



Underestimate the worry of deferred donors.

The Donation Experience

71



We assume what donors want.



The emotional needs of donors.



Donors take something away.



Ask donors what matters to them.

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Loyalty Assessment

Donation Experience Survey

• Reliability Index

• Random selection of donor population

• Retention rate

• Score out of 10 for the whole experience

• Frequency rate

• About how they were looked after

• New donor intake

• About how they were valued

• Donor attrition rate

• Specifically about the needle insertion

• Age and gender group profiles

• Specifically about waiting times

• Satisfaction measures

• Specifically about staff attitude 73

74

The New Donor

Personal Contribution Reinforces Partnership • All donor’s comments are opportunities

• Should be made to feel extra special.

• Actively look for opportunities

• May be frightened and overwhelmed.

• Interaction with donor’s makes a difference

• May have incorrect information.

• Assess and understand the needs

• Requires careful observation all through. • Needs clear reassuring explanation.

• Modify approach in response to feedback 75

76

The Deferred Donor

Staffing

• Think how the donor may be feeling.

• Selection

• Upset, rejected, concerned and confused.

• Professionalism

• Reassure and handle with empathy.

• Donor clinical care skills • Donor/customer services skills

• Give adequate explanation.

• Dedication and conviction • New vision and new approach

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Documentation • Each activity in the donor area, and the outcome of each activity, should be documented Write what you do, do what you write

X. Record Maintenance

• Donor records enable to: Record and analyze successes and failures Monitor staff compliance Assist in error analysis Enable a document trail for audit purposes

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79

Donor Records

Donor Records

• Donor records are vital in maintaining the safety of the blood supply. • Minimum donor record keeping requirements:  Record of blood donors. o o o o o o

 Record of blood donation. o Date of blood donation o Donation number (Identification number) o Physical examination record ‐ pulse, temperature, and blood pressure

Blood donor consent for donation Donor's name and father/husband name Donation ‐ voluntary or replacement Date of birth (age), Sex and weight Address (office & residence) and telephone number History of illness

o Hemoglobin o ABO and Rh(D) group o Results of HBsAg, anti‐HCV, anti‐HIV 1&2. VDRL/RPR and malaria tests o Disposal: issued for transfusion or discarded 81

82

Confidentiality • Confidentiality is critical in the management of blood donors.

XI. Monitoring and Evaluation

• All donor information is privileged and must be kept confidential. • Records must be kept secure at all times. • Only designated staff should have access to records. 83

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Monitoring and Evaluation

Monitoring and Evaluation

• It is necessary to periodically evaluate each part of the donation program in order to ascertain the extent of its influence and success as opposed to what it was spent .

• Number of regular and new donors. • Number of permanently deferred donors. • Number of donors experienced adverse reactions.

• Developing indicators with which to measure the success or failure of the program in a simple sentence , "the program is considered successful if the provision of safe blood throughout the year”.

• Number of organizations and communities involved in motivating voluntary blood donation. • Donor feedback system. 85

86

Finally – what to do?

Recommendation

• Set Goals

• Leave it to the professionals…

• Hire Professionals • Educate the Public • Treat Donors Well & Enforce Donation Behavior • Ask Them to Come Back • Manage Donors With Their Best Interest in Mind 87

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Blood Components Description


Blood Component Preparation and Storage

2

Anticoagulant‐Preservative Solutions (mg in 63 mL) for 450 mL Collections

Whole Blood

CPD CP2D CPDA‐1

• **Fresh Whole Blood contains all blood elements plus the anticoagulant‐preservative in the collecting bag.

Ratio (mL solution to blood) 1.4:10 1.4:10 1.4:10

• **After 24‐hour storage, it essentially becomes red cells suspended in a protein solution equivalent to liquid plasma.

Sodium citrate 1660 1660 1660

FDA‐approved shelf life (days) 21 21 35 Content

Citric acid 206 206 206 Dextrose 1610 3220 2010 Monobasic sodium phosphate 140 140 140

• **It is used commonly as a source for component production.

Adenine 0 0 17.3 With 500 mL collections, the volume is 70 mL and the content 10% to 11% higher. 3

4

Whole Blood Unit

Blood Components Separation ‐ Goals

After centrifugation

• Decrease harmful effect of blood transfusion

WB separates into

plasma & platelets &

• Giving patients specific component needed

PRBCS • Allow a longer survival for components • More than one patient will use the unit 6

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What are the Components of Blood?

Red Blood Cells

► Packed Red Blood Cells (PRBCs)

• Red Blood Cells (RBCs) are units of Whole Blood with most of the plasma removed.

► Fresh Frozen Plasma (FFP)

• The final hematocrit must be ≤80%. • Additive Solution supports red cell survival and function up to 42 day.

► Platelet Concentrates (PC)

• Additive Solution allows blood centers to use or recover a maximum amount of plasma, yet still prepare a red cell component

► Cryoprecipitate (CRYO)

• with a final hematocrit between 55% and 65%. • RBCs can be prepared at any time during their shelf life, but AS must be added within the time frame specified by the manufacturer, generally within the first 72 hours of storage.

► Leukoreduced Red Blood Cells

8

7

Content of Additive Solutions (mg/100 mL)

PRBCs

AS‐1 AS‐3 AS‐5 (Adsol) (Nutricel) (Optisol)

How to make (PRBCs)?

 RBCs have higher specific gravity than plasma, it moves to

Dextrose 2200 1100 900

lower portion of the bag by centrifugation

Adenine 27 30 30

 WB (Light spin) ‐ Two products:

Monobasic sodium phosphate 0 27 6 0 Mannitol

1) PRBCs

750 0 525

2) Platelet Rich Plasma (PRP)

Sodium chloride 900 410 877 Sodium citrate 0 588 0 Citric acid 0 42 0

9

10

PRBC 1

2

• A PRBC unit contains ~ 200ml RBC and 50ml Plasma

3

• All RBC transfusion must be ABO/RH compatible, or in emergency, transfuse type O PRBCs • RBC units must by stored at 2‐6°C • PRBC are indicated for – Patients with anemia – Surgery – Newborn exchange transfusion 11

12

329

Platelets

Platelets Concentrate (PC)

• Platelet concentrates (Platelets) are prepared from units of whole blood that have not been allowed to cool below 20 C. • • Platelet‐rich plasma (PRP) is separated within 4 hours after completion of the phlebotomy or within the time frame specified in the directions for the use of the blood collecting, processing, and storage system—typically 8 hours

How to prepare PC? • Platelet Rich Plasma (PRP) centrifuged using (heavy spin), this will produce: – Fresh frozen plasma (FFP) – Platelets concentrate (PC)

• The final component should contain resuspended platelets in an amount of plasma adequate to maintain an acceptable pH; generally,40 to 70 mL is used

•

PC are stored at room temperature on platelet agitator (prevent platelets clumping)

•

PC have a 3‐5 days expiration date

13

14

Platelets Concentrate

1

Indications: 1. To prevent bleeding due to thrombocytopenia or dysfunction 2. To a patient undergoing an operation, if the platelet count is less than 20,000/µL

2

3

15

16

Fresh Frozen Plasma(FFP)

Plasma

• Fresh Frozen Plasma (FFP) is plasma prepared from whole blood, either from the primary centrifugation of whole blood into red cells and plasma or from a secondary centrifugation of PRP. • The plasma must be frozen within 8 hours of collection.

• Plasma can be separated at any time during storage, up to 5 days after the expiration date of the Whole Blood. • When stored frozen at –18°C or colder, this component is known as Plasma and can be used up to 5 years after the date of collection. • If not frozen, it is called Liquid Plasma, which is stored at 1 to 6°C and transfused up to 5 days after the expiration date of the Whole Blood from which it was prepared.

17

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330

Cryoprecipitated AHF

Plasma Cryoprecipitate Reduced

• Cryoprecipitated antihemophilic factor (AHF) is the cold‐ insoluble portion of plasma that precipitates when FFP is thawed between 1 to 6°C.

• If cryoprecipitate has been removed from plasma, this must be stated on the label.

• It contains ≥80 IU FactorVIII (AHF), >150 mg of fibrinogen, and Factor XIII .

• When stored at –18°C or colder, this component has a 12‐month expiration date from the date of collection.

• CRYO contains both the procoagulant activity (Factor VIII) and the von Willebrand factor of the FactorVIII vonWillebrand complex.

• This component is used primarily in the treatment of thrombotic thrombocytopenic purpura.

• Once separated, CRYO is refrozen within 1 hour of preparation and stored at –18°C or colder for up to 1 year after the date of phlebotomy. 19

20

Expiration Dates for Selected Blood Components

Leukoreduced Red Blood Cells

1‐Whole Blood ACD/CPD/CP2D –21 days CPDA‐1 – 35 days 2‐Red Blood Cells (RBCs) ACD/CPD/CP2D – 21 days CPDA‐1 – 35 days Open system – 24 hours Additive solutions – 42 days RBCs Washed 24 hours 3‐Platelets 24 hours to 5 days, depending on collection 4‐Platelets pooled or open system 4 hours 5‐FFP 12 months (–18 C) 7 years (–65 C), as approved by the FDA 6‐FFP Thawed 24 hours 7‐FFP Thawed – Open System 24 hours 8‐Plasma Frozen within 24 hours 12 months 9‐Plasma Cryoprecipitate 12 months Reduced Frozen 10‐Plasma Cryoprecipitate 24 hours to 5 days Reduced Frozen 11‐Cryoprecipitated AHF 12 months 12‐Cryoprecipitated AHF Thawed 4 hours if open system or pooled, 6 hours if single unit





What are Leukoreduced RBCs? PRBCs that have WBCs removed by special filters or by a machine Advantage of Leukoreduced RBCs: 



May prevent febrile nonhemolytic transfusion reactions (FNHTR) Reduces risk of cytomegalovirus (CMV) transmission it resides within cytoplasm of WBCs

21

22

23

24

Leukoreduced Red Blood Cells Preparation methods: • In Line filtration • Prestorage: Filter used to remove leukocytes before storage of RBCs, this allows up to 42 days before expiry • Poststorage: filteration is done within 3 days of storage • Bedside filtration :Use a special filter during transfusion of the unit to the patient (rare).

331

Summary of Blood Components

Summary of Blood Components

Blood Component

Storage Blood Component

1) PRBCs

2) PC

Indication Temperature

Time

2‐6°C

+ SAGM 42 days

‐ Anemia ‐ Newborn exchange transfusion

R.T.

3‐5 days

‐ Bleeding ‐ Operation if plt. Less than 2000/ul

18°C

1 year

‐ Clotting factor deficiencies

65°C

7 years

‐ Severe burns

3) FFP

Centrifugation

Storage

Indication

Temperature

Time

‐30°C

1 year

a)WB special heavy spin – 3500rpm at 4°C RBC + plasma  11 min 4) Cryo

25

b)Plasma  Store at 18°C Thaw at 4°C Heavy spin at 4°C

‐ Hemophilia ‐ A Von Willebrand disease

26

332


QC of Whole Blood  Frequency of control: 1% of all units with minimum of 4 units per month


 Storage :‐ 2°C to 6 °C, for CPDA‐1 the storage time is 35 days, CPD & CD2D – 22days.


 On expire date :‐ measure HCT, pH, total Hb , K+ and perform sterility assays Quality Control of Blood Component Preparation

2

QC of Whole Blood

Red Cell Concentrates

• Volume : 450ml ± 10 % of body volume excluding anticoagulant

• Perform the same assay as for Whole blood on the expiry date

• HCT : 40±5%

• Storage : 2‐6°C, for 35 days if prepared from WB collected in CPDA‐1

• pH > 6.5

• Quality Assurance:

• K < 27mmol/L

• • • • • •

• Hb minimum 45g/unit • Sterility : no growth

Volume: 280ml± 50ml, frequency of control 1% of all units HCT: 0.65‐0.75 pH > 6.5 K < 78 mmol/L Hb: minimum 45g/unit Sterility: no growth

3

4

QC of Platelet Concentrates

Platelet Concentrates

• Volume > 40ml

• Prepared within 6Hrs of blood collection • Must evaluate at least 4 platelet preparations monthly for platelet count, pH and plasma volume • Platelets should be selected from each centrifuge in use • The T° at which pH is measured should be the same as stored • Label the volume, the actual volume by measurement must be 10% of the stated volume • Storage : 20‐24°C • T° should be recorded at least every 4Hrs during storage.

• pH: 6.8‐7.4 • Plt count: at least 5.5 x 1010 /bag in at least 75% of the units tested at the end of the storage. By apheresis : minimum 3 x 1011/bag platelets in at least 75% units tested • WBC contamination: < 2 x 103/bag • RBC contamination: < 2 x 109/bag • Macroscopic appearance: no visible platelets aggregates • Sterility: no growth

5

6

333

QC OF FFP

Cryoprecipitate

 Volume: 220‐250ml

• Assayed on at least 4 bags/ month – for factor VIII

 Factor VIIIc : > 0.7IU/ml‐ every 2 months

• Storage:

 No leakage after pressure in plasma extractor, before freezing and after thawing o Macroscopic : no abnormal color or visible clots o Residual cell:

– 12 months at below ‐18 °C

• Must be thawed at 37°C and used within 6Hrs

Red cell: < 6.0 x 109/l Leukocyte: < 0.1 x 109/l Platelets: < 50 x 109/l 7

8

QC Of Cryoprecipitate

Transportation • A system must be used to ensure that all blood and blood components shipped by or received into a blood bank or blood transfusion service have been maintained within T required.

 Volume: 10‐20 ml  Factor VIII : > 80 IU/unit

• All liquid RBC components kept at T of 1‐10°C during transport

 Fibrinogen: > 150 mg per unit

• All component routinely stored at 20‐24°C should be maintain T during shipment

 Macroscopic: homogenous

• All frozen components should be transport in frozen state at –18°C or colder

 Sterility: no growth

• Periodic T check and documented to ensure the transportation adequate to meet the criteria 9

10

Red Blood Cells Quality Control Form UNIT NO.

HCT

Volume

PH

K

Hb

Platelets Quality Control Form Culture

UNIT NO.

** AT LEAST 4 UNITS TESTED MONTHLY Volume : 280ml± 50ml HCT : 0.65‐0.75 pH > 6.5 K < 78 mmol/L Hb : minimum 45g/unit Sterility : no growth

WEIGHT/gm

VOLUME/ml

PH

** AT LEAST 4 EXPIRED (6TH DAY) UNITS TESTED MONTHLY ** ACCEPTABLE LIMITS: * PLATELETS COUNT >.5.5 × 1010 platelets per unit * PH > 6.2

11

334

Platelets count/ul

PLATELETS COUNT/UNIT

12

Centrifuge Calibration Form

Cryoprecipitate Quality Control Form UNIT NO.

WEIGHT

VOLUME

FIBRINOGEN Mg/BAG

UNIT NO.

FACTORVIII IU/BAG

UNIT NO.

WHOLE BLOOD PLT COUNT

VOL/ ml

PLT/UNIT

Platelets rich plasma SPEED

TIME

PL/Ul

Volume

PLATELETS CONCENTRATES Plt/unit

Yield %

speed

TIME

PLT /uL

VOLUME

PLT / UNIT

PLATELETS COUNT / UNIT PLATELTS RICH PLASMA PLATELETS YIELD‐ PLATELETS RICH PLASMA =‫* ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ 100 PLATELETS COUNT/ UNIT WHOLE BLOOD

PLATELETS COUNT /UNIT platelets c. PLATELETS YIELD‐ PLATELETS CONCENTRATE = ‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬ PLATELETS COUNT/ UNIT PRP

**ACCEPTABLE LIMITS: *FIBRINOGEN : > 150mg/BAG *FACTOR VIII > 80IU/BAG

** ACCEPTABLE VALUE : ** PLATELETS YIELD in PLATELETS RICH PLASMA >75% ** PLATELETS YIELD in PLATELETS CONCENTRATE > 90%

13

335

* 100

14



Leukoreduced blood component


Leukoreduced and irradiated blood components 2

Etymology The name "white blood cell“ is derived from the fact that after centrifugation of a blood sample, the white cells are found in the buffy coat, a thin, typically white layer of nucleated cells between the sedimented red blood cells and the blood plasma. The scientific term leukocyte directly reflects this description, derived from Ancient Greek λευκό (white), and κύτταρο (cell).

3

4

• Leukocyte content of whole blood averages two billion ( 2 x109 ) leukocytes per 500 mL of whole blood. • During blood component preparation: – 90% of leukocytes fractionate with the red blood cells (RBCs). – 8% is retained within Platelet concentrates. – 2% are present in the plasma before freezing.

5

6

336

Leukocyte reduction can be achieved by various techniques, including : • • • • • •

Leukocyte Reduction Filters

Centrifugation Leukocyte filtration Sedimentation Washing Freeze‐thawing Apheresis

Generation

Pore Size

Mechanism

First

170–260 um

Screen filter

Second

20–49 um

Screen filter

Third

Not applicable

Adhesion filter

Purpose No leukocyte filtration; “standard” blood filter Micro-aggregate filter; leukocyte filtration, 90% Adsorption filter; leukocyte filtration 99.9%

7

8

Adverse Effects Associated with Donor Leukocytes 1. Nonhemolytic febrile transfusion reactions 2. Transmission of leukocyte‐associated viruses cytomegalovirus 3. Alloimmunization 4. Immunomodulatory effects 5. Cancer recurrence 6. Postoperative infections 7. Transfusion storage time for red blood cells 8. Transfusion storage time for platelets 9. Transfusion‐related acute lung injury 10. Transfusion‐associated graft‐versus‐host disease 10

9

2. Transmission of leukocyte‐associated viruses (e.g.cytomegalovirus)

1. Febrile Nonhemolytic Transfusion Reactions • Definition: as a temperature increase of 1°C after an allogeneic blood transfusion.

• Transfusion‐associated CMV infection is a significant cause of morbidity and mortality in immuno‐ compromised patients and especially in organ transplant recipients.

• Cause: alloantibodies in the recipient’s plasma against antigens present on donor leukocytes and/or platelets

• After either kidney or liver transplants, more than 60% of patients develop antibodies against CMV.

• Incidence:

– 0.5% in patients receiving a first transfusion – 60% in Chronically transfused patients

11

12

337

3. Platelet Refractoriness and Alloimmunization

4. Immunomodulation and Postoperative Infectious Complications

• Alloimmunization can reduce the clinical effectiveness of platelet transfusions by 50%. Especially prevalent among those:

• Contaminating leukocytes in RBC transfusions might be responsible for down‐regulation of : – – – – –

– Patients receiving pooled random donor – Platelet concentrates – Who are Pregnant

Natural‐killer (NK) cell activity, T cell proliferation T lymphocyte antitumor activity CD‐4 helper to CD‐8 suppressor ratio Lymphocyte blastogenesis

13

14

5. Cancer Recurrence

6. Postoperative infections

• An association between allogeneic blood transfusion and colorectal cancer recurrence after surgery.

• Transfusion of blood components containing bacteria may lead to potentially fatal sepsis.

• Blood transfusions in colorectal surgery patients have been reported to increase cancer recurrence by 37% also have been associated with increased recurrence of breast, lung, kidney, prostate, stomach, cervical, laryngeal, soft tissue, and bone malignancies.

• Cause : inadequate skin preparation before venipuncture. • Common pathogens : include Gram‐negative endotoxin producing organisms such as Yersinia enterocolitica, pseudomonas and enterobacter 15

16

6. Postoperative infections

7. Transfusion storage time for red blood cells

• Optimal storage time before filtration to allow for maximal leukocyte ingestion of bacteria appeared to be between 2 and 12 hours.

• • • •

Decrease ATP. Glucose consumption. Increase lactate and K+ production. The presence of leukocytes in blood components reduces glucose availability. • Leukocyte lysis leads to release of cytokines that reduce RBC survival.

• The beneficial effect of leukocyte reduction may lie in removal of leukocytes containing ingested bacteria.

17

18

8. Transfusion storage time for platelets • • • • •

9. Transfusion‐related acute lung injury

Decreases in pH Increases in glucose consumption Lactate production Lactic dehydrogenase release Platelets stored with leukocytes express decreased quantities of glycoprotein Ib (GPIb) receptor, resulting in a bleeding disorders.

• Stored blood contains micro‐aggregates of degenerated leukocytes, platelets and fibrin • These micro‐aggregates have been associated with pulmonary insufficiency due to agglutination of donor leukocytes by recipient antibodies • C/P : severe dyspnea, non‐cardiogenic pulmonary edema 19

20

10. Transfusion‐associated graft‐versus‐host disease (GVHD)

Adverse Effects of Leukocyte Reduction • Few side effects have been reported

• GVHD is a potentially lethal condition.

1. Hypotension : due to release of bradykinin like vasoactive substance esp. in patients receiving ACEI prolong intravascular half life of bradykinin : by decreasing bradykinin degradation. 2. Complement activation and formation of platelets aggregate

• Cause : donor T lymphocytes. • Mechanism : immunocompromised recipients, host defense mechanisms fail to suppress viable transfused donor lymphocytes, which engraft within the recipient’s marrow, ultimately resulting in death. • Occurrence :

– 2‐8% decrease in potency of cellular components of blood.

– When the donor and recipient share an HLA haplotype. – Use of directed‐donor blood from first degree relatives.

• Prophylaxis : gamma‐irradiation. 21

22

Outline • Irradiation Source • Radiation Dose

Irradiation of Blood Component

• Submission Contents – Standard Operating Procedures (SOPs) – Records – Labeling

23

24

339

Irradiation Source

Radiation Dose

• Cesium-137 – sealed source irradiator

• 2500 cGy targeted to container’s central portion

• Cobalt-60 – sealed source irradiator

• 1500 cGy minimum dose at any other point of the container

• Linear accelerator

• If product is irradiated more than once, document total (additive) dose • An indicator should be used with each batch that is irradiated

• X-ray – irradiator

– Follow manufacturer’s instructions for use, including temperature control 25

26

Submission Contents

Submission Contents (cont.)

• Cover letter

• Labels for each product with Form FDA 2567 • Two months of irradiation records

• Form FDA 356h

• Most recent dosimetry map

• SOP for manufacturing irradiated blood products

• Contractor information, if applicable – Contractor who performs irradiation must register with FDA

• Typically, SOPs for equipment maintenance and personnel training

27

28

SOPs

SOPs (cont.)

• Description of the irradiator (e.g., radiation source)

• Indication of the maximum number of units to be irradiated at one time

• Description of the dose delivered to the center of the container

• Description of procedures for monitoring to determine actual dose delivered • Validation

• Length of time required to deliver irradiation

– – – – –

• Maximum irradiation dose limits • Description of procedures for re-irradiation, if applicable

29

Initially Annually for Ce‐137 Semi‐annually for Co‐60 After mechanical repairs Use Thermoluminescent Dosimeter (TLD) chips or other direct methods of measurement 30

340

SOPs (cont.)

SOPs (cont.)

• Dating period for Red Blood Cell products

• Maintenance of irradiator

– Not more than 28 days from the date of irradiation but no more than the dating period of the original product

• Procedure for personnel training • Staff safety

• Dating period for platelets remains unchanged

• If contract facility used for irradiation, your SOPs should: – Describe what steps are performed by you and by the contractor – Ensure manufacturing steps are performed according to your specifications and are in compliance with all applicable regulations 31

32

SOPs (cont.)

Records

Quality control (QC) (review considerations) • Irradiator

• Strength of source • Irradiation Records

– Daily QC (e.g., check of turntable rotation) – Monthly comparison of irradiator timer and back‐up timer, if available, with certified stop watch

• QC • Equipment maintenance

• Irradiation indicators – Shipping and storage temperature checks – Expected results of each new lot – Investigation of failures and corrective actions 33

34

Irradiation Records/QC

Irradiation Records/QC (cont.)

• Operator ID

• Level/dose of irradiation

• Site of irradiation

• Documentation of QC for irradiator and irradiation indicators

• Date and time of irradiation • Duration of irradiation

35

36

341

Labeling

Labeling (cont.)

• Container Label (21 CFR 606.121)

• Circular of Information (cont.)

– Container label must include proper name of product, and modifier, if applicable (e.g., RBCs, Irradiated) – Change expiration date for RBCs if appropriate

– Removal of residual supernatant plasma prior to transfusion may reduce risks associated with elevated plasma potassium

• Circular of Information should include:

• License Number should not appear on the container unless the product has been licensed by the FDA

– Indications for use in treating patients at risk of transfusion‐associated GVHD – Side effects and hazards of irradiating RBCs • Higher supernatant potassium levels than non‐ irradiated RBCs due to cell membrane damage

37

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The Antiglobulin Test • Antiglobulin serum (Coombs’ Serum) was discovered by Coombs in 1945. • The antiglobulin test can be used to detect red cells sensitized with IgG alloantibodies, IgG autoantibodies or complement components. • Sensitization of red cells can occur in vivo or vitro. • The use of AHG serum to detect sensitization of red cells in vitro can be:


– One stage technique , the direct antiglobulin test (DAT). – Two stage technique , the indirect antiglobulin test (IAT).

Basic Immunology: Direct & Indirect Antiglobulin Test

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Principle

Principle

• Normal human red blood cells, in presence of antibody directed towards the antigen they possess, may fail to agglutinate when centrifuged and become sensitized. This may be due to the particular nature of the antigen and antibody involved. • Sensitization of RBC’s may be with IgG or complement. • In order for agglutination to occur an additional of anti‐antibody or anti‐complements, which reacts with the Fc portion of the IgG antibody, or with the C3b or C3d component of complement alternatively.

• This will form a "bridge" between the antibodies or complement coating the red cells, causing agglutination. • The coating (sensitization) of red cells can occur in vivo or in vitro following incubation at 37°C with serum containing antibody.

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Production Methods of Anti‐Human globulin (AHG or Coombs) Reagent

Types of AHG reagent  Polyspecific Anti‐human Globulin: blend of Anti‐IgG and Anti‐C3b, ‐C3d  Monospecific reagents: Anti‐IgG alone or Anti‐C3b,‐C3d alone

• May be made by injecting rabbits , goats or sheep with purified human IgG or C3, then harvesting the antibodies produced by the rabbit. • Monoclonal technology may be used to make monoclonal antiglobulin reagent.

Note: Reagent does not contain antibodies to IgM. Information about IgM coating of cells comes from the presence of C3 coating the cells since IgM is a strong complement activator.

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Direct Antiglobulin Test (DAT)

DAT • The direct antiglobulin test (DAT) detects sensitized red cells with IgG and/or complement components C3b and C3d in vivo. • In vivo coating of red cells with IgG and/or complement may occur in any immune mechanism is attacking the patient's own RBC's. • These mechanism could be: – Autoimmunity – Alloimmunity – Or a drug‐induced immune‐mediated mechanism 7

Examples of autoimmune hemolysis

Examples of alloimmune hemolysis • Hemolytic transfusion reaction • Hemolytic disease of the newborn (also known as HDN or erythroblastosis fetalis)

• Warm antibody autoimmune hemolytic anemia – Idiopathic – Systemic lupus erythematosus – Evans' syndrome (antiplatelet antibodies and hemolytic antibodies)

– Rhesus D hemolytic disease of the newborn (also known as Rh disease) – ABO hemolytic disease of the newborn (the indirect Coombs test may only be weakly positive) – Anti‐Kell hemolytic disease of the newborn – Rhesus c, E hemolytic disease of the newborn – Other blood group incompatibility (RhC, Rhe, Kidd, Duffy, MN, P and others)

• Cold antibody autoimmune hemolytic anemia – Idiopathic cold hemagglutinin syndrome – Infectious mononucleosis – Paroxysmal cold hemoglobinuria (rare) 9

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Drug‐induced immune‐mediated hemolysis

Blood Sample

• Methyldopa (IgG mediated type II hypersensitivity)

• Whole Blood Sample ‐ It should be as fresh as possible not more than 24 hours old

• Penicillin (high dose) • Otherwise, the sample should be taken in EDTA. • Quinidine (IgM mediated activation of classical complement pathway and Membrane attack complex)

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Procedure of DAT

Indirect Antihuman Globulin Test (IAT)

1. 2.

Indications • The IAT is done to determine the presence of sensitization of red cells with IgG and/or complement in vitro in the following conditions.

3. 4. 5. 6.

Take 2‐3 drops of blood to be tested in a clean labeled tube. Wash the red cells 3‐4 times in a large volume of saline to remove free globulin molecules. Remove all supernatant after each wash. Completely decant the final supernatant wash. Add 2 drops of polyspecific AHG serum in 1 drop of sensitized washed red cells or in 1 drop of 3‐5 % suspension of sensitized cells immediately. Mix, Centrifuge at 1000 rpm for 1 minutes immediately. Gently shake the tube to dislodge the cell button and see for agglutination, use optical aid if needed, Record the result. Add 1 drop of IgG coated red cells to a negative test. Mix, centrifuge at 1000 rpm for 1 min. Immediately look for agglutination. If a negative result (no agglutination) is obtained the test result is invalid and whole test should be repeated. If agglutination is obtained, the result is valid.

1. Compatibility testing. 2. Screening and detection of unexpected antibodies in serum. 3. Determination of red cells phenotype K, Lea, Fya Fyb, Jka, Jkb and sub‐group of Rh etc by using known sera.

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Indirect antiglobulin test

Procedure: 1.

2. 3. 4. 5. 6.

Place 2‐3 drops of the test serum in a tube. Serum should be fresh for detecting complement components and complement binding antibodies, otherwise, fresh AB serum should be added to it. Add 1 drop of 3‐5% suspension of washed O Rh (D) positive red cells to the serum in the tube. Mix and incubate at 37°C for 30‐40 minutes. Centrifuge at 1000 rpm for 1 minutes. Examine for hemolysis and/or agglutination. Use optical aid if necessary. Agglutination at this stage indicates the presence of saline (complete) antibodies. If no agglutination is seen, wash cells 3‐4 times in large volume of saline. Decant supernatant in each wash as completely as possible.

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Procedure: 7. Add 2 drops of AHG serum to the cells. 8. Mix and centrifuge at 1000 rpm for 1 minutes immediately. 9. Gently shake the tube to dislodge the button and examine for agglutination, using optical aid. Record the result. 10. Add 1 drop of IgG coated red cells to any test that is negative. Mix and centrifuge at 1000 rpm for 1 minutes. Look for agglutination. If there is no agglutination, the test result is invalid and the whole test is repeated. If agglutination is obtained the result is valid. 11. Auto control should be kept with IAT.

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BOVINE ALBUMIN(22%)‐IAT

Sources of Error in AHG tests

One Stage Method - Additive method

False negative results: General DAT & IAT • Failure to wash red blood cells adequately, since globulins not bound to RBCs will neutralize the AHG reagent.

Procedure: 1. Two drops of albumin 22.5% are added in step (2) of saline‐IAT 2. Mix and incubate for 20‐30 minutes at 37°C 3. Proceed further as in saline‐IAT procedure.

– The washing process and the addition of AHG reagent must be undertaken as quickly as possible to minimize loss of bound antibodies by elution.

• Improper storage, bacterial contamination and contamination with human serum will impair the AHG reagent activity. – – – – –

Not adding the AHG reagent Improper centrifugation Number of cells present in the test: Too many cells give weak reactions Too few cells will impair the reading of the agglutination

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Antigen‐Antibody Ratio The optimum ratio is 80 parts antibody to 1 part antigen. There are specific terms for variations in this ratio. – Prozone ‐ antibody excess: Antibodies saturating all antigen sites; no antibodies forming cross‐linkages between cells; no agglutination – Zone of equivalence: antibodies and antigens present in optimum ratio, agglutination formed – Zone of antigen excess (Post‐zone): too many antigens ‐ any agglutination is hidden by masses of unagglutinated antigens

False negative results

False positive results:

DAT • All samples negative at the AHG phase should be incubated at room temperature for 5 minutes to achieve maximal sensitivity needed for complement detection.

DAT and IAT ; • In specimens containing potent cold‐reactive antibodies agglutination may occur before adding the AHG reagent. • Dirty glassware may cause clumping of cells. • Over centrifugation

IAT • Serum and/or RBCs lose reactivity if improperly stored. • Plasma used instead of serum can lead to failure to detect antibodies depending on presence of active complement (anti‐Jka, ‐Jkb) • Temperature and incubation time affect attachment of antibody or complement to cells. • An optimal proportion of serum to cells should be achieved: usually 2‐3 drops serum to one drop of 5% cell suspension.

DAT • A positive DAT from a clotted sample should be repeated on an EDTA sample • Samples collected from infusion lines may have complement present on the cells. IAT • Cells with a positive DAT will give a positive result in any indirect antiglobulin procedure. 23

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Coomb’s Cells

Preparation of Coomb’s cells

• To show that test cells were properly washed and that no neutralization or reagent deterioration has occurred, antibody‐coated cells are used as a positive indicator. • In a negative antiglobulin test the anti‐human globulin should remain active and this can be demonstrated by the addition of IgG sensitized cells. • Agglutination of the IgG sensitized cells after mixing and centrifuging confirms that the anti‐human globulin was added to the test, that the test cells were properly washed and all free globulin molecules were removed and that the anti‐human globulin was active. • Failure of the IgG sensitized cells to agglutinate indicates that the original negative antiglobulin test result is not valid and testing must be repeated.

• Preparing Coombs control cells is very easy. To about 10 drops of washed O Positive red cells add 5‐6 drops of anti‐D antisera. Incubate at 37C for 15 minutes. Wash 4 times then prepare a 3 to 5% cell suspension. • To verify reaction, add two drops of AHG into test tube and one drop of newly prepared Coombs cells. • Centrifuge on High speed for 15 seconds, You should get 1‐2 + reaction.

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Objectives ‐ Abo Discrepancies •

Training Program for Health Institute Graduates Laboratory Technician • • • •

Blood Grouping Discrepancies: ABO Discrepancies

Describe the reactions, list the clinical situations in which they may occur, and explain how to resolve each of the following causes of ABO discrepancies: *Decreased immunoglobulin levels *Weak subgroups of A with anti‐A1 *Passively transfused anti‐A1 *Unexpected alloantibody reacting at room temperature *Loss of A or B antigen *Acquired B antigen *Rouleaux *Cold agglutinins Explain what must be done if an ABO discrepancy cannot be resolved before the patient requires a transfusion. Explain what must be done when a discrepancy arises in a blood donor. List causes of technical or clerical errors that may cause ABO discrepancies. List causes for mixed‐field agglutination. 2

Definition • ABO Discrepancies must be resolved

Any deviation from the expected pattern of antigen on the cell and the opposite antibody in the serum .

• In recipients the discrepancies must be resolved before any blood component is transfused. If not resolved before blood is needed, transfuse Group O (O NEGATIVE if there is a discrepancy in the Rh type also). • In donors the discrepancies must be resolved before any blood is labeled with a blood type. 3

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General Rules to Resolve

Kinds of Discrepancies

• Always re‐test first.

• Clerical errors (transcription errors)

• Check for clerical/technical errors

• Technical errors

• Weakest reaction is usually the one in doubt.

• Problems with serum testing

• Check results of the screening cells. • Check the patient’s age.

• Problems with red cell testing

• Check the diagnosis

• Problems with both cells and serum

• Check the transfusion history. 5

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Clerical Errors (Transcription Errors)

Technical Errors

• Clerical errors are the most common.

There are a number of technical errors that may also occur: 1. Sample: mix‐up 2. Reagents: o Failure to add serum or reagent. Remember for both ABO and Rh always add your reagent antisera and serum before adding cells. o Addition of wrong reagent o Contaminated reagents could result in either false negative or false positive results

• Record the results as you read each tube • On the right worksheet. • One patient or donor at a time. • Record on the right spot of worksheet (this is why labeling procedure uses capital A and B for the forward type and a1C and bC for the reverse typing) 7

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3. Cell suspension: Too many cells in your cell suspension can lead to decreased or negative reactions since there are too many cells for the number of antibodies present in the reagents. Remember we want to be in the zone of equivalence for our reactions. 4. Centrifugation: Under or Over 5. Incubation: Warming the test could result in a false negative reaction since ABO antibodies are IgMs that react better in the cold.

6. Interpretation: o o o

Failure to detect weak results can occur if you are not watching the reactions while you are shaking them out or if you shake too hard. Failure to detect hemolysis can be a definite problem. Remember a positive reaction can be hemolysis as well as agglutination since the antigen‐antibody reaction can bind complement. When complement is bound it can lead to hemolysis that is also an indication of a positive reaction.

7. Dirty glassware can cause the cells to artificially clump.

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Problems With Serum Testing

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1. Weak or Missing Antibody (ies)

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1‐Weak or Missing Antibody(ies)

1‐Weak or Missing Antibody(ies)

Causes:

An extreme example would be no reaction for the forward and reverse typings. The steps to follow to resolve this discrepancy is to: • Check birth date since newborns and the elderly are more likely to demonstrate this discrepancy. Newborn antibodies are not present until at least 6 months. DON'T ATTEMPT TO SERUM‐CONFIRM NEWBORNS. As individuals ages they may also lose their ability to maintain their antibody levels. Therefore, the very elderly have decreased antibody levels.

1. Extreme age 2. Immunocompromized

• Check diagnosis since patient conditions such as: Immune deficiencies, Chemotherapy, Radiation Therapy, and Bone marrow transplantation may explain the missing antibodies. 15

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Resolution of Missing Antibodies • Add two more drops of serum just in case you forgot to add them the first time and centrifuge. If negative then incubate in cold (4‐18oC) 15‐30 MINUTES • Include autocontrol to rule out interference from natural anti‐I when incubating at (4‐18oC). (At 4oC Anti‐A and Anti‐B enhanced since they are saline, cold‐acting antibodies as seen in this example for an O individual.)

Compare this with a 4oC Auto‐Anti‐I enhanced would have a positive autocontrol as seen in the example below:

Group A or Group B can serve as its own negative control. 4oC Anti‐B enhanced is shown below:

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4oC Anti‐I enhanced on the other hand would have a positive autocontrol.

If anti‐I enhanced along with anti‐A or anti‐B, can re‐set up and incubate at 18oC. As seen in this example of 18oC: Anti‐B enhanced, anti‐I nonreactive

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A. Presence of Unexpected Anti‐A when the Immediate Spin Antibody Screening is Negative

2. Presence Of Excess Antibodies • 1‐Subgroup A • 2‐Passive transfused Ab

The presence of Anti‐A1 should be suspected when the antibody is reactive against the A cells but not the screening cells at immediate spin as seen in the example below

• 3‐Alloantibodies (cold reacting) • 4‐Excess serum protein(Rouleaux)

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Naturally anti‐A1 occurs in subgroups of A or are passively‐ transfused from Group O platelets and other blood products. How to Resolve the Issue of Unexpected Anti‐A: 1. Check recent transfusion history for group O products, (especially platelets) that would explain the presence of this antibody.

3. Test patient serum with three A1 cells and three A2 cells and if it is an anti‐A1 the following reactions will occur: Anti‐A1: SERUM + A1 CELLS = + SERUM + A2 CELLS = 0 Anti‐A1 will react only with the A1 cells but not with the A2 cells

2. Test patient cells with lectin‐A1. Subgroups will be negative with this reagent but A1cells will be positive. Lectin + A1 CELL = 4+ Lectin + A subgroups CELLS = 0

4. In the case of passive Anti‐A from Group O Platelets the reactions would be the following: SERUM + A1 CELLS = + SERUM + A2 CELLS = + In this case if the antibody is strong enough you may need to transfuse group O blood . 25

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How to Resolve the Issue of Unexpected Anti‐A that is probably another antibody due to the results of the Antibody Screening: (Alloimmunization) • Identify the antibody by performing an identification panel at room temperature. • Pre‐warm away (use caution) the effect of this antibody by doing the reverse typing with prewarmed serum and reagent cells. • Type reagent A1 or B cell for the corresponding antigen once the antibody is identified. • For example, if the patient had an anti‐N that was showing up at room temperature according to the antibody identification process, you would then type for N on the reagent cells used for the reverse typing. If anti‐N is causing your problem, then the cells should have N antigen present.

B. Unexpected A Or B Antibody when the Immediate Spin Antibody Screening is Positive You may have a positive reaction with the reagent A1 or B cell that is due to a room‐ temperature antibody reacting with an antigen other than A or B on the cells

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Rouleaux Formation Giving Unexpected Agglutination in all Serum Tests Rouleaux may also give false positive cell typing if strong enough and cells are insufficiently washed. This phenomenon is due to alteration in serum protein concentration such as:

Rouleaux can give unexpected agglutination in all serum tests

– – – –

Multiple myeloma Macroglobulinemia Liver disease (decreased albumin) Also seen with volume expanders

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Problems with Cell Typing Characteristics of rouleaux is that it: • Looks like agglutination macroscopically • Microscopically it appears as "stacks of coins" How would you resolve rouleaux problems? • Do saline replacement technique: – – – – –

Re‐centrifuge the test tube. Draw off serum without disturbing cell button Add two drops of saline Resuspend Rouleax disperses in saline; TRUE AGGLUTINATION REMAINS

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1. Mixed‐Field Agglutination

Mixed‐Field Agglutination

Mixed‐field agglutination is seen as large or small agglutinates with many un‐agglutinated cells. Usually mixed‐field agglutination means a MIXED‐CELL POPULATION The causes of mixed‐field agglutination can be:

1. Massive transfusion of another blood group ("O" red blood cell) 2. Bone marrow transplant patients 3. Weak subgroups of A3 . 4. Chimerism due to intrauterine exchange of erythrocyte precursors between twins or 2 fertilized eggs fuse into one individual. Check the patient's transfusion records and clinical history. If it appears to be a weak subgroup performed the tests discussed under Unexpected Anti‐A. 33

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How would you resolve a weak, or missing, antigen?

2. Weak or Missing Antigen

• Obtain recent transfusion history and any clinical history of bone marrow transplant • Read forward grouping microscopically • Use anti‐A,B and incubate at 4‐22oC at least 15 minutes • Use monoclonal antisera that is known to react with antigens like Ax and Bx • Perform specialized tests if the above steps do not resolve the problem: • Specialized tests would include absorption/elution techniques and saliva studies.

May be due to : 1) Very weak subgroup of A or B, 2) Loss of transferase in acute leukemia, 3) Massive transfusion of GROUP O, or 4) Bone marrow transplant

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Acquired B

3‐Acquired B Antigen Acquired B antigens are seen in problems with the colon or infections with Gram‐negative rods

• Bacteria (E. coli) have a deacetylating enzyme that effects the A sugar….

Bacterial enzymes modify the "A" antigen to a "B" antigen and the patient forward types as an AB but reverses as an A.

Group A individual

N‐acetyl galactosamine

Acquired B Phenotype

Galactosamine now resembles D‐ galactose (found in Group B)

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How would you resolve a possible acquired B antigen?

4‐Polyagglutinable cells

• Set‐up an autocontrol. The patient's own anti‐B will not agglutinate their own AB cells.

Most monoclonal anti‐A and anti‐B will show problems with polyagglutinable cells if it is a problem with the cell membrane that leads to the agglutination. • The most likely causes of due to : 1‐Wharton's Jelly, found in cord blood, and strong positive direct antiglobulin test due to a cold agglutinin. 2‐Strong positive DAT, it would appear to be an AB in the forward type and an O on reverse.

• Check clinical history to evidence of colon problems or Gram‐ negative rods. • Check monoclonal anti‐B product inserts since some will not react with B acquired antisera • Acidify some reagents anti‐B to pH 6 and re‐test. Modified (acquired) B antigens will not react in the acidified antiserum, normal B antigens will still react. 39

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Wharton's jelly • Coats newborn cord cells and the child's type may appear AB. You do not do a reverse on newborn blood since they have not made any anti‐A or anti‐B yet. • If the baby types as an AB recheck by washing cells several times and re‐ testing since you need to make sure you have removed the Wharton's Jelly and the baby is truly an AB. Better yet Always wash cord blood at least 4 TO 5 X's before determining the type of the baby. Strong positive DAT • May be seen in cold auto‐immune hemolytic anemia • If due to cold agglutinin, wash several times in warm saline and re‐test • Cells washed 3X at 37oC would probably look like this: 42

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Problems with Both Cells and Serum

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Strong Cold Auto‐Agglutinins A strong cold auto‐agglutinin is most often due to strong auto‐anti‐I. 1. To resolve cell typing difficulties: • Wash cells 3‐4X with warm (37oC) saline • Re‐test warm‐washed cells 2. To resolve serum typing difficulties: • Perform serum testing at 37oC (Use caution that weak isoagglutinins (anti‐A and anti‐B) are not missed using this technique) • Autoabsorb cold agglutinins onto patient cells at 4oC.

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Statement of Purpose • The Blood Transfusion Service performs tests for serologic compatibility between patient and donor blood prior to transfusion, except in case of urgent blood need.


• To minimize the risk of hemolytic transfusion reaction and maximize post‐transfusion red cell survival. Guidelines Of Pretransfusion Compatability Procedures and Neonatal Transfusion Policy

2

Transfusion request/ Samples

Transfusion Request/ Samples

• The Majority of ABO‐ incompatible transfusion are due to documentation/Identification errors. • Transfusion requests must be prescribed by a medical officer. • The request form and sample contain the following minimum identification:

• Information concerning the sex of the patient and obstetric and recent transfusion history should be obtained . • Requests should include the date and time required, the number or volume and type of components required, the reason for request and any other specific requirements relating to the patient or request i.e. irradiated, filtered blood. • Samples received from trauma or from unconscious accident or emergency patient must contain at least one identifier like trauma or emergency number and sex of the patient and must be signed by medical officer. • If one identifier not available and in‐life threatening situation so give him (O)Negative blood.

– – – –

(a) Surname (b) First name(s) (c) Date of birth (d) Hospital number/accident and emergency number. 3

Sample Requirements

Storage of Samples

• Clotted or EDTA sample may be used for pretransfusion testing. • Whole blood sample deteriorate due to red cell lysis, loss of complement, decrease potency of red cell antibodies and bacterial contamination. • In situations in which patients are being repeatedly transfused it is not necessary to require daily antibody screening as it is valid for 72 h. and make only immediate spin cross‐matching • Patients with no history of transfusion or pregnancy in the last 3 months antibody screening can be valid for one week

EDTA whole blood

Serum

5

18 – 25°C

4°C

‐ 30°C

Up to 48 h.

Up to 7 days

N/A

N/A

Up to 7 days

Up to 6 months

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Receiving Blood Request • Receptionist must sure the request data and sample full filled the requirements mentioned. • The request form and sample tube shall carry identical patient identification information., in case of discrepancy or doubt, the officer‐in‐charge of the blood bank shall be noted. Unlabelled samples shall be discarded. • Search for the history of the patient in computer system if available or in type& screen file which contains the results of blood group and antibody screening of all patients receiving blood in the last 72h • If there is any data for the recipient (patient) in the records of blood bank must write blood group and result of antibody screening and last time received blood. • Receptionist must write the time of receiving the request and sign.

Pre‐transfusion Compatibility Tests for Patients> 4 Months Old

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1. ABO&Rh grouping

2. Antibody screening

1. Cell and serum typing using ID Gel card (see Sop#CBB035) and if not available, use tube method 2. Controls must be included in each beginning of shift, using new reagents or new lot number of ID‐Gel card. 3. The ABO&Rh grouping must be verified against previous results from type&screen file or from computer records if available 4. Any discrepancies must be resolved prior transfusion of red cells and in the 5. In case of emergent request choose (O) RH negative units. 6. In Rh negative patient not make weak D test and in case of partial D consider

• Antibody screening more reliable and sensitive than crossmatching against donor red cells so should performed in all pretransfusion testing . • Antibody screening is valid for 72h in negative result. so if negative can give patients blood without repeating antibody screening only repeat ABO&Rh grouping then immediate spin crossmatching

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3. Antibody Identification

4. Selection of Blood

• When irregular antibody is detected in the antibody screening identification must be done to determine its specificity and clinical significant.

• In case of transfusion of whole blood must be ABO RhD identical. • Red cell components of the same ABO and RhD group as the patient must be selected whenever possible.

• If the antibody screening is positive must repeat identification for each request of blood transfusion

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5. Immediate Spin Crossmatch

6. IAT crossmatch

• Done when antibody screening is negative from sample taken within 72 h. which is the time limit for the validity of the test. • A short incubation time of 2‐5 min. before centrifugation is recommended. • Done only when the blood in need so do not begin selection and immediate spin crossmatch until blood in need and made within 10 minutes. • It is recommended all procedures of compatibility done by one person but in case of stand by request every technician sign for work done by him.

• Done when antibody screening result is positive and must be repeated for every request after making identification but if request is urgent can be made without identification using trials units. • There must be a compatibility label which should be securely attached to the blood bag and include patient name, hospital number ,blood group and the date blood required/crossmatched. 13

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Visual inspection of red cell unit • Before the unit is placed in blood issue refrigerator it should be inspected for:

Pre‐transfusion Compatibility Tests for Neonates (0‐ 4 Month Old)

– (a) Integrity of the pack by checking for leaks – (b) Evidence of hemolysis in plasma (c) evidence of discoloration – (d) Presence of large clots

• If there is any evidence of the above the unit should be not used

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1. ABO&Rh Grouping &DCT

2. Antibody screening

1. Cell typing and DCT using ID‐ gel card for neonate and if not available use tube method

• Make Antibody screening using mother serum which is the first choice and if not available use neonatal eluate or neonate serum.

2. Verify the result against previous result from type & screen file or from computer record

• Antibody screening is valid until new born is 4 months old

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3. Selection of Blood

4. Crossmatching

• If the DCT is negative and blood group of the mother is known so give him PRBCS according to the following:

• Antibody screening result is valid until new born is 4 months old. • If antibody screening is negative no need for doing crossmatching and give ABO&Rh compatible blood only . • If antibody screening is positive Antibody Identification must be done and select blood unit negative for antigen which can react with antibody specified by antibody identification followed by AHG crossmatching

– If DCT is negative and blood group of mother is unknown so give him (O) and Rh of neonate. – If DCT is negative and blood group of mother unknown and PRBCS blood goup (O) not available so must make reverse grouping until AHG phase for neonate serum to detect Anti‐ A or/and Anti‐B which may be transferred to him from mother and choose blood group compatible with that. – If DCT is positive you must select (O) Rh negative units

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5. Special Notes

References

• Blood should be not more than 5 days old for exchange transfusion. • Packed red cells should be reconstituted with AB plasma at the time of issue for exchange transfusion. • If the mother’s antibody is reactive against a high frequency antigen and no compatible blood is available Mother’s siblings can be tested for compatible blood. A unit of blood can be collected from mother if the obstetrician agrees that is safe. Mother’s red cells should be constituted in AB plasma

• AABB Technical Manual, 14th Edition; 2008. • AABB Standard 23rd Edition; 2005

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The Basics….. • As you recall,


– Antibody Screens use 2 or 3 Screening Cells to “detect” if antibodies are present in the serum – If antibodies are detected, they must be identified…


present

Not present

Antibody Identification 2

Why do we Need to Identify?

Key Concepts

• Antibody identification is needed for transfusion purposes and is an important component of compatibility testing

• In blood banking, we test “knowns” with “unknowns”

• It will identify any unexpected antibodies in the patient’s serum • If a person with an antibody is exposed to donor cells with the corresponding antigen, serious side effects can occur

Unknown

Known

Patient serum

+ Reagent RBCs

Patient RBCs

+ Reagent antisera

• When detecting and/or identifying antibodies, we test patient serum (unknown) with reagent RBCs (known)

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Reagent RBCs

Antibody Panel vs. Screen

• Screening Cells and Panel Cells are the same with minor differences:

• An antibody panel is just an extended version of an antibody screen • The screen only uses 2‐3 cells:

– Screening cells • Antibody detection • Sets of 2 or 3 vials – Panel cells • Antibody identification • At least 10 vials per set

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Antibody Panel

Panel

• An antibody panel usually includes at least 10 panel cells:

• Group O red blood cells

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Panel

Panel

• Each of the panel cells has been antigen typed (shown on antigram)

• An autocontrol should also be run with ALL panels

– + refers to the presence of the antigen – 0 refers to the absence of the antigen

Autocontrol Patient RBCs + Patient serum Example: Panel Cell #10 has 9 antigens present: c, e, f, M, s, Leb, k, Fya, and Jka

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Panel

Antibody ID Testing

• The same phases used in an antibody screen are used in a panel

• A tube is labeled for each of the panel cells plus one tube for AC:

IS 37°

1

2

3

4

5

6

7

8

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10

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AHG

  11

1 drop of each panel cell

AC

+

2 drops of the patients serum 12

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IS Phase • •

(LISS) 37°C Phase

Perform immediate spin (IS) and grade agglutination; inspect for hemolysis Record the results in the appropriate space as shown:

• 2 drops of LISS are added, mixed and incubated for 10‐15 minutes • Centrifuge and check for agglutination

2+ 0 0

• Record results

Last tube

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IAT Phase (or AHG)

(LISS) 37°C Phase

• Indirect Antiglobulin Test (IAT) – we’re testing whether or not possible antibodies in patient’s serum will react with RBCs in vitro • To do this we use the Anti‐Human Globulin reagent (AHG)

2+ 0 0 0 0 0 2+ 0 0 2+ 0 2+ 0 0

– Polyspecific – Anti‐IgG – Anti‐complement

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AHG Phase

AHG Phase

• Wash cells 3 times with saline (manual or automated) • Add 2 drops of AHG and gently mix

2+ 0 0 + 2 0 0 2+ 0 2+ 0 0 0

– Centrifuge – Read – Record reactions

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

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IS

And don’t forget….

2+ 0 0 2+ 0 0 2+ 0 2+ 0 0

….add “check” cells to any negative AHG ! 21

LISS AHG 37° 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0

CC           

All cells are negative at AHG, so add “Check” Cells

22

Interpreting Antibody Panels

You have agglutination…now what?

There are a few basic steps to follow when interpreting panels

CC

2+ 0 0 2+ 0 0 2+ 0 2+ 0 0 0

??

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

 

1. “Ruling out” means crossing out antigens that did not react 2. Circle the antigens that are not crossed out 3. Consider antibody’s usual reactivity 4. Look for a matching pattern

         

23

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363

Always Remember:

1. Ruling Out

An antibody will only react with cells that have the corresponding antigen; antibodies will not react with cells that do not have the antigen

2+ 0 0 + 2 0 0 2+ 0 2+ 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

           

Cross out antigens that show NO REACTION in any phase; do NOT cross out heterozygous antigens that show dosage. 25

26

2. Circle antigens not crossed out

3. Consider antibody’s usual reactivity

2+ 0 0 2+ 0 0 2+ 0 2+ 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

2+ 0 0 + 2 0 0 2+ 0 2+ 0 0 0

         

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

           

Lea is normally a Cold‐Reacting antibody (IgM), so it makes sense that we see the reaction in the IS phase of testing; The E antigen will usually react at warmer temperatures

 

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4. Look for a matching pattern

Interpretation

E doesn’t match and it’s a warmer rx Ab

2+ 0 0 + 2 0 0 2+ 0 2+ 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

 

anti‐ Lea

         

…Yes, there is a matching pattern! 29

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Guidelines

About reaction strengths……

• Again, it’s important to look at:

• Strength of reaction may be due to “dosage”

– Autocontrol • Negative ‐ alloantibody • Positive – autoantibody or DTR (i.e.,alloantibodies) – Phases • IS – cold (IgM) • 37° ‐ cold (some have higher thermal range) or warm reacting • AHG – warm (IgG)…significant!! – Reaction strength • 1 consistent strength – one antibody • Different strengths – multiple antibodies or dosage

– If panel cells are homozygous, a strong reaction may be seen – If panel cells are heterozygous, reaction may be weak or even non‐reactive

• Panel cells that are heterozygous should not be crossed out because antibody may be too weak to react (see first example)

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Guidelines (continued)

Rule of three

• Matching the pattern

• The rule of three must be met to confirm the presence of the antibody • A p‐value ≤ 0.05 must be observed • This gives a 95% confidence interval • How is it demonstrated?

– Single antibodies usually shows a pattern that matches one of the antigens (see previous panel example) – Multiple antibodies are more difficult to match because they often show mixed reaction strengths

– Patient serum MUST be: • Positive with 3 cells with the antigen • Negative with 3 cells without the antigen

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Our previous example fulfills the “rule of three”

3 Positive cells

3 Negative cells

2+ 0 0 2+ 0 0 2+ 0 2+ 0 0 0

What if the “rule of three” is not fulfilled? 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0

• If there are not enough cells in the panel to fulfill the rule, then additional cells from another panel could be used • Most labs carry different lot numbers of panel cells

           

Panel Cells 1, 4, and 7 are positive for the antigen and gave a reaction at immediate spin 35 Panel Cells 8, 10, and 11 are negative for the antigen and did not give a reaction at immediate spin

36

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Phenotyping

Remember Landsteiner’s Rule

• In addition to the rule of three, antigen typing the patient red cells can also confirm an antibody • How is this done?

Individuals DO NOT make allo‐antibodies against antigens they have

– Only perform this if the patient has NOT been recently transfused (donor cells could react) – If reagent antisera (of the suspected antibody) is added to the patient RBCs, a negative reaction should result…Why?

37

38

Multiple antibodies

So what is a tech to do?

• Multiple antibodies may be more of a challenge than a single antibody • Why?

• Several procedures can be performed to identify multiple antibodies – Selected Cells – Neutralization – Chemical treatment • Proteolytic enzymes • Sulfhydryl reagents • ZZAP

– Reaction strengths can vary – Matching the pattern is difficult

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Selected Cells

Selected Cells

• Selected cells are chosen from other panel or screening cells to confirm or eliminate the antibody • The cells are “selected” from other panels because of their characteristics • The number of selected cells needed depends on how may antibodies are identified

• Every cell should be positive for each of the antibodies and negative for the remaining antibodies • For example: – Let’s say you ran a panel and identified 3 different antibodies: anti‐S, anti‐Jka, and anti‐P1 – Selected cells could help…

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366

Selected Cells Selected cells

Neutralization

S

Jka

P1

IS

LISS 37°

AHG

#1

+

0

0

0

0

2+

#5

0

+

0

0

0

3+

#8

0

0

+

0

0

0

• Some antibodies may be neutralized as a way of confirmation • Commercial “substances” bind to the antibodies in the patient serum, causing them to show no reaction when tested with the corresponding antigen (in panel) 

These results show that instead of 3 antibodies, there are actually 2: anti‐S and anti‐Jka 43

44

Neutralization

Neutralization

• Manufacturer’s directions should be followed and a dilutional control should always be used

• Common substances – – – –

– The control contains saline and serum (no substance) and should remain positive – A control shows that a loss of reactivity is due to the neutralization and not to the dilution of the antibody strength when the substance is added

P1 substance (sometimes derived from hydatid cyst fluid) Lea and Leb substance (soluble antigen found in plasma and saliva) I substance can be found in breast milk Sda substance derived from human or guinea pig urine

**You should be aware that many of these substances neutralize COLD antibodies; Cold antibodies can sometimes mask more clinically significant antibodies (IgG), an important reason to use neutralization techniques

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Enzymes (proteolytic)

Enzymes

• Can be used to enhance or destroy certain blood group antigens • Several enzymes exist:

• Enzymes remove the sialic acid from the RBC membrane, thus “destroying” it and allowing other antigens to be “enhanced” • Antigens destroyed: M, N, S, s, Duffy • Antigens enhanced: Rh, Kidd, Lewis, I, and P

– Ficin (figs) – Bromelin (pineapple) – Papain (papaya)

• In addition, enzyme procedures may be – One‐step – Two‐step

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Enzyme techniques

Enzyme techniques

• One‐stage

• If there is no agglutination after treatment, then it is assumed the enzymes destroyed the antigen

– Enzyme is added directly to the serum/cell mixture

• Two‐stage – Panel cells are pre‐treated with enzyme, incubated and washed – Patient serum is added to panel cells and tested

49

Enzyme treatment

50

Enzyme treament

Sulfhydryl Reagents • Cleave the disulfide bonds of IgM molecules and help differentiate between IgM and IgG antibodies • Good to use when you have both IgG and IgM antibodies (warm/cold)

Anti‐K

– Dithiothreitol (DTT) is a thiol and will denature Kell antigens – 2‐mercaptoethanol (2‐ME) Perfect match for anti‐Fya

•Duffy antigens destroyed •Kell antigens not affected

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ZZAP

Autoantibodies….

• A combination of proteolytic enzymes and DTT • Denatures Kell, M, N, S, Duffy and other less frequent blood group antigens • Does not denature the Kx antigen • Good for adsorption techniques

• Warm & Cold Reacting

– “frees” autoantibody off patient’s cell, so that autoantibody can then be adsorbed onto another RBC

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Autoantibodies

Getting a positive DAT

• Autoantibodies can be cold or warm reacting • A positive autocontrol or DAT may indicate that an auto‐antibody is present • Sometimes the autocontrol may be positive, but the antibody screening may be negative, meaning something is coating the RBC

• We have focused a lot on the IAT used in antibody screening and ID, but what about the DAT? • The direct antiglobulin test (DAT) tests for the in vivo coating of RBCs with antibody (in the body) • AHG is added to washed patient red cells to determine this

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What can the DAT tell us?

Identifying autoantibodies

• Although not always performed in routine pretransfusion testing, a positive DAT can offer valuable information

• Auto‐antibodies can sometimes “mask” clinically significant allo‐antibodies, so it’s important to differentiate between auto‐ and allo‐antibodies

– If the patient has been transfused, the patient may have an alloantibody coating the transfused cells – If the patient has NOT been transfused, the patient may have an autoantibody coating their own cells

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Cold autoantibodies

Cold autoantibodies

• React at room temperature with most (if not all) of the panel cells and give a positive autocontrol • The DAT is usually positive with anti‐C3 AHG (detects complement) • Could be due to Mycoplasma pneumoniae, infectious mono, or cold agglutinin disease

• Mini‐cold panels can be used to help identify cold autoantibodies • Since anti‐I is a common autoantibody, cord blood cells (no I antigen) are usually included Group O individual with cold autoanti‐I

Group A individual with cold autoanti‐ IH Anti‐IH is reacting weakly with the cord cells (some H antigen present) 59

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Avoiding reactivity

Other techniques

• Cold autoantibodies can be a nuisance at times. Here are a few ways to avoid a reaction:

• If the antibodies remain, then prewarmed techniques can be performed: – Red cells, serum, and saline are incubated at 37° before being combined

– Use anti‐IgG AHG instead of polyspecific. Most cold antibodies react with polyspecific AHG and anti‐C AHG because they fix complement – Skipping the IS phase avoids the attachment of cold autoantibodies to the red cells – Use 22% BSA instead of LISS

• Autoadsorption is another technique in which the autoantibody is removed from the patients serum using their own red cells – The serum can be used to identify any underlying alloantibodies

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62

Warm autoantibodies

Warm autoantibodies

• More common that cold autoantibodies • Positive DAT due to IgG antibodies coating the red cell • Again, the majority of panel or screening cells will be positive • The Rh system (e antigen) seems to be the main target although others occur

• Cause warm autoimmune hemolytic anemia (WAIHA)…H&H • How do you get a warm autoantibody? – Idiopathic – Known disorder (SLE, RA, leukemias, UC, pregnancy, infectious diseases, etc) – Medications

• Several techniques are used when warm autoantibodies are suspected…

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Elution (whenever DAT is positive)

Elution • The eluate is a term used for the removed antibodies • Testing the eluate is useful in investigations of positive DATs

Elution techniques “free” antibodies from the sensitized red cells so that the antibodies can be identified

– HDN – Transfusion reactions – Autoimmune disease

Y

Y

Y

• The red cells can also be used after elution for RBC phenotyping if needed • When tested with panel cells, the eluate usually remains reactive with all cells if a warm autoantibody is present

Y Positive DAT

Y

Sensitized RBC

Frees antibody

Antibody ID 65

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Elution Methods

Adsorption

• Acid elutions (glycine acid)

• Adsorption procedures can be used to investigate underlying alloantibodies • ZZAP or chloroquine diphosphate can be used to dissociate IgG antibodies from the RBC (may take several repeats) • After the patient RBCs are incubated, the adsorbed serum is tested with panel cells to ID the alloantibody (if present)

– Most common – Lowers pH, causing antibody to dissociate

• Organic solvents (ether, chloroform) – Dissolve bilipid layer of RBC

• Heat (conformational change) • Freeze‐Thaw (lyses cells)

ABO antibodies

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Adsorption Remove serum and test for alloantibody

Two types:

2 tubes

1. Autoadsorption • No recent transfusion • Autoantibodies are removed using patient RBCs, so alloantibodies can be identified 2. Allogenic (Differential) adsorption • If recently transfused • Uses other cells with the patients serum

Wash x3 after incubation

Centrifuge after incubating; and transfer serum to 2nd tube of treated cells; incubate and centrifuge again

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More reagents….

Chloroquine diphosphate

• Many of elution tests can damage the antigens on the RBC • Choroquine diphosphate (CDP) and glycine acid EDTA reagents can dissociate IgG from the RBC without damaging the antigens

• Quinilone derivative often used as an antimalarial • May not remove autoantibody completely from DAT positive cells • Partial removal may be enough to antigen type the cells or to be used for autoadsorption of warm autoantibodies

– Very useful if the RBC needs to be antigen typed

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The Infectious Diseases Transmitted by Blood Transfusion:


The infectious microbes that transmitted by blood transfusion: 1‐ Hepatitis B and C 2‐ HIV 3‐ HTLV 4‐ Cytomegalovirus (CMV) 5‐ Epstein‐Barr virus 6‐ Human Parvovirus (B19) 7‐ Human Herpesvirus 8‐ Bacterial contamination 9‐ Syphilis 10‐ Malaria 11‐ West Nile Virus (WNV)


Transfusion Transmitted Diseases (TTD)

2

Factors That Play a Role in Establishment of Blood Transfusion Infection

Blood Borne Pathogents

1‐ Viral Window Period is the period between the onset of viral infection and the appearance of detectable antibodies to the virus.

What are blood borne pathogens? – Micro‐Organism: • Hepatitis B (HBV) • Hepatitis C (HCV) • Human Immunodeficiency Virus (HIV)

2‐ The Genetic Vertical transmission of viruses. 3‐ Donor immune status (Asymptomatic immuno‐competent patients).

– Substances that are carried by the blood or other body fluids and cause illness or injury to the body.

4‐ Laboratory and personal error.

– Virus and bacteria are pathogens and many are “Blood borne”.

5‐ Bacterial contamination. 3

4

Types of Blood Borne Pathogens

Blood Borne Pathogens Hepatitis ‐ An inflammation of the liver usually caused by drugs, toxins, autoimmune disease, or infectious agents. ‐ Potentially life threatening blood borne pathogen. ‐ Potential for carriers to pass disease to others. ‐ Effects can be both acute and chronic.

Include: Execution

Hepatitis: A, B, C, D, E

Viruses or bacteria that are carried in blood and cause disease in People.

Human Immunodeficiency Virus (HIV)

Brucellosis

Malaria

Hepatitis A Hepatitis D

Syphilis 5

372

Hepatitis B Hepatitis E

Hepatitis C

6

Blood Borne Pathogens

Hepatitis A (HAV)

The most commonly concerned of Hepatitis are:

• Transmitted via contaminated food or water which contains fecal matter containing the virus. There is a vaccine to prevent HAV. • Two Types of HAV: 1. Infectious (transmitted person to person by the fecal‐oral route) or 2. Serum (transmitted by transfusion of blood products). 7

8

Hepatitis B (HBV)

Hepatitis B

• Transmitted by injections transporting a virus‐ bearing serum, most often during blood transfusions and by contaminated needles and syringes. • Transmitted primarily through “Blood to Blood” contact. • Very durable, and it can survive in dried blood for up to seven days. • This virus is the primary concern for housekeepers, custodians, laundry personnel and other employees who may come in contact with blood or potentially infectious materials in a non first‐aid or medical care situation.

• • • • • •

Transmitted through parenteral, sexual exposure Mean incubation time 90 days 50% of infections are symptomatic 1/500 infections are lethal 6 ‐10% of infections become chronic Vaccination makes donor anti‐HBs+, HBsAg‐, anti HBc‐ • Risk of transmission 1/66,000

9

10

Hepatitis B Virus Geographic Distribution

Symptoms of HBV • • • • • • • • • 11

Mild flu‐like symptoms Fatigue Yellow Eyes and Skin Possible stomach pain Loss of appetite Fever and Vomiting Nausea Jaundice Darkened Urine 12

373

HBV‐Seroconversion in Early Infection

Markers of HBV Infection

13

14

Hepatitis C (HCV)

Hepatitis C Virus – Geographic Distribution

• Transmitted in blood or body fluids. No vaccination exists for HCV. • Chronic liver disease develops in about 70% of persons who become infected with HCV and nearly all (85%‐100%) persons with acute HCV infection become persistently infected. • Antibody appears in serum 54 – 192 days post‐ infection 15

16

Hepatitis C Transmission in Developed Countries

Hepatitis C Virus – Mode of Transmission  Injection drug use

In developing countries, the primary sources of HCV infection are unsterilized injection equipment and infusion of inadequately screened blood and blood products.

 Receipt of donated blood, blood products, and organs.  Needlestick injuries in healthcare settings (3%).  Birth to an HCV‐infected mother.  Sex with an HCV‐infected person.  Sharing personal items contaminated with infectious blood (razors or toothbrushes).

17

18

374

Viraemia and Seroconversion During Early HCV Infection Anti-HCV EIAs

Hepatitis D (HDV)

1st gen 150 d 2nd gen 80 d 3rd gen 70 d

Plateau phase viremia: 105-108 gEq/mL

• One of the newer types and it is transmitted primarily through injected drug use and sexual contact.

HCV RNA

Ramp-up phase Viral set-point: 102-107 gEq/mL

- HCV Ag EIA - HCV MP-NAT

Pre-rampup blip viremia 0

• Prevention: – Education to reduce risk behaviors for those with chronic HBV infection.

ALT

- HCV ID-NAT 10

20

30

40

50

60

70

80

90

100

Days following infection Glynn et al, Transfusion 2007

19

20

21

22

Hepatitis D Virus – Defective single stranded RNA virus. – Only in patients with HBV infection. – Requires HBsAg in order to synthesize an envelope protein.

– Screening donors for HBV infection eliminates the risk for HDV.

Human Immuno‐deficiency Virus (HIV)

Hepatitis G Virus

• HIV is the virus associated with AIDS

• Also called GBV‐C is a Flavivirus distantly related to HCV.

– There is no specific treatment for it – There is no cure – There is no preventative vaccine

• Recent reports do not implicate HGV/GBV‐C as a cause of hepatitis or any other disease manifestation.

• HIV attacks the body immune’s system, weakening it so that it cannot fight other deadly disease. • HIV is very fragile and will not survive very long outside of the human body. 23

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Symptoms of HIV

Transmission of HIV

Symptoms of HIV infection can vary, but often include: – Weakness – Fever – Sore throat – Nausea – Headaches – Diarrhea – White coating on the tongue – Weight loss – Swollen lymph glands

• Sexual intercourse (0.11‐ 1.7%). • blood or blood products (90%). • Needle stick injury of Health care workers (0.3% transmission). • Intravenous drug users ( 0.67%). • From mother to child (25 – 35%): – Transplacentally – During birth – Breast feeding 25

26

Adults & Children Estimated to be Living with HIV in 2009

Incubation Period of HIV • Time from exposure to HIV until onset of acute clinical illness is 1– 4 weeks. • After primary infection there is a period ranging from a few months to more than 10 years with no or mild symptoms before the appearance of severe immunodeficiency.

27

28

Human T‐Cell Lymphotropic Viruses (HTLV‐I & II)

HIV Viraemia Markers

Mode of Transmission:

Peak viremia: 106-108 gEq/mL HIV RNA (plasma)

Ramp-up viremia DT = 21.5 hrs

 Sexual transmission.

HIV Antibody

 Intravenous drug abuse.

HIV p24 Ag

 Blood transfusion.

p24 Ag EIA HIV MP-NAT -

1st

HIV ID-NAT -

“blip” viremia 0

10

16 20

gen

 Breast feeding.

2nd gen

3rd gen 11

Viral set-point: 102 -105 gEq/mL

22 30

40

50

60

70

80

90

100

Closing the WP through improved screening tests 29

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Human T‐Cell Lymphotropic Viruses (HTLV‐I & II)

Laboratory Diagnosis • ELISA for screening and Western blot for confirmation .

Symptoms and signs of ATL: Is Fatal associated with acute infiltration of skin and visceral tissue with monoclonal proliferation of CD4 bearing T‐lymphocytes & the following clinical manifestations:

• There is extensive cross‐reactivity between HTLV‐I and HTLV‐II, making it difficult to distinguish between these two viruses.

 Skin lesions due to infiltrating leukaemic cells.  Interstitial pneumonia.  Hepatosplenomegaly.

• PCR methods can also be used to detect HTLV‐I and HTLV‐II in peripheral blood and spinal fluid.

 Bone lesions. 31

32

CMV and EBV

Parvovirus

• Seropositivity rate : 50‐90% among blood donors.

• Benign and /or transient nature of most parvovirus disease.

• 1% of cellular components transmit the viruses. • Effective treatment :IV Immunoglobulin. • Hepatitis is rare & generally mild in the absence of severe immunosuppression (e.g. high risk neonates and transplant recipients).

• Extreme rarity of transmission by blood components.

• Removal of leukocytes from donated blood = reduce if not prevent post transfusion transmission. 33

34

Brucellosis

Syphilis

• An infectious disease caused by the bacteria of the genus Brucella.

• A Sexually Transmitted Disease (STD) caused by a bacteria • It can also pass through broken skin on other parts of the body • Signs of Syphilis include:

• These bacteria are primarily passed among animals, and they cause disease in many different vertebrates.

– – – – – – –

• Commonly transmitted to susceptible animals by direct contact with infected animals or with an environment that has been contaminated with discharges from infected animals. 35

Chancres" ("shan‐ker"), or sores. Skin rash. Mild fever. Feeling very tired. Headache. Sore throat. Hair loss. 36

377

Syphilis

Malaria

 Causes:

Causes: • Most frequently plasmodium falciparum. • Risk rate is <1:1,000,000

– Risk of disease transmission by blood transfusion is exceedingly rare. – Causative agents Treponema pallidum diagnosed by examination of microbe by dark field or by immunofluorescent microscopy.

Signs and symptoms: • Of malaria infection Management: • Antimalaria medication

 Prevention: – Prevented by VDRL or RPR testing for blood donors. – All positive cases confirmed by TPHA testing

Prevention: • Exclusion and deferral of high risk donors during donor selection. • Testing thick blood film for malaria for all blood donors. Or use more sensitive tests for detection of malaria if available.

38

Malaria

Babesiosis Causes: • Infectious agent is Babesia microti Risk factor <1: 1,000,000 Prevention: • Defer donors from endemic areas or those who have previous infection.

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West Nile Virus

West Nile Virus

Transmitted by arthropod exposure (incidental) Birds are primary hosts 50nm spherical, lipid‐enveloped flavivirus Single‐stranded positive sense RNA genome (11,000 nts) • Encephalitis, meningitis • Asymmetric flaccid paralysis (poliomyelitis‐like)

• • • •

2002: 4156 cases reported in U.S. (284 fatalities) 5‐10 % mortality Initial screening by questionnaire Voluntary NAT testing: through 2004, 1017 units withdrawn due to presumptive WNV infection • Peak exposure in August‐September • Viremia 6.5 to 56.4 days

• • • •

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Prion Diseases: Transmissible spongiform encephalopathies (TSEs)

Chagas Disease Causes: • Risk factor is 1:42,000 • Infectious agent is Trypanosoma cruzi.

• Fatal neurodegenerative diseases of man and mammals. • Long incubation period with little or no host immune response. • Associated with the conversion of normal host prion protein to a disease‐associated isoform (PrPSc). • Characteristic neuropathology with spongiform changes in the grey matter of the brain, usually concurrent with deposition of PrPSc.

Prevention: • Defer donors from endemic areas or those who have previous infection.

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Transmission of vCJD by Blood Transfusion

Transmission of vCJD by Blood Transfusion

• First reported case in 2004. Recipient clinical onset of vCJD 6.5 yrs after blood transfusion. Donor developed vCJD approx 40 months after donation (non‐leucodepleted red cells) • Second case later in 2004 (Peden et al: Lancet; 364:527). Recipient died of aortic aneurism 5 yrs post transfusion. No signs of vCJD but abnormal prion accumulation in spleen and one lymph node. Blood Donor developed vCJD approx 18 months after donation (non‐leucodepleted red cells) • Third case 2006 (Wroe et al; Lancet; 368:2061). Recipient clinical onset of vCJD 7.8 yrs after blood transfusion. Donor developed vCJD 21 months post donation (non‐ leucodepleted red cells)

• Fourth case 2007 (HPA press release). Developed vCJD 9 yrs after transfusion. Blood donor had already been implicated in previous transmission case • Hemovigelance Report review in UK: No cases yet from transfusion of plasma or leucodepleted blood. • Also no evidence that donors who develop sCJD have transmitted vCJD in blood • Blood appears to be highly infective. Some indication that leucodepletion may reduce risk. • Asymptomatic patient with prion accumulation in spleen suggests carrier status is possible 45

46

Measures to Protect the Blood Supply From Transmission of vCJD

Potential Transmission

1. Leucodepletion of blood for transfusion. 2. Blood donations not accepted from donors whose blood was transfused to patients who later developed vCJD. 3. Blood donations not accepted from recipients of blood transfusions in the UK since January 1980. 4. Filtration of red cells to remove or reduce prion titre. 5. Defer donors with family history of (CJD), exposure to risk factors, or residence in endemic areas with (CJD), for more than 6 months (UK).

Bloodborne Pathogens may also transmitted by: • Contact between broken or damaged skin and infected body fluids. • Contact between Mucous Membranes and infected body fluids. • Example: Eyes, Nose, and Mouth • Anytime there is blood to blood contact with infected blood or body fluids, there is a slight potential for transmission. • Accidental puncture from contaminated needles, broken glass, or other sharps is potentially how transmission could occur for custodial employees. 47

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Compliance Control Methods

Compliance Control Methods Standard Precaution

Personal Protective Equipment

Universal Precaution Precaution

Personal Hygiene

Work Practice Controls

– Treat all blood and bodily as if they are contaminated. – All body fluids must be considered as potentially infectious materials. – Proper cleanup and decontamination – Always wear appropriate PPE – Replace PPE that is torn or punctured – Remove PPE before leaving the work area – Change PPE between patients and wash hands each time after removal of glove.

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PPE Controls • PPE must be used to prevent potentially infectious materials from coming in contact with work clothes, street clothes, undergarments, skin or mucous membranes. • Employees must wear gloves when there is potential contact with blood, potentially infectious materials, mucous membranes or broken skin. • Remove gloves promptly after use, and before touching non‐contaminated items and environmental surfaces.

Safe Work Practice Controls • Remove contaminated PPE or clothing as soon as possible • Clean and disinfect contaminated equipment and work surfaces • Thoroughly wash up immediately after exposure • Properly dispose of contaminated items, including contaminated PPE 51

52



Personal Hygiene • Do not touch anything that is contaminated, such as sharps or body fluids. • Take care to minimize splashing of all infectious materials. • Eating, drinking, smoking, applying cosmetics or lip balm, and handling contact lenses are prohibited in areas where there is a potential for occupational exposure.

Personal Hygiene Cont. • Use CDC guidelines for hand hygiene: • If hands are not visibly soiled, use alcohol gel. • When hands are visibly soiled, wash hands with soap and water. • Always wash your hands before eating and after using the restroom.

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Summary • Always know what you are working with. • Always wear appropriate Personal Protective Equipment (PPE) when handling any type of bodily fluid. • Always wash your hands after handling any type of bodily fluid, even when wearing gloves. • Do not handle sharps or broken glass with your hands and without protection. • Properly dispose of pathogen waste, including PPE. • Always report all suspected exposures. 55

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Bacterial Contamination in Blood Products • What is the Problem? • What are the Risks? • What Organisms are Associated with Bacterial Contamination? • What are the Sources of Contamination? • What Options exist to Detect Bacterial Contamination? • What Corrective Actions (Prevention and Management) are Planned?


Bacterial Contamination in Blood Products: Risks, Prevention and Detection

2

Bacterial Contamination in Blood Products


• What is the problem?

• First recognized infectious risk of blood transfusion. • Risk greatly reduced in the 1960s by the use of closed, sterile systems for the collection and storage of blood. • Recent dramatic improvements in safety from viral screening and testing have reduced the risks from Hepatitis and HIV. • Bacterial sepsis is now the most common infectious disease event following transfusion. 3

4



• Bacterial contamination occurs primarily in room‐ temperature stored products (platelets) but can occur in red blood cells and plasma also. • The blood banking community is taking steps to improve prevention and detection of bacterial contamination. • The American Association of Blood Banks (AABB), as well as the College of American Pathologists (CAP) have established compliance criteria for transfusion services.

• The American Association of Blood Banks has issued two new standards (March, 2003): – “5.1.5.1 The blood bank or transfusion service shall have methods to limit and detect bacterial contamination in all platelet components.” – “5.6.2 The venipuncture site shall be prepared so as to minimize the risk of bacterial contamination. Green soap shall not be used”

5

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College of American Pathologist’s Accreditation Checklist (December, 2002): • “TRM.44955 Phase 1 Does the laboratory have a system to detect the presence of bacteria in platelet components?”

• What are the risks?

7

8

Bacterial Contamination Reaction

Clinical Manifestation

• Although uncommon, but this type of specific reaction can have a rapid onset and high mortality in recipients.

• Usually appear rapidly during transfusion or within about 30 minutes after transfusion with dryness, flushing of skin.

• The presence of bacteria in transfused blood may lead either to febrile reactions in the recipient ( due to pyrogens ) or serious manifestations of septic or endotoxic shock.

• Fever, Hypotension, Chills, Muscle pain, vomiting, Abdominal cramps, Bloody diarrhoea, Hemoglobinuria, Tachycardia, Shock, Renal failure, DIC.

• Commonly caused by endotoxin produced by bacteria capable of growing in cold temperatures such as Pseudomonas species, E. coli, Yersinia enterocolitica.

9

10

Bacterial Contamination

Bacterial Contamination

• Bacterial Contamination occurs at a much higher frequency than any other infections (Incidence: 0.3%) and is associated with substantial mortality. • Rate of bacterial infection/contamination:

1. Bacterial testing of apheresis products initiated in 2004 2. Assessment of risk based upon reporting – Pre testing:  Septic reactions 1:40,000  Fatalities 1:240,000 – Post testing:  Septic reactions 1:75,000  Fatalities: 1:500,000

– RBCs 1 in 30,000 – Random Platelets 1 in 2,000

• The higher rate with platelets is because they are stored at room temperature and the units are generally pooled between 6 and 10 donor units. 11

12

Residual Risk of Bacterial Sepsis After Bacterial Culture of Aphaeresis Platelets 1‐ American Red Cross ‐ Eder et al. (Transfusion 2007) • 20 septic reactions (3 fatal) • 1,004,000 tested products ~1:59,000 2‐ Canada ‐ Ramírez‐Arcos et al. (Transfusion 2007) • 2 septic reactions (1 fatal) • 82,004 tested products ~1:41,000 3‐ Holland ‐ deKorte et al. (Transfusion 2006) • 2 septic reactions • 113,092 tested products ~1:56,500 4‐ Germany ‐ Schmidt et al. (Vox Sanguinis 2007) • 2 septic reactions (1 fatal) • 52,243 products tested ~1:26,000

Bacterial Contamination in Blood Products • What Organisms are Associated with Bacterial Contamination?

13

14

Organisms Involved Exogenous

Organisms Involved Normal skin

Endogenous

Staphylococcus epidermidis Staphylococcus aureus Diphteroids spp Micrococcus spp Pseudomonas spp Bacillus cereus Propionibacterium acnes Flavobacterium spp

Staphylococcus spp Teeth Streptococcus viridans Serratia liquefaciens Osteomyelitis Staphylococcus S. cholera suis

Yersinia enterocolitica Intestines Salmonella spp Campylobacter spp

15

16

Bacterial Species in Plts Implicated in Clinical Sepsis S. S. E. B. S.

Bacterial Species in Plts Implicated in Septic Fatalities Reported to the FDA (1976‐1998)

epidermidis, 30.2% aureus, 10.5% coli, 9.3% cerus, 9.3% cholerae-suis, 8.1%

S. epidermidis 9.6% S. aureus 17.3% E. coli 5.7% Bacillus 5.7% Salmonella 7.7% Enterobacter 5.7% Streptococcus 7.7% Klebsiella 17.3% Serratia 15.4% P. mirabilis 2.2%

B-hem. Strep, 5.8% E. aerogenes, 2.3% 10 others, 1.3% each

Compilation of data from Clin Micro Rev 1994; 7:290-302; Transfusion 2001;41:149399; www.shot.demon.co.uk/toc

n = 52 n = 86

17

18

384

Differences Between the Species Implicated in Septic Morbidity & Mortality in Platelet Components

Organisms Implicated in Sepsis From Platelets

• S. epidermidis is less commonly observed in septic fatalities and more commonly observed in septic reactions.

• Approximately 30% are associated with normal skin flora. • Approximately 56% are gram positive. • All are aerobic or facultative anaerobes;

• Klebsiella is commonly observed in septic fatalities. • Gram negative organisms are implicated in more fatalities (60%) than gram positive organisms (40%); gram positives cause a majority of septic reactions (56%).

– A rare (single case) exception: Clostridium perfringens fatality from a pooled platelet unit Trans Med 1998;8:19‐ 22.

19

20

Bacterial Contamination of Blood Products

Sources of Bacterial Contamination

• What are the sources of contamination?

• • • • •

Skin Surface Contamination. Phlebotomy Core. Donor Bacteremia. Containers and Disposables. Environment.

21

22



• Infection of stored blood is extremely rare.

• Healthy donor who are bacteremic at the time of donation. The majority are due to Yersinia enterocolitica, which grows well in red cell components due to its dependence on citrate and Iron.

• Skin contaminants are not infrequently present in freshly donated blood but these organisms (predominantly staphylococci ) do not survive storage at 4°C although they will grow profusely in platelet concentrates stored at 22° C.

• Gram negative, endotoxin – producing contaminants found in dirt, soil and faeces may rarely grow in the storage condition of blood. 23

24

385

AABB Association Bulletin #03‐07 May 16, 2003

Bacterial Contamination of Blood Products • What Options exist to Detect Bacterial Contamination?

Methods to Detect Contamination: • Culture methods optimal. Two approved products cited. Other culture methods can be validated. No label claims allowed • Due to insensitivity, staining and dipstick methods should be used as close in time to issue as possible • Validation of all methods is required • “Swirl” procedure useful for inspection but does not by itself meet AABB Standard 5.1.5.1 25

26

Bacterial Detection Options in Platelet Products

Bacterial Detection Tests

• Visual examination for discoloration, clumping or abnormal morphology

• 3 devices are cleared for quality control monitoring of platelet collection

• Microscopy – Gram stain – Acridine orange

process of leukoreduced platelets:  BioMeriuex BacT/ALERT®

• Measuring Biochemical changes – Lowered pH – Reduced Glucose

 Pall eBDS  hemoSystems Scansystem™

• Bacterial culture – Detection through oxygen consumption – Detection through CO2 production

• Use of non‐validated tests ( glucose and pH by dipstick, swirling ). • Non‐standardized methodology even with culture‐based devices.

27

28

29

30

Bacterial Detection Options in Platelet Products Visual Examination • Inspect product prior to transfusion for discoloration or abnormal clumping. • Perform “swirl” procedure to detect morphologic changes in platelets. – Normal shaped platelets will align with fluid flow and “shimmer” when swirled. – Contaminated platelets, among others, lose discoid shape and do not “shimmer” when swirled– Not a specific marker for contamination.

386


Swirling Alignment with flow

Microscopic Methods • Gram Stain or Acridine Orange preferred methods • Limitations: – Must be performed by the Transfusion Service prior to product issue for transfusion – Lack sensitivity with low bacterial load

SENSITIVITY: 75% SPECIFICITY: 95% No alignment with flow Low pH Metabolic disturbance

Leach MF et al. Vox Sang 1998;74(suppl 1):1180. 31

32


Metabolic Changes (pH)

Measuring Biochemical Changes • Measure changes in glucose consumption against a control. Variances of >2 S.D. may indicate bacterial contamination. • “Dipstick” testing. • Limitations:

• The majority of proliferating bacteria will metabolize glucose and produce acid, lowering the pH. Measurement of glucose and pH has been evaluated and, indeed, utilized in the USA for the detection of bacteria in platelet concentrates (Burstein et al,. 1997; Choo et al., 2004; Cocco et al., 2004; Hahn etal., 2004; Werch et al., 2002). An advantage of this system is that it is extremely rapid and conclusive , PH of all platelet Units must be  6.2.

– Both this method and staining methods are subjective, require high levels of contamination, and must be performed prior to issue by the Transfusion Service. 33

34

Chemical Tests ‐ Dipsticks

Validation of Bacterial Detection Methods

Must be performed immediately before issue because of its relative insensitivity and the need for high bacterial counts.

• pH & glucose tests are analytically insensitive (Yomatovian R, Brecher ME. Transfusion 2005;45:647‐8) – Validation required: • Variable plastic bags. • Variable anticoagulants. • Variable handling.

• Gram stain insensitive unless 106 CFU/mL. • Facilities may not have FDA approved equipment. • Validate for QC use – Sensitivity – Types of Use – Growth time 35

36

387

BioMeriuex BacT/Alert System

BioMeriuex BacT/ALERT®

• Detects bacterial growth in culture bottles by measuring CO2 production. • Automated reader continuously monitors samples. • Sampling interval of >24 hours post phlebotomy • Culturing interval of >24 hours post sampling (aerobic and anaerobic cultures). • Cultures incubate for 5‐7 days; may identify positive cultures post‐transfusion. • FDA‐Approved for Q.C. purposes only on Leukoreduced Aphaeresis Platelets.

• Colorimetric technology/Sensor Culture bottles – CO2 release causes sensor bottle to turn yellow – Instrument measures & detects color change, analyzes data to determine positivity, alerts when positive culture

37

38

Pall Biomedical BDS System

Pall eBDS

• Detects bacterial contamination by measuring O2 consumption. • Automated reader measures O2 levels in headspace of culture pouch. • Sampling interval of >24‐48 hours. • Culture performed for >24‐30 hours. • FDA‐Approved for Q.C. on leukoreduced platelet concentrates and leukoreduced apheresis platelets.

Sample set/Oxygen Analyzer • Sterile weld platelet component to set • Fill pouch with ~3 mL of product • Disconnect sample pouch from set and incubate at 35°C for 24‐30 hrs • Measure the O2 content in the air above the plasma sample with insertion of analyzer probe into pouch • LED display will read PASS or FAIL 39

40

Limitations of Blood Culture Methods


• Early sampling/testing may not detect small # bacteria per bag. Approved methods require 24‐30 hour wait before sampling • Two FDA‐Approved methods require bacteria to grow up after sampling to detectable levels, so culture must be done well before planned transfusion (Blood Center) • The two time intervals (collection to sampling and sampling to release/transfusion) dominate the logistic considerations

• Both options require leukoreduced platelets. • BacT/Alert requires continued culture after product release. • Release and recall (BacT/ALERT) or hold to end of culture to release (PALL BDS). • Need to balance the risk of platelet shortages versus the risk of platelet contamination. • Complex and expensive.

41

42

388


New Device for Transfusion Services Verax rapid Platelet PGD® Test

• The two available devices are FDA‐Approved for Q.C, and not approved as pre‐release tests. • Probable negative impact on outdates. • Possible extension of platelet storage to seven days or pooling/storing whole blood derived platelets. • False positives, indeterminants, false negatives, Follow‐up of suspected products. • Limited effectiveness: delayed bacterial growth

Single‐use disposable test 1. Rapid ~ 25 minutes (3 min hands on) 2. Positives typically < 10 minutes 3. Sensitivity ~ 103 CFU/ mL 4. Specificity > 99.7%

43

44

Methods Comparison Verax PGD

BacT/ALERT

Pall eBDS

Conserved Bacterial Ag Immunoassay

Culture CO2 Measure

Aerobic Culture O2 measure

Sample Volume

500 uL

4‐20 mLs

3‐5 mLs.

Time to Result

10 – 30 min (positives typically within 10 minutes)

24 – 96 hours

24 – 30 hours

Detect Aerobic and Anaerobic bacteria?

Yes

Yes, but time varies

Misses Anaerobes

Clinical Specificity

99.7%

99.2‐99.8%

~ 99%

Technology

Source: Abbott, Biomerieux and Pall Medical web site. 46

45


Prevention

• What Corrective Actions (Prevention & Management) are Planned?

• Strict adherence to policies & procedures regarding blood component collection, storage, handling, and preparation is essential to reduce the risk. • Visual Inspection of components before release from the transfusion service include any discolouration, visible clots, or hemolysis. • Ensure the blood components are infused within standard time limits (4 hours).

47

48

389

Precautions to be Observed in Preparing Components

Prevention • Blood packs should never be opened for sampling, if any open method of preparation has been used, the unit should be transfused within 24 hours.

In collection of blood • Proper selection of donor • Clean & aseptic venepuncture site to minimize bacterial contamination • Clean venepuncture with minimum tissue trauma and free flow of blood • The flow of blood should be uninterrupted and continuous. If any unit takes more than 10 minutes to draw, it is not suitable for preparation of blood components.

• Blood should always be kept in accurately controlled refrigerators (with alarms), maintained strictly at 2 – 6°C, the blood should never be removed and taken to the ward or OT until it is required.

49

50

Precautions to be Observed in Preparing Components (contd)

AABB Association Bulletin #03‐07 May 16, 2003

• A correct amount of blood proportionate to anti coagulant should be collected in primary bag that has satellite bags attached with integral tubing. • Monitor the collection of blood with automatic mixer which is used for collecting the desired amount of blood and mixing the blood with anticoagulant • If platelets are to be harvested the blood bag should be kept at room temperature 20‐24°C until platelets are separated. Platelets should be separated within 6 hours from the time of collection of blood.

Methods to Limit Contamination: • Careful phlebotomy – No green soap prep. • Iodine based scrub recommended. • Consider phlebotomy diversion – “sample first” technologies. • Consider increased use of aphaeresis platelets.

51

52

Impact of Skin Disinfection on Surface Bacteria

Skin disinfection methods • Some agents may reduce the number of surface bacteria more than others. • Method of application and applicator may have some impact on the extent of reduction of surface bacteria. • Minimum scrub of 30 seconds required to be effective.

CFU per plate

PVPI

Isopropyl Alcohol + Tincture of Iodine

Chlorhexidine Gluconate

Green soap + Isopropyl alcohol

0

34-40%

63%

60%

0%

1-10

35-43%

34%

25%

17%

11-100

10-14%

2%

12%

47%

>100

0-13%

1%

3%

36%

Goldman et al, Transfusion 1997;37:309-12

53

390

54

Avoiding Skin Contamination

Diversion of Initial Blood Flow

• Diversion of the initial blood flow.

• Diversion of initial blood flow into sampling tubes • Reduces the load of skin‐associated bacteria entering blood container • Phlebotomy “core” directed into sampling pouch instead of blood container

• Improvement in pre‐phlebotomy skin cleansing.

55

56

Clinical Data Supporting Diversion of Initial Blood Flow

Inspection of Donor Blood

de Korte et al. Vox Sang 2002;83:13‐16 ‐ Collected blood normally or diverted the first 10mL of whole blood into a satellite bag ‐ Performed bacterial testing by automated blood culture (BacT/Alert) in a laminar flow hood Total bacterial prevalence

S. epidermidisprevalence

Standard Collection

0.35% (0.27-0.44)

0.14%

Collection with diversion

0.21% (0.12-0.35)

0.03%

P-value

<0.05

<0.02

• Blood should be inspected before transfusion for possible bacterial contamination, hemolysis, visible clots, brown or red plasma. • Segment closest to unit is hemolyzed. May indicate bacterial contamination.

57

58

59

60

Donor Blood Inspection and Disposition • If a unit's appearance looks questionable do the following: – Quarantine unit until disposition is decided. – Gently mix, allow to settle and observe appearance.

• If bacterial contamination is suspected the unit should be cultured and a gram stain performed. • Positive blood cultures usually indicative of: – Inadequate donor arm preparation. – Improper pooling technique. – Health of donor ‐ bacteremia in donor.

• If one component is contaminated, other components prepared from the same donor unit may be contaminated.

391

Summary • Bacterial contamination of blood components continues to pose a threat to transfusion recipients • Progress to prevent adverse reaction to blood transfusions due to bacterial contamination continues to be seen • Microbiologists now play a major role in this progress

61

392


Screening & Confirmatory Tests for TTDs


Screening & Confirmatory tests for TTDs

TTDS

SCREENING TEST

CONFIRMATORAY TEST

NAT

HIV

ELIZA Chemiluminescent

WESTERN BLOT

NAT

HCV


RIBA TEST

NAT

HBV


NEUTRILIZATION

NAT

HTLV


WESTERN BLOT

‐‐‐‐‐

SYPHILIS

ELIZA, RPR, TPHA Chemiluminescent

TPHA

‐‐‐‐‐‐‐

MALARIA

ELIZA, THICK FILM

THICK FILM

‐‐‐‐‐‐ 2

ELIZA Techniques

Definitions • Antibodies (also known as immunoglobulins abbreviated Ig) are gamma globulin proteins that are found in blood and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. 3

4

Definitions (cont)

Enzyme Linked Immuno Sorbent Assay

• Antigens A substance that when introduced into the body stimulates the production of an antibody

• Used to detect the presence of Ag, Ab from the Serum • By measuring the enzymatic activity which interacts with substrate that chemically changed to produce color. • The density of color is either directly or indirectly proportional to target according to the type of ELISA. • This Enzyme may be linked to Ag or Ab. • Performed as a solid phase assay in which the target to be detected is bound to solid surface (micro‐titiration well)

• Immunoassay A laboratory technique that makes use of the binding between an antigen and its homologous antibody in order to identify and quantify the specific antigen or antibody in a sample 5

6

393

Types Of ELISA Techniques

Competitive ELISA

The techniques are divided into:‐ 1‐ Competitive ELISA 2‐ Sandwich ELISA (also called direct ELISA) 3‐ Indirect ELISA

The labelled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). The more antigen in the sample, the less labelled antigen is retained in the well and the weaker the signal). 7

8

Indirect ELISA protocol (for screening monoclonal antibodies

Sandwich ELISA Protocol

9

10

Chemiluminescent Immunoassays

Chemiluminescent Immunoassays • The process of chemiluminescence occurs when energy in the form of light is released from matter during a chemical reaction. • Light emission ranges from quick burst or flash to light which remains for a longer time. • Different types of instruments are required based on emission.

11

12

394



• Can be used for heterogeneous or homogeneous assays. • Heterogeneous assays use competitive and sandwich assay. • Competitive assays used to measure smaller analytes. • Sandwich assays are used to measure larger analytes.

• • • •

Many applications. Can measure antigen or antibody. Add chemiluminescently tagged analyte. Measure light which is emitted which is directly related to concentration although competitive binding assays are available. 14

Advantage of Chemiluminescent Immunoassays

Rapid Plasma Reagin (RPR) Test

• Not required long incubation time. • No addition of stopping reagent (colorimetric method is required). • More economical compared to conventional colorimetric method (Ex: ELISA) • More sensitive compare to the colorimetric method.

• The RPR test is a non treponemal card aggluitination test for serologic screening for syphilis. • The known RPR antigen consists of cardiolipin and cholesterol bound with charcoal particles. • Charcoal makes the reaction visible. • If the donor has syphilis , the antilipid antibodies (reagin) in his serum will cross react with the known RPR lipid antigens giving a visible clumping of the charcoal particles. 15

16

Types of Samples

Interpretation & Use of Confirmatory Results

• Serum. • Plasma (centrifuge to remove fibrin before testing). • Samples not suitable for testing:

• Confirmatory testing is primarily concerned with the status of the donor and the subsequent action to be taken.

‐ Haemolysed. ‐ Lipemic. ‐ High conc. of Billirubin.

• Donations that are repeat reactive may be confirmed as being of negative, inconclusive or positive status.

17

18

395

Negative Conclusion

Inconclusive Outcome

• A negative conclusion on confirmatory testing indicates that the donor is not infected with the specific infection. • However, a donor showing repeat reactive results on screening and negative results on confirmatory testing should be counseled and temporarily deferred until screen non‐reactive on follow‐up. • The donor can then be accepted for future donations.

• An inconclusive outcome is usually due to non‐ specific reactivity not related to the presence of the infectious agent. • The donor should be counseled, deferred for blood donation and followed‐up for further investigations.

19

20

21

22

Positive Conclusion • A positive conclusion confirms that the donor is infected and should be deferred from future blood donation. • The donor should be counseled and referred for appropriate medical care.

Western Blot •

• •

•

Western Blot • Indeterminate Result: some, but not all, bands are present.

A Western Blot looks for the presence of antibodies in the patient’s blood to certain components of the HIV virus. When those antibodies are present, a band of color appears on the test result. To be considered a positive test, a certain number of different antibodies, and consequently color bands, need to appear. A test is considered indeterminate when some, but not all, color bands appear.

• Causes: recent infection, advanced HIV, certain strains of HIV, cross reaction to other antibodies, HIV vaccine Or Lab error. • Reset in > 6 weeks. Risk counseling if indicated.

23

24

396

Western Blot

Disadvantage of Western Blot • • • •

Expensive. Technically more difficult. Visual interpretation. Lack standardisation: ‐ performance ‐ interpretation ‐ indeterminate reactions

25

26

Recombinant Immunoblot Assay (RIBA)

Results of RIBAs

• Contains the structural and non‐structural HCV antigens.

• A positive RIBA assay requires at least two reactive bands. • Tests with only one reactive band are considered indeterminate.

• Individual HCV antigens are displayed on a nitrocellulose strip.

• The sensitivity of RIBA tests are not higher than sensitivity of EIA tests

• Antibodies against specific HCV antigens can be identified.

• If NAT test gives the negative result, RIBA tests can be used to distinguish false‐positive EIA results from prior exposure to HCV. 27

28

Disadvantage of RIBA Assay

Neutralization Test

• Detect only IgG antibodies.

• In the first stage: Sample allowed to react with specific Ab bound to a solid phase, where upon either the complex solid

• RIBAs are technically more demanding than ELISA.

phase Ab/Ag or the complex solid‐phase Ab/interfering substance is formed.

• Many intermediate results.

• In the second stage: A specific unlabeled anti HBs Ab is allowed to react with one of the two duplicate series, while a

• High cost.

serum non‐reactive for anti HBs Ab is allowed to react with the second duplicate series. 29

30

397

TPHA Test For Syphilis

Neutralization Test cont..

• the Syphilis TPHA test is a classic, indirect hemagglutination test used for the detection and titration of antibodies against the causative agent of syphilis, Treponema pallidum. • In the test red blood cells (erythrocytes) are sensitized with antigens from T. pallidum. The erythrocytes will then aggregate together to form distinctive patterns on the surface of a microplate wells when exposed to syphilitic serum.

• In the third stage: The labeled anti HBs Ab is added to both duplicate. If the positive reaction in the screening test was caused by an interfering substance, the unlabeled specific Ab will not inhibit the reaction of the labeled Ab with the solid phase complex. Conversely, if the positive reaction in the screening test was caused by the presence of HBsAg , the unlabeled specific Ab will inhibit the binding between the labeled Ab and the solid phase complex. • A reduction of the value of the neutralized sample with respect to the value of the non‐neutralized sample will confirm the presence of HBsAg. 31

32

Preparing thick films

Thick Blood Film

1. The second or third finger is usually selected and cleaned.

• Numerous ring form of Plasmodium can be seen as indicated by the arrows. • Note size of neutrophils (for comparison • The only definitive diagnosis that can be made from this film is that Malaria is present.

2. Puncture at the side of the ball of the finger. 3. Gently squeeze toward the puncture site.

• Thick film considered “gold standard” for detection of parasites due to being able to use larger volume (10µl of blood).

4. Slide must always be grasped by its edges. 5. Touch the drop of blood to the slide from below.

6. Spread the first drop to make a 1 cm circle.

33

Thick blood film

34

A drop of blood is spread over a small area. When dry, the slide is stained with Field’s or Giemsa stains. The red cells lyse leaving behind the parasites.

Microscopy – The Gold Standard • Benchmark diagnostic standard for over 100 years.

• Used to detect parasites, even if parasitaemia is low • Less useful for speciation

• In expert hands: highly sensitive, specific. • Results provide a wealth of clinically important data. • Stained slide serves as permanent record.

Back

35

36

398

Microscopy Limitations • Microscopy skills may be lacking in areas not routinely doing malaria evaluations – Smear preparation, staining – Interpretation • Mixed infections ‐ can be difficult to diagnose. • Low parasitemia ‐ can be difficult to diagnose. • Hands on time is very high. 37

399


Introduction • Transfusion of blood and blood components is an important issue of any health care system.


• It is necessary to ensure that blood & blood components administered are 100% safe.


• Transfusion transmitted infections are by far, the more complex and vexing problem faced by BTS.

NAT & Blood Safety 2

Transfusion Transmitted Infections Are A Real Concern

Improving Blood Safety Worldwide

• More than 92 million blood donations are collected globally per year1

• “Screening for TTIs to exclude blood donations at risk of transmitting infection from donors to recipients is a critical part of the process of ensuring that transfusions are as safe as possible” – WHO, 2010.

• Annually, unsafe blood transfusions are estimated have been responsible for up to : – 16 million new HBV infections – 5 million new HCV infections – 160,000 cases of HIV infections

The Lancet

“Global Blood Safety and Availability: Facts and Figures from the 2007 Blood Safety Survey,” WHO Fact Sheet #279, Nov. 2009.

3

4

TTI Risks & Implications in Developing Countries

Blood Supply Safety

• Developing countries are more likely to use blood that is contaminated than industrialized nations:‐

The Goal • Preventing transfusion‐transmission of blood‐borne pathogens.

o Higher disease prevalence o Use of paid, family/replacement donors o Concealing medical history and/or risky behavior in questionnaire o Inadequate serology‐based screening

Overall Impact • A single whole blood donation can be transfused to 4 recipients. • May be added to pools of more than 1,000 units to manufacture blood derivatives.

Asian J Transfusion Sci.; v4(2), July 2010

5

6

400

What is NAT?

Advantages of NAT

Nucleic Acid Amplification Testing (NAT) :

• • • •

Highly sensitive & specific . Targets specific viral nucleic acid sequences. Direct detection of low level of viral RNA or DNA. Shortens the Window Period from infection to detection. • Helps prevent transfusion transmitted disease. • Provides additional layer of safety to the blood supply. • Improves confidence in blood supply.

• NAT is a molecular technology that focused on the detection of viral DNA or RNA of intended viruses. • NAT utilizes either Polymerase Chain Reaction (PCR) or Transcription Mediated Amplification (TMA) methods permitting the amplification of viral sequences in vitro. • Highly sensitive and specific technique. • Fully automated technique, either based on individual testing or pooling system. 7

8

Why do we do NAT?

Window period “WP” • The most important factor for TTIs residual risk. • The WP is defined as the time from infectivity to test reactivity. • The chance of transmission is a function of both incidence and length of WP. • Blood transfusion authorities and blood banks were concerned about the ability to close the gap of ‘window‐period’ by additional steps to ensure quality and safety of blood and blood products

Antibody negative window

NAT negative window

INFECTION

Detection Single donation

Detection minipool

Seroconversion 9

10

Progress in Pathogen Detection Transfusion‐Transmitted Pathogens

Steps to Safety

Shorter window period to detection All volunteer donors 25

HBsAg test

Surrogate Marker

Antibody Testing

Viral Antigen Detection

Serum ALT

Anti‐HIV

HIV p24 Ag

T‐cell count

Anti‐HBc

HBsAg

Anti‐HCV

HCV Ag

Viral RNA/DNA Detection

% Recipients Infected By Blood Borne Diseases

20

Anti-HIV test AIDS high-risk exclusions

15

Anti‐HTLV

ALT/HBcAb tests 10

Anti-HCV test Improved HCV tests

0 1965

NAT is the only direct test for the infectious agent.

NAT Implementation

5

1970

1975

1980

1985

1990

NAT HIV‐1/2 HCV HBV

1995

2000

Year of Transfusion 11

12

401

Window Period • Residual risk = Incidence rate × window period duration

• Incidence rate = Seroconversions / Person / Years Source: Busch et al. Transfusion.2005;45(2):254-264. Kleinman and Busch. Transfusion. 2006;36:S23-S29

13

Sources of Residual Risk

Pre‐requisites to performing NAT

• Window period donations.

• Serological screening in place and effective. • Technical development at molecular level (trained staff). • In depth training of staff. • Sufficient blood supply to afford quarantining. • Donated or affordable equipment. • Supply, transport & storage of reagents cold chain. • Methods adapted to small or large numbers, affordable.

• Viral variants not detected by traditional serological tests. • Immunosilent donors. • Laboratory testing errors.

15

16

Testing modes for NAT

Calculated Risk / Unit Transfused

1. Individual sample screening - Duplex HCV/HIV-1 RNA - Triplex HCV/HIV/HBV

. Expensive. . Sensitive. . Specific.

Serology testing

Mini Pooled NAT

Single Unit NAT

HIV

1:1,300,000

1:1,900,000

1:3,000,000

HCV

1:230.000

1:1,600,000

1:2,300,000

HBV

1:180,000

1:210,000

2. Minipool screening (pools of 96, 48, 24, 16, 10, 8, 6) - Duplex HCV/HIV, Triplex HCV/HIV/HBV . Less expensive . Less sensitive . Less specific?

1:410,000 Data from FDA December 2001 Workshop: Presented by M. Busch, M.D. (Current risk estimates in bold)

All commercial duplex or triplex require identification of positives with three discriminatory single virus assays except Roche. 17

18

402

Published Yield Cases ‐ Demonstrated NAT Yield in Developing Countries (Ekiaby et al. 2010) Country

Author

Serology

Mode

NAT Yield

Brazil

Levi

HIV

MP6-12

-

Mexico

Chiquete

-

Mexico

HBV

IDT

HCV

IDT

-

Garcia-Montalvo

HBV

IDT

<1:865

Lebanon

El-Zaatari

HBV

IDT

1:501

Lebanon

Ramia

HBV

IDT

1:358

Mongolia

Tsatsralt-Od

HBV

IDT

1:81

China

Ren

HBV

MP8

1:1,430

Malaysia

Lam

HBV

IDT

1:3,616

HBV

IDT

1:2,037

India

Makroo

HIV

IDT

1:6,112

HCV

IDT

<1:617

India

Chaudhuri

HBV

IDT

Iran

Behzad-Behbahani

HBV

IDT

1:125

Kuwait

Al Radwan

HBV

-

1:24,275

TMA Vs PCR Transcription Mediated Amplification

Polymerase Chain Reaction

Idea

The amplification is performed by making many RNA transcriptions at 1 fixed temperature (41.5 C ‐ Isothermal Reaction)

The amplification is performed by chain of reactions at 3 different temperatures (94 C then 50 – 60 C then 72C – thermo Cycling)

Amplified product

RNA (Labile in environment Minimal contamination issues.)

(stable in environment Very prone to contamination)

Amplified Product for 1 Cycle

100 ‐ 1000 Amplicons

pilcons

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TMA Vs PCR

TMA Vs PCR TMA

PCR

After 1 Hour of Amplification

Approximately: a billion‐fold of RNA copies

Approximately: 1 million of copies

Detection

Chemi‐luminescent detection (2 times of magnitude more sensitive than Fluorescence)

Fluorescence detection

There are billions sites of detection labeled with Chemiluminescence Molecule. (more detection sensitivity)

There are million site of detection labeled with Fluorescence Molecule.

RESULT

Limit of Detection 95%

/ ULTRIO Plus

Cobas S201 / MPX

HCV (All Genotypes)

3.1 IU/mL

11 IU/mL

HBV (All Genotypes)

2.1IU/mL

3.8 IU/mL

HIV‐M

49 IU/mL

HIV‐O

27.6 IU/mL

152 Copies/mL

Not Available

2.2 Copies/mL

Not Available

HIV‐N HIV‐2 21

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Limitations of current HIV NAT

Hepatitis B virus (HBV)

• The Ultrio Assay Detects only HIV‐1 RNA • HIV‐N RNA are not detected by the Taqscreen MPX (Roche) • HIV NAT does not detect HIV DNA • Current anti‐HIV assays cover all HIV types

• Hepatitis B virus (HBV) is a major human pathogen that causes acute and chronic hepatitis in humans. • Most primary infections in adults are self‐limited, the virus is cleared from blood and liver, and individuals develop a lasting immunity.

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Definition of OBI (Taormina workshop 03/2008)

Chronic Hepatitis B

• Presence of HBV DNA in the liver (± serum) of individuals with undetectable HBsAg with currently available assays

• Less than 5 percent of infected adults develop persistent infections that can be asymptomatic (i.e. a carrier state).

– Seropositive OBI (anti‐HBc+; ± anti‐HBs) – Seronegative (anti‐HBc and anti‐HBs negative)

• Twenty percent of chronically infected individuals can develop cirrhosis.

• To be distinguished from: – Primary infection window period

• Chronically infected subjects have 100 times higher risk of developing hepatocellular carcinoma than non carriers.

– S protein escape mutants undetected by HBsAg assays – Genomic assay contamination

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Mechanisms for OBI occurrence

Testing for blood HBV safety

• Lack of HBsAg production: primary OBI • Incomplete control of host immune system

1.

HBsAg o o o

– In HBV genotype A2, B, C, D – Escape mutants when anti‐HBs present – Accumulation of random mutations in anti‐HBc

2.

Anti‐HBc o o o

• Deficiency or inhibition of viral replication/gene expression 3.

– In genotype A1 and E for deficiency – In all genotypes for inhibition of HBsAg expression

low specificity, recently improved need confirmation with alternative test(s) Inexpensive

NAT o o o

• Vaccine breakthrough acute infections

wide variation in sensitivity generally excellent specificity Inexpensive

variable sensitivity 5‐50 IU/ml or 35‐350 copies excellent specificity expensive, delicate, contamination, time consuming.

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Efficacy of anti‐HBc vs NAT Type of HBV Infection

HBsAg

Anti‐HBc

NAT*

Window Period

No

No

Yes

Primary OBI

No

No

Yes

2d WP

No

Yes

Yes

Chronic

No

Yes

Yes

Anti‐HBc+ OBI

No

Yes

Yes

Anti‐HBs only OBI

No

Yes

Yes

Chronic NAT‐

Yes

Yes

No

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Results

Conclusions

• HBsAg+ or anti‐HBc +/‐ anti‐HBs = Window period • Repeat initial results = confirm preliminary diagnosis • HBV DNA undetected

• • • • • • • •

– Fluctuation – Contamination

• Anti‐HBs undetected = potential fluctuation • All negative = contamination

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Blood safety can be improved by NAT. NAT can not be the only blood screening test. Multiplexing increases cost‐efficiency. Pooling decreases cost but also sensitivity. NAT detects Window Period for HIV, HCV, HBV. NAT also detects HBV OBI. OBIs are multifacet & difficult to diagnose. OBIs can be infectious even when anti‐HBs present. 32

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Hematology

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Kingdom of Saudi Arabia – Ministry of Health General Directorate of Training and Scholarship Training Program for Health Institute Graduates Hematology

Introduction to Hematology - Slide 8 – Stability of anti-coagulated blood Accuracy of results for EDTA bloods reduces dramatically after 4 hours in the fridge

407


Content outline Main Function of hematology • Development of blood cells • Anticoagulants used in hematology • Stability of anticoagulated blood • Blood Film : Value Preparation Staining


Introduction to Hematology 2

Continued •Nutrients absorbed from the digestive tract, e:g. monosaccharides (especially glucose), amino acids, fatty acids, glycerol, and vitamins; are transported to •Waste products of metabolism are transported from the tissues to site of excretion, e.g carbon dioxide produced from cellular activity is carried to the lungs for excretion, and the waste products of protein metabolism (urea, creatinine, uric add) are transported to the kidneys for excretion. •Hormones are carried from endocrine glands to the organs where they are needed. 3

4

Development of blood cells

. Anticoagulants Used in Hematology

•EDTA (ethylene diamine tetra-acetic acid), also. called sequestrene, and tri-sodium citrate. These chemicals prevent blood from clotting by removing calcium. EDTA anticoagulated blood can be used for most tests, e.g. haemoglobin, PCV,WBC count platelet count reticulocyte count; and reporting blood cell morphology. It is not suitable for coagulation tests. The ICSH, (International Committee for Standardization in Haematology) recommends the use of dipotassium EDTA at a concentration of 1.5 ±O.25' mg\ml of blood. 6

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Continued

Stability of anti-coagulated blood EDTA anticoagulated blood

•Trisodium citrate 32 g\l is used to anticoagulate blood for:

•When EDTA anticoagulated blood cannot be tested within 1-2 hours it must be refrigerated at 4-8 C to prevent cellular changes affecting test results.

•Measuring the ESR, with 1.6 - ml of venous blood (or previously collected EDTA blood) being mixed with 0.4 ml of sodium citrate anticoagulant.

•Manual or automated blood cell counts, reticulocyte counts, and PCV change little in EDTA blood at 4-80C when stored for up to 24 hours .

•Coagulation tests, with 9 ml of venous blood being mixed with 1 ml of sodium citrate anticoagulant

•Hemoglobin concentration is stable for 2-3 days at 4-8 °C providing there is no hemolysis

.

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Continued

Continued Important

Some of the blood cell changes which occur in EDTA blood include:

Blood which has been refrigerated must be allowed to warm to room temperature and be well mixed before being tested.

•. Neutrophil degeneration with neutrophils becoming more irregular in shape, nudear lobes separating, and vacuoles .appearing in the cytoplasm. There is also loss of granules.

In EDTA anticoagulated blood, morphological blood cell changes occur soon after blood is collected when is stored at room temperature (18-250C) and within 3 hours when stored at 4-8 C

•Segmentation (budding) of the nucleus of lymphocytes and monocytes and vacuoles appearing in the cytoplasm.

•Erythrocytes becoming crenated and spherocytic. Platelets disintegrating

It is therefore recommended that blood films be made and methanol-fixed as soon as possible after blood is collected and never made after overnight storage.

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Continued Citrate anti coagulated blood:

Even when citrated blood is stored at 4-8 C there is a decrease in the ESR due to changes in erythrocyte shape affecting rouleaux. The ESR should be measured within 4 hours. of collecting the blood. Coagulation tests should be carried out as soon as possible after blood is collected into citrate anticoagulant.

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Continued

Blood films staining Romanowsky stains

Important :

These stains contain eosin Y which is an acidic anionic dye and azure B and other thiazine dyes (derived from the oxidation, or polychroming, of methylene blue) which are basic cationic dyes. When diluted in buffered water, ionization occurs. Eosin stains the basic components of blood cells, e.g. haemoglobin stains pink‐red, and the granules of eosinophils stain orange‐red. Azure B and other methylene blue derived dyes, stain the acidic components of cells.

Reliable blood film reporting is only possible when laboratory staff are trained adequately in the recognition of blood cells and follow standardized procedures for preparing and staining blood films, reporting morphological changes and performing a differential white cell count.

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Continued

Continued Note:

Nucleic acids and nucleoprotein, stain various shades of mauve‐ purple and violet

There is some variation between batches of Romano sky stains due to the different thiazines and iron ties they contain. A highly . purified Romano sky stain been developed which contains only azure B and eosin Y.I recommended by ICSH for standardized Romano staining but it is very expensive and not needed for rout work.

The granules of basophils stain dark blue‐violet, and cytoplasm of monocytes and lymphocytes stains blue or blood grey. The staining reactions of Romano sky stained are dependent which is why the stains are diluted in buffered water of specific pH

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Continued

Continued Staining results

Leishman staining technique cont. Many of the difficulties in reporting blood films,particularly red cell morphology, are due to variable staining. It is important therefore for laboratories use a reproducible standardized staining technique.

Red cells …….Pink‐red cont. Nucleus of cells…… Purple violet Cytoplasm Neutrophils, eosinophils Pale pink Large Lymphocytes Clear blue Small lymphocytes .... Darker dear blue Monocytes Grey‐blue

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Continued

Continued Inclusions:

Granules

Malaria pigment (in monocytes)' Brown‐black Howell‐Jolly body , Purple‐violet

Eosinophils ..................... Orange‐red Neutrophils …………………. Mauve‐purple Toxic granules ……………… Dark Violet Basophils …………………….. Dark blue‐violet Platelets : ……………………. Purple

Auer body (in myeloblast) : Purple‐red

Wright's stain is similar to Leishman stain* * A different polychroming technique is used in the production Of Wright's stain and nuclear staining is usually paler than

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Red Cells


(erythrocytes) form the main cellular cormponent of blood, i.e. about 450/0 of total blood volume in an adult, giving blood its red coulor. Each litre of blood contains about 5 x 1012 red cells, the exact number varying with age, gender, and state of health


Red Cells

Production of red cells

Continued

• Red cells are produced in the bone marrow Tissue hypoxia (lack of oxygen) leads to the release of the hormone erythropoietin which stimulates progenitor cells BFU‐E and CFU‐E, to develop into pronormoblasts (proerythroblasts). • Development in bone moitiw (about 5 days)

Continued BFU‐E CFU‐E Pronormoblast

Reticulocyte count Early Normoblast

• Value of test : Reticulocytes are immature, red cells normally present in small numbers in the blood (up to 2%). Reticulocyte numbers increase when. there is an increase in erythropoietin activity. • A reticulocyte count assesses bone marrow activity, e.g. whether there is an effective erythropoietic response when there is a reduction in the number of red cells due to haemolysis or haemorrhage. • A reticulocyte count is also of value in monitoring the erythropoietic response of an anaemic patient following treatment

Late normoblast

Reticulocyte Blood circulation Normal blood also contains Up to 2% reticulocytes

Erythrocyte • Note : Normal erythropoiesis is dependent on there being adequate amounts of erythropoietin, protein and particularly item vitamin B12 and folic acid which are essential for hemoglobin synthesis.

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Hemoglobin synthesis

Continued

• The main function of red cells is to carry O2 to the tissues and to return carbon dioxide (C02) from the tissues to the lungs. In order to achieve this gaseous exchange they contain the specialized protein haemoglobin. Each red cell contains approximately 640 million haemoglobin molecules. Each molecule of normal adult haemoglobin (Hb) A (the dominant haemoglobin in blood after the age of 3‐6 months) consists of four polypeptide chains, a.2~2' each with its own haem group. The molecular weight of Hb A is 68 000. Normal adult blood also contains small quantities of two other haemoglobins: Hb F and Hb A2These also contain a chains, but with y and I) chains, respectively, instead of B

• Haemoglobin synthesis in the developing red cell. The mitochondria are the main sites of protoporphyrin synthesis, iron (Fe) is supplied from circulating transferrin; globin chains are synthesized on ribosomes.b‐Al.A, l)..aminolaevulinic add; Coa, coenzyme A.

Normal Hemoglobin Types

PCV and red cell indices • Value of test: The packed cell volume (PCV) also called haematOOit, is used to calculate the mean cell haemoglobin concentration (MCHC and mean cell volume (MCV). These red cell indices are used in the investigation of anaemia. TheTV is also used to screen for anaemia whf not Possiblle to measure haemoglobin, and to. diagnose polycythaemia vera and to monitor its treatment is suitable for screening farge dinic Populations, e.g. antenatal clinics.

Continued

Measuring red cells indices

• The packed cell volume is that proportion of whole blood occupied by red cells, expressed' as a' ratio, (Iitrellitre). when using an electronic cell analyter which computes the value from the MCV and red cell Count • (PCV = MCV x RBC).

• Red cell indices most frequently' used in the invesgation of anaemia are: • Mean cell haemoglobin concentration (MCHC) • Mean cell volume (MCV) • Mean cell haemoglobin (MCH)

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MCHC

MCV

• The MCHC gives the concentration of haemoglobin gld in litre of packed red cells. It is calculated from the haemoglobin (Hb) and PCV as follows:

• The mean red cell volume (MCV) • provides information on red cell size. It is measured in femtolitres (fl) and is determined from the PCV and electronically obtained RBC count .It can be calculated as follows:

– Hbg/l = MCHC g/l – PCV (l/I)

• PCV (l\l) = MCV fl RBC×1012/l .

MCV

Disorder of red cells The main disorders of red cells are: • Anaemia • Haemoglobinopathies (thalassaemias, abnormal haemoglobins) . • Disorders due to red cell enzyme defects,eg G6PD deficiency • Disorders due to red cell membrane defects,eg hereditary spherocytosis • Polycythaemia

• The MCH gives the amount of hemoglobin in picograms (pg) in an average red cell. It is calculated from the hemoglobin and electronically obtained RBC count: Hb in g\l RBC x 1012/1 = MCH pg • A picogram (pg) is 10‐‐12 of a gram.

414


Anemia • Worldwide anaemia is the commonest red cell disorder. It occurs when the concentration of haemoglobin falls below what is normal for a persons age , gender, and environment, resulting in the oxygen carrying capacity of the blood being reduced.


• Haemoglobin values are lower in women than men probably due to menstrual loss and the influence of hormones on erythropoiesis. Hemoglobin levels fall in normal pregnancy due to an increase in plasma volume. Anemia 2

Haemoglobinizeltion of red cells with the terms:

Morphologically anemia can be classified by:

• Normochromic, describing normal staining of red cells as seen when haemoglobinization is adequate. The cells contain a small area of central pallor (no more than one third of the cell's diameter) due to the biconcavity of red cells. • Hypochromic, describing pale staining of red cells, as seen when haemoglobinization is inadequate. Hypochromic cells show an increased area of central pallor.

• Red cell size with the terms: ‐ Normocytic referring to normal size red cells (approximately 8 um in diameter) ‐ Microcytic referring to smaller than normal red cells ‐ Macrocytic referring to larger than normal red cells.

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Macrocytic Anemia Caused by:

Microcytic hypochromic anemia caused by: • • • •

Megaloblastic changes: • Folate deficiency • Vitamin B12 deficiency

Iron deficiency (commonest cause) Thalassaemia syndromes Some anaemias of chronic disease Sideroblastic anaemia*

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Normocytic changes caused by :

Normochromic Anemia • In a normocytic normochromic anemia the red appear normocytic and normochromic in a strained blood film and the MCHC; MCV and MCH are normal.

• Liver disease • Alcoholism • Haemolytic anaemia (associated with raised reticulocyte count.)

• A normocytic normochromic anaemia may, found in: Acute blood loss Anaemia of chronic disease Aplastic anaemia

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Blood film findings

Haemolytic anaemias

• Haemolytic anaemias are characterized by a falling hemoglobin, jaundice, dark urine, increasing reticulocytosis (when there is effective erythropoiesis) and usually splenomegaly.

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Red blood cell (RBC) inclusions • May be seen in the peripheral blood film in various conditions • The reticulocyte RNA and Heinz bodies are only demonstrated by supravital staining (e.g. with new methylene blue • Heinz bodies are oxidized denatured haemoglobin. Siderotic granules (Pappenheimer bodies) contain iron. They are purple on conventional staining but blue with Perls' stain • The Howell‐Jolly body is a DNA remnant. • Basophilic stippling is denatured RNA.

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HEREDITARY RED CELL MEMBRANE DISORDERS

Continued

• Hereditary spherocytosis: Which usually causes only a mild chronic haemolytic anaemia with splenomegaly. Obstructive jaundice due to gall stones is a common complication. Red cells have a reduced life span. Anaemia can become more severe when there is also folate deficiency , anaemia of chronic disease and aplastic crisis induced by parvovirus B19 infection.

• The blood film shows spherocytes (which may be scanty) with polychromasia. • The osmotic fragility test shows increased fragility of the cells, particularly following 24 h incubation. • The direct antiglobulin test (DAT) is negative which helps to differentiate this condition from immune causes of spherocytosis in which the DAT is positive.

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Hereditary elliptocytosis

Hereditary ovalocytosis

• May cause mild anaemia In homozygotes. It is found in West and North Africa. A hemolytic crisis may occur during severe infections, eg malaria The blood, typically shows elliptocytes (elongated red cells) and also red cell fragments

• A common disorder in Southeast Asia, Indonesia, Philippines and Melanesia. It is usually an asymptomatic condition. The blood film contains large numbers ovalocytes

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(a) The bone marrow aspiration needle and a smear made from a bone marrow aspirate. (b) The bone marrow trephine (biopsy) needle and normal trephine section.

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Abnormal Haemoglobins • HbS which has a wide distribution in tropical Africa, parts of India, the Caribbean Mediterranean region, Arabian peninsula, arid elsewhere in people of African descent


• HbC which is found only in West Africa and else‐ where in people of West African descent • HbE which is found in Southeast Asia and the Indian subcontinent. Abnormal Hemoglobins 2

Continued

Alkaline cellulose acetate electrophoresis • Used to separate and idetify the different hemoglobins by there migration within an electric field . Hemoglobin variants separate at different rates due to differences in their surface electrical charge as determined by their amino acid structure

Several techniques are available to separate haemoglobin variants by electrophoresis. For routine work, electrophoresis in an alkaline buffer at pH 8.4‐8.6 using a cellulose acetate membrane is adequate. This gives good separation of HbA, HbF, Hbs, and HbC Cont.

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Continued

An electrophoresis chamber (tank), model

• On alkaline electrophoresis HbD and HbS have the same mobility and HbC, HbE and HbO also co‐migrate. In specialist laboratories agarose gel electrophoresis at an acid pH (6.0) can be used to separate these haemoglobins and also to provide a clear separation of HbF from HbS and HbC

Plate S.S(a) Helena BioSciences Zip Zone electrop ,. chamber suitable for alkaline cellulose acetate haem electrophoresis

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Continued

G6PD Deficiency • G6PD deficiency is the commonest cause of the inherited red cell enzyme disorders. Deficiency of the enzyme glucose‐6‐phosphate dehydrogenase (G6PD) in red cells reduces the ability of the cells to withstand lysis from oxidant damage, particularly during infections and following exposure to oxidant drugs or chemicals.

• (b) Applicator system used with the electrophoresis chamber. Courtesy of Helena Bio Sciences

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Granulocytes, monocots


The White Cells 2

Normal blood counts

White blood cells

Adults Total leucocytes Neutrophi1s Eosinophils Monocytes Ba50phils Lymphocytes

Blood count

Children

Blood count

Total leucocyies 4.00‐11.0xH)9/L* 2.5‐7.5 x 109/L*. 04‐0.4 x 109/L 0.2‐0.8 x 109 /L

Neonates 1 year 4‐7 years 8‐12 years

10.0‐25.0 X 109/L 6.0‐18.0 x 109/L 6.0‐15.0 x 109/L 4.5‐13.5 x 109/L

0.01‐0.1 x 109/L 1.5‐3.5 x 109/L

4

Abnormal white blood cells.

Continued •

(a) Neutrophil leucocytosis:

Toxic changes shown by the presence of red‐purple granules in the band form neutrophils.

Dahle body can be seen in the cytoplasm of the neutrophil.

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Continued

Continued (e)Pelger‐Huet anomaly: Coarse clumping of the chromatin in configuration.

(c) Megaloblastic anaemia: hypersegmented oversized neutrophil in " peripheral blood.

(f) Chediak‐Higashi syndrome: Bizarre giant granules in the cytoplasm of a monocyte

(d) May‐Hegglin anomaly the neutrophils contain basophilic inclusions 2‐5 in diameter

(g) Alder's anomaly:

there is an associated mild thrombocytopenia with giant

Coarse violet granules in the cytoplasm of a neutrophil.

platelets. 7

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Lymphocytes and their benign disorders • Lymphocytes :


In postnatal life, the bone marrow and thymus are the primary lymphoid organs in which lymphocytes develop. The


secondary lymphoid organs in which specific immune responses are generated are the lymph nodes, spleen and lymphoid tissues of the alimentary and respiratory tracts. Lymphocytes and their benign disorders 2

Lymphocytosis Lymphocytes:

• Lymphocytosis often occurs in infants and young

(a) small lymphocyte;

children in response to infections that produce

(b) activated lymphocyte; (c) large granular lymphocyte;

neutrophil reaction in adults.

(d) plasma cell.

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Causes of lymphocytosis

The Spleen functions of the spleen

• Infections • Acute: infectious mononucleosis, pertussis, mumps, acute infectious lymphocytosis, infectious hepatitis' cytomegalovirus, HIV, herpes simplex or zoster

• The spleen is the largest filter of the blood it and several of its functions are derived from • Control of red cell integrity – The spleen has an essential role in the control of red cells.

• Chronic: tuberculosis, toxoplasmosis, brucellosis. syphilis Chronic lymphoid leukaemias Acute lymphoblastic leukemia Non‐Hodgkin's lymphoma (some) Thyrotoxicosis 5

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Continued

Continued

Extramedullary haemopoiesis • The spleen like the liver undergoes a transient period of haematopoiesis at around 3‐7 months of fetal life but is not a site of erythropoiesis in the adult. However,haemopoiesis may be restablished in both organs as extramedually haemopoiesis

In disorders such as myelofibrosis or in chronic severe haemolytic and megaloblastic anaemias. Extramedullary haematopoiesis may result either from reactivation of dormant stem cells within the spleen or moving of stem cells from the bone marrow to the spleen

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Continued • The lymphoid tissue in the spleen is in a unique position to respond to antigens filtered from the blood and entering the white pulp. Macrophages and dendritic cells in the marginal zone initiate an immune response and then present antigen to B and T cells to start adaptive immune responses

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Leukemia • The leukaemias are a group of disorders characterized by the accumulation of malignant white cells in the bone marrow and blood.


Leukemia 2

Classification of leukemia

Continued

Acute Acute myeloid leukaemia (AML): Mo‐M7 Acute lymphoblastic leukaemia (ALL): L1‐L3

AML • • • • • • •

Chronic Chronic myeloid leukaemias (CML) Chronic lymphoid leukaemias (CLL)

Cytogenetic Abnormality

Mo undifferentiated MI. without maturation M2 with granulocytic maturation t(8,21) t(15%17) M3 acute promyelocytic inv(16) M4 granulocytic and monocytic maturation M5 monoblastic (M5a) or monocytic (M5b) M7 megakaryoblastic.

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Continued

Differentiation of ALL from AML

ALL : • L1: blast cells small, uniform high nuclear to cytoplasmic ratio. • L1: blast cells larger, heterogeneous, lower nuclear to cytoplasmic ratio . • L3: vacuolated blasts, basophilic cytoplasm (usually B‐ALL)

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Acute myeloid leukemia

Continued

•

The French‐American‐British (F AB) classification of acute myeloid leukaemia.

•

(a) M1 blast cells show few granules but may show Auer rods, as in this case

•

(b) M2 cells show multiple cytoplasmic granules

•

(e) M3 blast cells contain prominent granules or multiple Auer rods

•

(d) M4 blasts have some monocytoid differentiation

•

(e) M5a monoblastic leukaemia in which 80% of blasts are monoblasts

•

(f) M5b monocytic but<80% of blasts are monoblasts.

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Continued •

(g) M6 showing preponderance of erythroblasts

• (h) M77megakaryoblastic leukemia showing cytoplasmic blebs on blasts.

Prognosis in acute myeloid leukemia (AML)

Favourable (t15; 17) t(S;21) Bone marrow response to remission inv (16) NPM mutation Induction 5% blasts after first course <60 years Age Onset Cytogenetics

Primary

Umfavourable Deletion. of chromosome 5 or 7 FLt-3 mutation 1lq23 1(6;9) abn(3q) Compiex.rearrmgeme nts >20% blasts after first course >60 years Secondary 11


Haematological Tests


Chart Main laboratory features of acute and chronic leukemia's


Chronic leukemia 2

Erythrocyte sedimentation rate (ESR) (Westergren technique)

Acute leukaemias Acute lymphoblastic Kaemia (ALL) Acute myeloblastic kaemia (AML)

Value of test: • The erythrocyte sedimentation rate (ESR) is a nonspecific test. It is raised in a wide range of infectious, inflammatory, degenerative and malignant conditions assciated with changes in plasma proteins, particularly increases in fibrinogen immunoglobulins, and C‐reactive protein.

Chronic Leukaemias

Chronic lymphocytic leukaemia (CLL)

Chronic myeloid leukaemia (CML) * Chronic stage

* Accelerated/acute stage2

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Continued

Equipment • Disposable plastic Westergren pipettes are used. It is graduated from 0‐200 mm. The diameter should not be less than 2.55 mm • Timer capable of timing accurately 1 hour

The ESR is also affected by many other factors including anaemia, pregnancy, haemoglobinopathies, hemoconcentration and treatment with anti‐ inflammatory drugs.

• Reagent • Tri‐Sodium citrate, 32 g/l • (3.2% w Iv) anticoagulant

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Specimen

Specimen Either venous blood collected directly into sodium citrate and tested within 2 hours

Either venous blood collected directly into sodium citrate and tested within2 hours, or EDTA anticoagulated blood diluted in sodium citrate can be used. If EDTA blood is used and kept refrigerated at 4‐8°C citrate dilution of the blood and testing can be delayed for up to 6 hours

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Quality control

Continued • Testing blood samples at the hottest time of the day, or leaving tests in direct sunlight. Temperatures over 25.C increase sedimentation.

• The most practical way of controlling ESR tests is to follow the test method exactly. • Problems (erroneous results) occur when: • Using the wrong volume of blood to anticoagulant

• Pipette not positioned vertically. Even slight variations from the upright increase sedimentation.

• Blood not sufficiently mixed with anticoagulant • Clots in the blood. Even the smallest fibrin dot in the sample will invalidate the test result • Air bubbles at the top of the column.

• Not checking whether the ESR stand is level on the bench. • Placing an ESR stand on the same bench as a centrifuge where vibration will interfere with sedimentation. • Measuring the ESR when a patient is dehydrated. 9

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Interpretation of ESR test results

Causes of a significantly raised ESR • Anemia due to any cause . • Acute and chronic inflammatory conditions and infections including. • HIV disease. • Tuberculosis. • Acute viral hepatitis. • Arthritis. • Bacterial endocarditis. • Pelvic inflammatory disease. • Ruptured ectopic pregnancy • Systemic lupus erythromatosis.

Reference range : • Men …………………….. Up to 10 mm/hour. • Women …………………. Up to 15 mm/hour. • Elderly …………………… Up to 20 mm/hour.

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Causes of bleeding disorders


Abnormal bleeding may be caused by: • Damage to vascular endothelium • Reduction in platelet numbers • Defective platelet function • Disorders of blood coagulation


Investigation of bleeding disorders 2

Laboratory investigation :

• • • •

APTT test method

• Test the control plasma and patient's plasma in duplicate • Reference APTT range : Normal plasma clots in 36‐45 seconds

Hemoglobin test Blood film report Platetet count Bleeding time test

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Sources of error when performing APTT tests (also and TT tests)

Continued

• Difficulty in obtaining a venous sample resulting in hemolysis or small clots. in the sample. • Delay in testing the plasma and leaving sample at room temperature. • Using tubes or pipettes which are not clean and dry or are contaminated with detergent. when ever possible use disposable tubes. • Not timing accurately the different stages of the test (a stop‐ watch must be used).

Causes of a prolonged APTT include: DIC (involving several clotting factors) . • Deficiency of clotting factors • On heparin anticoagulant

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Continued

Continued

• Using unsatisfactory reagents (will be detect when testing control plasma). • Not pipetting correct volumes of plasma and reagents • Not correctly filling tubes to specified line • Clotting in collection tube • Inadequate mixing

Reconstituting reagents and control plasma contaminated deionized water. This is a common cause of incorrect coagulation tests (usually shorter times are obtained). Whenever possible fresh distilled water should be used. When it is possible to obtain good quality water, pure distilled water from a pharmacy or manufacture of the coagulation reagents being used.

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Prothrombin time (PT) test

Continued

• The PT is a screening test for the extrinsic clotting system,

• INR =ISI i.e prothrombin ratio to the power of the ISI Cont.

i.e. factor VII. It will also detect deficiencies factors, prothrombin, V, X and fibrinogen. It is mainly used to monitor patients receiving warfarin anticoagulation.

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Reference PT range • Normal plasma samples (patients not on anticoagulant) in 11‐16 seconds. Each laboratory should establish its own normal reference range.

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Continued


The normal hemostatic response to vascular damage depends on closely linked interaction between the blood vessel wall,


circulating platelets and blood coagulation factors

Platelets , blood coagulation 2

Thrombocytopenia

Continued

Infections, e.g. typhoid and other septicaemias . Deficiency of folate or vitamin B12 Aplastic anaemia . Drugs (e.g. cytotoxic, quinine , aspirin), chemicals (e.g. benzene), some herbal remedies. • Leukaemias, lymphoma, myeloma, myelofibroses, carcinoma. • Hereditary thrombocytopenia (rare condition). • • • •

The involvement of blood vessels, platelets and blood coagulation in haemostasis. ADP. adenosine diphosphate

3

4

Continued

Thrombocytosis Causes of an increase in platelet numbers include: • Chronic myeloproliferative diseases. E.g. essential thrombocythaemia, polycythaemia vera, chronic myeloid leukaemia, myelofibrosis. • Carcinoma (disseminated). • Chronic inflammatory disease, e.g. tuberculosis • Haemorrhage. • Sickle cell disease associated with a non functioning spleen or after splenectomy. • Iron deficiency anaemia, associated with active bleeding.

Increased destruction or consumption of platelets. • Infections, e.g. acute malaria, dengue, trypanosomiasis, visceral leishmaniasis. • Disseminated intravascular coagulation (DIC) • Hypersplenism. • Immune destruction of platelets

5

6

430

Thrombin time (TT) test • The TT test is sensitive to a deficiency of fibrinogen or inhibition of thrombin. It measures the formation of a fibrin clot by the action of thrombin on fibrinogen.


Thrombin time (TT) test 2

Reference TT range

Fibrinolytic system • Fibrinolysis is the enzymatic process used by the body to remove a fibrin thrombin to restore normal blood flow once damaged endothelium is repaired.

Normal plasma samples clot within 12‐15 seconds

3

4

Continued

Continued • Laboratory tests are available to detect and semi‐quantify,

During the clotting process, tissue, plasminogen activator (t‐PA) released from the blood vessel wall. and the plasma proenzyme

FDPs (D‐Dimers) in plasma. Most are simple to perform

plasminogen bind to the forming fibrin thrombus. When

slide tests using latex particles coated with anti D‐Dimer.

activated plasminogen is converted to plasmin which degrades

FDP titres in excess of 500 ng/ml can be found in DIC also in

the fibrin network,. Causing the clot to dissolve, During this

thrombosis, phelibitis, and embolism.

process fibrin degradation products (FDPs) i.e fragments called D‐ Dimers are produced

5

6

431


Principle of impedance analyzers • Blood cells are diluted in a buffered electrolyte solution.


A measured volume of the sample passes through an aperture tube (e.g. 100 !‐tm in diameter) between two


electrodes. Interruption of the current by the non‐ conducting blood cells alters the electrical charge and a pulse‐is produced.

Automated blood cell counting using an electronic blood cell analyzer

Continued

Continued

• The amplitude of each pulse is proportioned to the volume of

Analysis of the pulse heights enables mean cell volume

the cell which caused it. A threshold circuit ensures only

(MCV) to be measured and the haematocrit to be

those pulses that exceed the pre‐set threshold level are

calculated from the MCV value and red cell count

counted, The cell count is determined from the. total number of pulses obtained from a measured volume of blood.

3

4

Continued

Semi Automated Instruments

• The haematocrit is determined from voltage pulse data and

• Which require blood samples to be externally diluted (with separate dilutions for counting RBCs and WBCs)

the MCV calculated from the haematocrit value. The haemoglobin concentration is used with the red cell count, MCV and haematocrit, to calculate the MCH and MCHC

5

6

432

Fully Automated Instruments

Continued

• With internal dilution and simultaneous counting of white

•

cells, red cells, and platelets. The more advanced analyzers •

In addition to determining haemoglobin, WBC, RBC, platelets, haematocrit, MCV, MCHC, and MCH, also provide red cell distribution width (RDW), platelet distribution width

In addition to determining haemoglobin, WBC, RBC,

(PDW) and a white cell differential

platelets, haematocrit, MCV, MCHC, and MCH, also provide red cell distribution width (RDW), platelet distribution width (PDWl) and a white cell differential

7

8

Use of an electronic blood analyzer • An electronic blood cell analyzer is appropriate to use when: •

The work load is sufficiently high.

• The capital cost and running costs of the analyzer are affordable.

9

433


Specimen Collection

434

Kingdom of Saudi Arabia – Ministry of Health General Directorate of Training and Scholarship Training Program for Health Institute Graduates Specimen Collection

Specimen Collection - Slide 8 – Blood Universal precautions (old term) has been relabeled as “Standard Precautions”. This should be for ALL patients with anticipated blood, body fluid, or pathogen exposure.

435


Introduction • As part of the decision process for diagnosing, confirming, treating or monitoring diseases, physicians rely heavily on the results of the clinical laboratory tests. So, physicians use laboratory tests to diagnose disease, to monitor its progress or its response to treatment, and to screen for disease in seemingly healthy individuals.


• Many physicians consider disease as the only possible cause of abnormal test results. Yet, many factors besides disease affect the composition of body fluids; these factors may be either preanalytical or analytical. Specimens Collection 2

Introduction

Introduction

• Whenever possible, preanalytical variability should be controlled so that correct interpretation of test results is facilitated.

• When factors are not controllable (e.g. genetic and other long‐term influences) their possible effect on test values should be recognized and considered in the evaluation of laboratory data. • The diagnostic testing process can be divided into three phases:

• The variability of test results due to biological factors is often greater than the variability due to analytical factors.

1. 2. 3.

• Control of biological variability begins with proper preparation of an individual before proper specimen collection.

Preparation of the patient and collection of a specimen (pre‐ analytical phase). Analytical measurement by the laboratory (analytical phase). Reporting of the testing results to the physician (post‐analytical phase).

• The preanalytical phase is being concerned in this lecture. 3

4

Specimen Labelling

Types of Specimens

• Before collecting a specimen of any type, specimen containers must be labeled with the patient’s name, hospital or identification number, location in the hospital, and date and time of collection.

There are different types of samples on which laboratory investigations will be done according to certain diagnostic purposes for respective diseases. These types include: 1. Blood. 2. Urine. 3. Other body fluids: 4. Stools. 5. Solid tissue (tissue biopsy). 6. Swabs. 7. Calculi. 8. Investigations done on the individual himself.

• All specimens from patients with dangerous infections should be labeled with a yellow “dangerous specimen” sticker. A similar label should be attached to the request form. Of most concern to the laboratory staff are hepatitis and HIV viruses, but all specimens should always be handled as potentially hazardous. 5

6

436

Types of Specimens

1. Blood

Other body fluids: 1. Cerebrospinal fluid (CSF). 2. Peritoneal (Ascitic) fluid. 3. Pleural fluid. 4. Pericardial fluid. 5. Synovial fluid. 6. Seminal fluid. 7. Amniotic fluid. 8. Saliva and sputum. 9. Wound exudates, pus and others.

• Before collecting any blood sample, a phlebotomist should put on disposable gloves. • If a patient is known to have an infectious disease, universal precautions should be observed. • These precautions include the wearing of a facemask, glasses or goggles, and gown in addition to gloves.

7

8

Blood specimen tubes for specific laboratory tests

Containing 1 : 4 Sodium citrate as anticoagulant.

7) Metal-free tubes

Containing no anticoagulant and are demineralized.

8) Heparinized syringe

Containing heparin as anticoagulant.

9) Blood culture bottle

Containing liquoid (sodium polyanethol sulphonate and nutrient broth) as anticoagulant and bacterial growth medium.

Blood culture bottle

Containing 1 : 9 sodium citrate as anticoagulant.

6) 1 : 4 citrate tube

Heparinized syringe

Containing EDTA as anticoagulant.

5) 1 : 9 citrate tube

1 : 4 citrate tube

4) EDTA tube

1 : 9 citrate tube

Containing sodium fluoride and potassium oxalate as anticoagulants.

EDTA tube

3) Fluoride/oxalate tube

Fluoride/oxalate tube

Containing lithium heparin as anticoagulant.

Lithium heparin tube

Containing no anticoagulant.

2) Lithium heparin tube

Plain tube

1) Plain tube

Blood specimen tubes for specific laboratory tests

9

10

Blood Forms for Investigations

Blood Forms for Investigations

Serum: • If blood is collected into a plain tube and allowed to clot, after centrifugation (within one hour of clotting) a serum specimen is obtained. It is the recommended specimen for many biochemical, serological, immunological and hormonal analyses.

Whole blood: • It is obtained by collecting blood into a tube containing anticoagulant as EDTA for CBC, glycosylated haemoglobin (HbA1C) and glucose‐6‐ phosphate dehydrogenase (G6PDH), as 1:4 sodium citrate for ESR, as heparin for arterial blood gases (ABGs), and as liquid for blood culture. Red blood cells: • This type of samples is obtained by collecting blood into a tube containing EDTA. Then after centrifugation, the supernatant is removed, a suitable amount of normal saline (NaCl 0.9%) is added with gentle mixing and after second centrifugation the supernatant is removed. This process will be repeated until the supernatant becomes clear and colourless where it is removed, and the deposit now contains only red blood cells (RBCs) which are recommended for G6PDH assay and haemoglobin electrophoresis.

Plasma: • The blood is collected into a tube containing an anticoagulant and when centrifuged, the supernatant is called plasma. It is preferred when the analyte in question is unstable and speed is necessary. – Lithium heparin tube: For most biochemical tests. – 1 : 9 citrate tube: For PT, PTT and INR, and other coagulation studies. – Fluoride/oxalate tube: For blood glucose and lactate.

11

12

437

Blood Sampling • • • •

A. Venipuncture Before performing a venipuncture, phlebotomist should: •Verify that the patient is fasting if this is necessary to ensure medically useful results. The patient should be comfortably seated for about 20 min. before the specimen is withdrawn. This standardization minimizes differences in concentrations of blood due to variations in the blood volume. •Estimate the volume of blood to be collected and select the appropriate number and types of tubes for the plasma or serum tests requested. An appropriate needle must also be selected. The most commonly used sizes are gauges 19 to 22 for adults (21 ‐ 23 for children). The larger the gauge, the smaller the bore.

Blood for analysis may be obtained from: Veins by venipuncture: Venous blood. Capillaries by skin puncture: Capillary blood. Arteries by arterial puncture: Arterial blood.

13

14

A. Venipuncture

A. Venipucture

• Venipuncture is accomplished by using a vacutainer set or a syringe.

• A tourniquet should not be left in position for more than 1 minute and the patient should not be allowed to pump his or her fist while the tourniquet is in place. • When blood collection is complete, the tourniquet should be released and the needle is withdrawn. Then, the patient should hold a dry gauze pad over the puncture site, with the arm raised to lessen the like‐hood of leakage of blood. The pad can subsequently be held in place by a bandage. • The phlebotomist must separate the needle from the syringe or vacutainer tube and discard it in a container (sharps container) designed specifically for this purpose. • Vigorous suction on a syringe during collection, or forceful transfer from the syringe to the receiving tube may cause haemolysis of blood. Haemolysis is usually less when blood is drawn through a small‐bore needle because turbulence of the blood is less than when a larger‐bore needle is used. • Do not collect blood from the arm with a drip attached. Use opposite arm.

• The median cubital vein in the anticubital fossa is the preferred site for collecting venous blood in adults because the vein is both large and close to the surface of the skin. Veins on the back of the hand or at the ankle may be used, although these are less desirable and should be avoided in diabetics and other individuals with poor circulation. In severely ill patients and those requiring many intravenous injections, an alternative blood drawing site should be chosen to preserve the good veins for the patient’s treatment. • After cleaning the skin by alcohol swab (by benzalkonium chloride in specimens for alcohol determinations), a tourniquet is applied about 10 cm above the intended puncture site. 15

16

B. Skin Puncture

B. Skin Puncture

• Skin puncture is the method of choice in paediatric age group although it is done when only small volume of blood is required for a blood test in adults (e.g. glucose estimation by glucometer). It is often preferred in geriatric patients because of thinness of skin and the loss of elasticity. • The puncture site is: – The centre of the palmer side of the tip of the third or fourth finger of the nonwriting hands in adults or grown children. – Earlobe in adults or grown children. – The lateral or medial plantar surface of the foot in infants younger than 1 year of age. – The plantar surface of the big toe in older children.

Acceptable sites for skin puncture to collect blood from an infant’s foot

17

18

438

B. Skin Puncture

C. Arterial Puncture

• After selection of the puncture site, it is warmed by warm and moist towel, cleaned by alcohol swab and then the puncture is made with sterile lancet (manually or automatically). The depth of incision should be less than 2.5 mm to avoid contact with bone. • The first drop of blood is wiped off and subsequent drops are transferred to the appropriate tube by gentle contact. Blood may be collected into capillary tubes by capillary attraction. Filling should be done rapidly to prevent clotting, and the introduction of air bubbles should be avoided. • Massage of the finger to stimulate blood flow should be avoided because it causes the outflow of debris and of tissue fluid which does not have the same composition as plasma. The finger should be held in such a way that gravity assists the collection of blood on the fingertip. • The site of puncture must not be oedematous or a previous puncture site. • Skin puncture is time‐consuming and there is a greater risk of infection than from venipuncture.

• Arterial blood is used to measure arterial blood gases (ABGs). • Arterial punctures require considerable skill and are usually performed only by physicians or specially trained technicians or nurses. • The preferred sites of arterial puncture are (in order) the radial artery at the wrist, the brachial artery in the elbow and the femoral artery in the groin. • Because leakage of blood from the femoral artery tends to be greater, especially in elderly, sites in the arm are most often used. • The artery to be punctured is identified by its pulsation and thick wall. The skin surrounding the puncture site should be cleaned. No tourniquet is required for arterial puncture. 19

20



• A needle with a gauge size 18 – 20 should be used for one of the larger arteries but a smaller needle with a gauge size 23 – 25 should be used to collect the blood from the smaller arteries. • The needle and syringe should be flushed out with heparin solution both to ensure adequate anticoagulation and to eliminate trapping of air in the needle and in the dead space of the nozzle. • Evacuated blood tubes should not be used for the collection of specimens for blood gas analysis because the residual air in the tube may cause false results. • Once an arterial puncture has been performed, firm pressure should be applied over the puncture site for at least 5 minutes to minimize bleeding.

• After collecting the specimen for blood gas analysis, the nozzle of the syringe should be sealed and the syringe should be placed in ice water for immediate transport to the clinical laboratory. • Analysis should be performed within 15 minutes because chilling will not completely inhibit the metabolic activity of white blood cells. • The best method for blood gas specimen collection in neonates is the indwelling umbilical artery catheter. • In older children or adults in whom it is impossible to perform an arterial puncture, a capillary puncture may be performed to obtain arterialized capillary blood. Such a specimen yields acceptable values for pH and pCO2 but not for pO2. 21

22

Abnormal Colour of Serum or Plasma


• Normally, serum or plasma is clear and its color is yellowish like the color of cooking oil. According to the abnormal color of the sample, there are 3 types of samples:

1) Haemolyzed sample: •Serum or plasma is reddish in color that results from hemolysis of red blood cells. This can affect some laboratory investigations as follows: – Direct effect: Some constituents in red blood cells or in their membranes go out into the plasma or serum affecting the concentration of its constituents e.g. potassium, lactate dehydogenase, acid phosphatase, etc. – Indirect effect: The color affects the investigations done by colorimetric method e.g. glucose and bilirubin, and interferes with the kinetic determinations of certain constituents e.g. creatinine, CPK and alkaline phosphatase.

1. Haemolyzed sample. 2. Lipaemic sample. 3. Icteric sample.

23

24



2) Lipaemic sample: •Serum is whitish in color and may be milky due to its high content of lipids especially triglycerides. The color of this sample interferes with most (if not all) the methods employed for biochemical tests except those for lipid profile. This sample should be divided into 2 parts:

3) Icteric sample: •Serum or plasma is greenish yellow in color that interfere with the colorimetric methods for many investigations e.g. glucose and cholesterol, and with the kinetic determination of creatinine that should be measured by deproteinization method.

– The first part is used directly for lipid profile assay. – To the second part, equal volume of ethyl acetate will be added and well mixed. Then, it is separated by centrifugation into 2 layers: the upper layer containing the ethyl acetate with fat, and the lower clear layer for all investigations except lipid profile.

25

26

Timing of Blood Sampling

Timing of Blood Sampling

• All investigations for metabolites with dietary sources will be assayed after fasting for 8 – 12 hours except for lipid profile; the fasting should be 12 ‐ 14 hours. • Since the most satisfactory specimen is a post‐ absorptive one, the best time for sampling is after fasting overnight. This helps the laboratory as regards planning the day’s work. • The time at which a specimen is obtained is important for those blood constituents that undergo circadian (diurnal) variation (e.g. cortisol, growth hormone, iron) and those used to monitor drug therapy (e.g. digoxin, lithium or prothrombin time) because the interval after drug administration affects the drug concentration.

• Filaria migrates into peripheral blood at night. So, midnight is the best time for diagnosis of filariasis. • Metabolic challenge studies (such as xylose absorption test and glucose tolerance test) require specimen collection at specified timed intervals. • It is preferred to collect blood sample for the assay of female sex hormones according to their relation to menstrual cycle, e.g. at the luteal phase for progesterone, at the follicular phase for oestrogen and FSH, and in the mid‐cycle for LH. 27

28

Transport and Storage

Transport and Storage

• Transport of blood samples from collection site to the laboratory is an important component of sample processing. Specimen should reach the laboratory without undue delay. • All laboratory specimens must be transported in a safe and convenient manner to prevent biohazard exposure or contamination of the sample. • Agitation of blood specimens should be avoided to minimize haemolysis. • Specimens should be protected from direct exposure to light, which causes breakdown of certain metabolites e.g. bilirubin which should be analyzed as soon as possible.

• For analysis of unstable constituents, such as ammonia, plasma rennin activity and acid phosphatase, specimens must be kept at 4°C immediately after collection and they should be transported on ice. • Some serum specimens as that of liver enzymes, can be stored at 2 ‐ 8°C for 24 ‐ 48 hours, and others, as glucose and lipids, can be frozen at –20°C for months or even years. • The effect of freeze‐thaw cycles on constituent stability is an important consideration. Once the frozen sample is thawed, it should be analyzed as soon as possible. 29

30

Important Advises for Patient Preparation and Sample Processing

Sampling Errors There is a number of potential errors that may contribute to the success or failure of the laboratory to provide the correct answers to the clinician’s questions. Some of these problems may arise when the clinician first obtains specimens from the patient. 1. Blood sampling technique:

• Per‐rectum examination, prostatic massage, urinary catheterization and chronic constipation should be avoided for about one week before collecting the sample for acid phosphatase assay. • Muscular exercise or straining and intramuscular injection should be avoided before collecting the sample for total CPK, total LDH, and aldolase assays. • Individuals for lipid profile estimation should be on lipid‐free diet for 72 hours before specimen collection. • Individuals for glucose tolerance test (GTT) should be on the ordinary carbohydrate diet for 72 hours before specimen collection. • Mouth pipetting of any sample must be avoided.

o

2.

Prolonged stasis during venepuncture: o

3.

Difficulty in obtaining a blood specimen may lead to hemolysis with consequent release of red cell constituents. Plasma water diffuses into the interstitial space and the serum or plasma sample will be concentrated. Protein and protein‐bound components such as calcium, total bilirubin, lipids and thyroxin will be falsely elevated.

Insufficient specimen: o

Each blood analysis requires a certain volume of specimen to enable the test to be carried out.

31

32

Sampling Errors

2. Urine

6.

• The type of urine specimen to be collected is dictated by the tests to be performed. • Single specimens of urine (random or morning samples) are used for ward examinations and qualitative tests. • A clean, morning, fasting specimen is usually the most concentrated specimen performed for microscopic examinations and for the detection of abnormal amounts of constituents such as proteins and glucose, or of unusual compounds such as chorionic gonadotropin (for pregnancy test). • The double‐voided specimen is the urine excreted during a time period after complete emptying of the bladder by 15 – 30 min. It is used, for example, to glucose excretion during a glucose tolerance test. Its collection must be timed in relation to glucose ingestion. • 24 hours‐urine collections are preferred for quantitative assays due to the diurnal variation in the excretion of some substances.

Errors in timing: o

7.

Incorrect specimen container: o

8.

For many analytes, the blood must be collected into a container with appropriate anticoagulant. If a sample is collected into wrong container, it should never be decanted into another type of tube.

Inappropriate sampling site: o

9.

The biggest error in the measurement of some analytes may arise from inappropriate time of sampling.

Blood samples should not be taken from an intravenous drip or from the arm into which a canula is connected.

Incorrect specimen storage: o

A blood sample stored overnight before being analyzed will show falsely high potassium, phosphate and red cell enzymes such as lactate dehydrogenase because of leakage into extracellular fluid from the cells.

33

34

2. Urine

2. Urine

The 24 hours‐urine collection is as follows: a) At a suitable time (e.g. 8:00am), the patient empties his bladder and the urine is discarded. b) All specimens passed during the following 24 hours are saved in an appropriate container. c) At the same time of the next morning the patient empties his bladder and the urine is added to the collecting one.

• Instructions for collection of urine specimens for 5‐ hydroxyindole‐acetic acid (5‐HIAA) measurements should specify avoidance of bananas, plums, walnuts, pineapples and eggplant, as well as, acetaminophen and cough syrup containing glyceryl guaiacolate for 72 hours before sampling. • The patient’s genitalia should be cleaned before urine voiding to minimize the transfer of surface bacteria to the urine. • Bacterial examination of the first 10 ml of voided urine is the most appropriate to detect urethritis, whereas the midstream specimen is the best one for investigating bladder disorders.

o

N.B.: The creatinine content of the urine sample can be used as a rough check on the reliability of the collection. 35

36

441

2. Urine

Storage and Preservation of Urine Specimens

• Catheter specimens are used for microbiological examination in critically ill patients or in those with urinary tract obstruction, but should not normally be obtained just for biochemical assays. The suprapubic tap specimen is a useful alternative, because the tap is unlikely to cause infection. • Collection of a timed specimen from an infant is difficult. But for a random sample, a plastic bag is placed around the infant’s genitalia and left in place until urine has been voided. First, the scrotal or perineal area should be cleaned and dried, and any natural or applied skin oils should be removed. • To obtain a sterile urine specimen for culture from an infant, a suprapubic tap is performed. • The collection of a specimen from older children is done as in adults, using assistance from a parent when this is necessary.

• It is satisfactory in most cases to use specimens collected in cool, clean containers. • It is particularly important to use freshly voided and concentrated urine to identify casts, RBCs, and WBCs. One should deliver specimens to the laboratory within one hour of collection. • One of the most satisfactory forms of preservation of urine specimens is refrigeration immediately after collection. This is more successful when combined with chemical preservation. • Acidification to below pH 3.0 is widely used to preserve 24‐hours specimens for catecholamines, vanillylmandelic acid (VMA), 5‐ hydroxyindole acetic acid (5‐HIAA), steroids, and calcium assays. This acidification is done by adding HCl (10 ml of 6 mol/l, per 24‐H excretion), sulfamic acid (10 ml/l urine) or by boric acid (5 mg/30 ml urine). This is unsuitable for measurement of uric acid that is precipitated by acidification. 37

38



• Sodium bicarbonate is used to adjust pH between 8.0 and 9.0 for sample preservation of porphyrins, porphobilinogen, urobilinogen and uric acid. • Specimens for porphyrins, porphobilinogen, urobilinogen and bilirubin are collected and stored in dark containers to avoid their degradation by direct exposure to light. • Toluene is the organic solvent that is still used as a preservative. It acts as a barrier between the air and surface of the specimen. Neither thymol nor chloroform is used nowadays. • Urine may be frozen to be assessed at a later time. When repeat testing is expected, the specimen is stored in multiple bottles to avoid specimen degradation as a result of repeated freeze thawing of a single specimen.

• Before a specimen is transferred into small containers for each of the ordered tests, it must be thoroughly mixed to ensure homogeneity because the specific gravity, volume and composition of the urine may all vary throughout the collection period. • Urine should not be collected at the same time for two or more tests for which different preservatives are required. • Urine is collected for cytological identification of malignant cells into equal volume of 50% alcohol. 39

40

3. Cerebrospinal fluid (CSF)

3. Cerebrospinal fluid (CSF)

• Cerebrospinal fluid may be obtained by lumbar, cisternal or lateral cervical puncture or through ventricular cannulas or shunts. • Up to 20 ml of spinal fluid can be safely aspirated from an adult, although this amount is not usually required. Clinician should provide clinical history to the laboratory. The puncture site (i.e., lumbar, cisternal, etc.) should be noted since cytologic and chemical parameters vary at different sites. • CSF must not be blood‐stained. The first 1.0 to 2.0 ml should be discarded. • CSF (3 – 4 ml) should be aspirated into 3 tubes: the first tube should be used for chemical and/or serological tests, the second for microbiological tests, and the third for microscopical and cytological examination.

• Tests on spinal fluid require rapid processing of the specimens to minimize cellular degradation, which begins within 1 hour of collection. • CSF may be evaluated in the laboratory to establish a diagnosis of infection (bacterial, fungal, mycobacterial, or amoebic meningitis), malignancy, subarachnoid haemorrhage, multiple sclerosis or de‐ myelinating disorders. • Antiglycolytic agents (sodium fluoride) usually are not added to the CSF tube for glucose measurement because rapid processing of the sample ensures that little metabolism of glucose occurs even in the presence of many bacteria. • Highly bloody sample for cell count should be collected in EDTA tube to prevent formation of clot. • Glass tubes should be avoided since cell adhesion to glass affects the cell count. • Refrigeration is contraindicated for culture specimens because organisms like Haemophilus influenzae and Neisseria meningitidis will not survive. 41

42

442

4. Peritoneal, Pleural and Pericardial Fluids

5. Synovial fluid

• These fluids are aspirated by paracentesis (peritoneocentesis, thoracentesis and pericardiocentesis) which should be performed only by skilled and experienced physicians. • Specimens are obtained for chemical (proteins and/or enzymes), microbiological and cytological examinations. • For most chemical evaluations, no additive is used and specimen is allowed to clot. Bacteriological and cytological specimens may be collected in sterile EDTA or sodium heparin containers without preservatives. • On sample processing for mycobacterium, anaerobic bacteria or viruses, safety precautions and special handling procedures should be applied prior to specimen collection.

• Synovial fluid is obtained by arthrocentesis, which should be performed by a physician using sterile procedures. The technique must be modified from joint to joint depending on the anatomical location and the size of the joint. • Sterile plain tubes should be used for culture and for glucose, uric acid and protein measurements. An EDTA tube is needed for total leucocytes, differential and erythrocytes count. Microscopic slides are prepared for staining with Gram’s or other stains indicated, and for visual inspection. • The physician should establish priorities for the tests to be performed in case the available volume of synovial fluid is in‐ sufficient for all tests. • Normally, only a very small amount of fluid is present in any joint, but this volume is usually very much increased in the presence of inflammatory conditions. 43

44

6. Saliva and sputum

7. Seminal fluid

• The individual is asked to rinse out his mouth with water and then chew an inert material as a piece of rubber or paraffin wax for five minutes. The first mouthful of saliva is discarded and the followed saliva is collected into a small glass bottle. • The clinical application of methods using saliva has been limited. However, saliva can be used for drug analysis. • On the morning for 3 successive days, the fasting patient is asked to expectorate sputum into 3 sterile containers respectively. • The 3 specimens will be stained by Ziehl‐Neelsen stain for the diagnosis of Mycobacterium tuberculosis.

• Sexual abstinence should be 3 – 5 days before semen analysis. • Seminal fluid should be collected by one method of the following: – Sheath: It may be ruptured causing loss of seminal fluid. – Coitus interruptus: The semen (some or all) may be ejaculated into the vagina or may be contaminated by vaginal secretions. – Masturbation: It is the preferred method. The individual should not use water and soap but masturbation should be done by dry or saliva‐lubricated hand.

• Time of ejaculation should be written on the label, and once the sample is received it should be placed in the incubator at 37°C to be examined within the next 3 hours. 45

46

8. Amniotic Fluid

9. Stools

• Amniotic fluid is withdrawn by amniocentesis, which should be performed by physician. • It is performed for prenatal diagnosis of congenital disorders (by measurement of α‐fetoprotein), to assess fetal maturity (by measurement of the lecithin / sphingomyelin ratio, or to look for Rh in – compatibility (by measurement of bilirubin) or intrauterine infection (by culture). • The specimen is collected in dark brown container, which should immediately be placed in ice.

• The investigations which can be done on stools include: – Parasitological examination for the detection of any infested parasite. The individual is considered free from parasites when this investigation is negative for 3 successive days. – Biochemical investigation for detection of occult blood, for measurement of fecal fat, reducing substances and nitrogen, and for estimation of trypsin activity in feces of children. – Microbiological studies for detection of the cause of diarrhoea by culture (and sometimes sensitivity tests) for Salmonella, Campylobacter, Brucella, Shigella or Cholera.

• No preservative is added to feces but the container should be kept refrigerated throughout the collection period and should be sent to the laboratory within a short time after completing the collection. • Care should be taken to avoid contamination from urine or water. • It is preferable to avoid using stools containing liquid paraffin or barium salts, or which have been collected after an enema has been given.

47

48

443

10. Solid Tissue (Tissue Biopsy)

11. Swabs

• The solid tissue is most often analyzed in the clinical laboratory for malignancy. It may be used for toxicological analysis. • At least 0.5 – 1.0 g tissue should be removed surgically and should be trimmed of fat and non‐tumor material. • The tissue (especially that from breast for estrogen and progesterone receptors) should be quickly frozen, preferably in liquid nitrogen or in a mixture of dry ice and alcohol. The time between collection and freezing should be less than 20 minutes. • Tissue biopsy can be preserved in 10% formalin until analyzed histologically. • The tissue biopsy should be accompanied by a report from the surgeon including patient’s name, sex and age, date of tissue removal, anatomical site of the biopsy and the probable diagnosis.

• A swab can be taken from a tissue or from an organ for cytological studies or for culture and sensitivity test. • Swabs include conjunctival swab, throat swab, wound swab, high vaginal swab (HVS), cervical swab and others. • If the swab or any specimen is for culture and sensitivity test, the patient must not be under antibiotic therapy for 3 days before sampling. 49

50

12. Calculi (Stones)

13. Investigations done on the individual

• Description of the received stone should be documented. • This includes the type (either renal or gall stone), number, color, shape, surface, size and consistency of the stone.

Some investigations can be done on the individual himself e.g.: 1. Skin allergy test: The patient should not take antihistaminics or corticosteroids for 3 days before the test. 2. Tuberculin test for failed‐ or non‐BCG vaccinated individuals.

51

52

444


Quality Control and Assurance

445


Aims


• To have an overall understanding of the key terms associated with medical laboratory quality control & assurance (QCA) • To be familiar with the importance of sensitivity and specificity


Quality Control Assurance I Key Terms plus Sensitivity & Specificity 2

Teaching methods

Learning outcomes

• Formal lectures • Small group problem‐based to develop team work

To have a brief knowledge and understanding of the main aspects of quality control & assurance including management

• Seminars or workshops

To have a knowledge and understanding of the key aspects & • On site laboratory or practical sessions

importance of clinical sensitivity & specificity

• When appropriate video presentations

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4

Quality assurance

Key definitions

• Quality control: checking the performance of a particular test each time it is run; it is to ensure that the operator, reagents & equipment are all working properly.

• Overall term for monitoring all practices and procedures within the laboratory to make sure that required standards are being maintained.

5

6

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Quality assessment (internal or external)

Quality control & assurance

• Testing the standard of the laboratory procedures usually by testing a ‘blind ‘ sample • This can be an internal sample or one supplied from an external laboratory • ( In this case the dept can measure its performance against national standards)

• Policies, processes & practices undertaken by a laboratory that are necessary to ensure a test on a clinical sample gives the correct result & that the finding is delivered to the appropriate clinician in a timely & efficient manner

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8

Quality

Quality audit

• Quality: this is essentially about consistency • Getting the procedures right first time and every subsequent occasion

• Internal check of the whole procedure from the patient through the laboratory and back to the patient

• Quality assurance: is all the planned & systemic actions necessary to give adequate confidence that a product or service satisfies the necessary level of quality

• Exercises can involve members of staff outside of pathology

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10

Clinical governance

Accreditation

• Procedure through which a department is given formal recognition that they meet certain minimum standards in their work as defined by an independent organisation

• A system of regulation and monitoring to make sure that the highest quality of patient care is provided within a healthcare organisation

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Standard operating procedures (SOPs)

Good Clinical Practice (GCP)

•

These specify in detail how a particular procedure or method will be carried out in clinical laboratory in accordance with good laboratory practice (GLP) and good clinical practice (GCP)

•

SOPs need to be very accurate and hence can be very difficult to write

•

They must contain clear instructions regarding the operation of specified equipment and how to use the appropriate consumables

•

Health and safety should also feature

1. A standard for design, conduct, performance, monitoring, auditing, recording and analysis in a clinical setting/laboratory 2. That provides assurance that the data & reported results are credible and accurate in a clinical setting/laboratory 3. That the rights, integrity and confidentiality of the patient are protected in the laboratory and hospital setting

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14

Guidelines for writing SOPs

Guidelines & standards (SOPs etc) • International conference of harmonization good clinical practice (ICHGCP)

• SOPs are the work instructions to ensure standardization of working practices & compliance with relevant guidelines & standards

• Food & drug administration (FDA) • Control of substances hazardous to health (COSHH)

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Quality management system

QMS Continued

• Overall system of procedures and policies intended to ensure that defined standards are met with clear plans to address failures to meet these standards (the documentation in this system must be “controlled”)

• In the clinical laboratory setting it is essential to ensure that associated clinicians, doctors and patients are involved in the laboratory service

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Clinical sensitivity & specificity

Quality management systems (QMS) • • • • •

• To be effective a medical test is expected to detect abnormalities with a high degree of confidence • How likely is it that an individual who has a positive test has the disease? • What are the chances that an individual has a certain disorder even though the test for it is negative?

Training staff Showing compliance with current regulations & standards Internal & external quality controls (QCs) Training & competency assessments Audits

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Specificity

Sensitivity

• A measure of the ability of a test to identify individuals who are healthy after testing negative OR • Measures the proportion of negatives which are correctly identified as such (e.g. the percentage of healthy people who are correctly identified as not having the condition)

• The measure of the ability of a test to identify those individuals who have a disorder after testing positive

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Important aspects of “objective” tests

Warning

• Accuracy & validity • Variability, reproducibility or precision

• Unfortunately clinical tests are neither 100% specific nor 100% sensitive

• Two indices used to evaluate the accuracy of a test ‐ sensitivity & specificity

• In fact the two factors show an inverse relationship with each other

• Not absolute values

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449

The relationship between a diagnostic test result & the occurrence of disease

Sensitivity & specificity

DISEASE

PRESENT

ABSENT

POSITIVE

TRUE POSITIVE a

FALSE POSITIVE b

NEGATIVE

FALSE NEGATIVE b

TRUE NEGATIVE c

TEST

Indices are usually determined by administering the test to one group of persons who have the disease and another group who do not have the disease

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26

True & false positives

True & false negatives

• Those who have the disease test positive

• Those who test negative & do not have the disease

• “True positives”

• “True negatives”

• Those who test positive but do not have the disease

• Those who test negative but have the disease

• “False positives”

• “False negatives”

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28

Sensitivity

Sensitivity & specificity

• Sensitivity is the proportion of truly ill people in the screened population who are identified as ill by the screening test

True positives

• Specificity is the proportion of truly healthy people who are so

Sensitivity =

identified by the screening test

true positives plus False negatives

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450

Sensitivity

Sensitivity

True positives Sensitivity = All those with the Disease

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Specificity

Specificity

True negatives

True negatives

Specificity =

Specificity =

All those without the disease

True negatives plus False positives

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Specificity

Sensitivity & specificity ‐ important

• Sensitivity ‐ probability of a positive test in people with the disease • Specificity ‐ probability of a negative test in people without the disease

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451

The relationship between a diagnostic test result & the occurrence of disease DISEASE

PRESENT

ABSENT

POSITIVE

TRUE POSITIVE a

FALSE POSITIVE b

NEGATIVE

FALSE NEGATIVE b

TRUE NEGATIVE c

TEST

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Diagnostic test characteristics & definitions

Sensitivity (Se) = a / a + c Specificity (Sp) = d / b + d Prevalence (PV) = a + c / a + b + c + d Likelihood Ratio (LR)+ = a/a + c / b/b + d Likelihood Ratio (LR)‐ = c/a + c /d/b + d

False positives & negatives

Predictive value

• (A) a low limit results in a more sensitive test

• Sensitivity & specificity provide information about the accuracy of the test

• (B) intermediate limit results in minimum total error • Do not provide information about the meaning of a positive or negative test results

• (C) a high limit results in a more specific test

• Predictive values • Positive & negative

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Positive predictive value

Positive predictive value

True positives Positive predictive = Value

True positives Positive predictive = Value

true positives plus False positives

All those with a Positive test result

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Negative predictive value

Positive Predictive Value

True negatives Negative predictive = Value true negatives Plus false negatives

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Negative predictive value

Negative Predictive Value

True negatives Negative predictive = Value

All those with a Negative test result

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The relationship between a diagnostic test result & the occurrence of disease

Predictive values ‐ important

DISEASE

PRESENT

ABSENT

POSITIVE

TRUE POSITIVE a

FALSE POSITIVE b

NEGATIVE

FALSE NEGATIVE c

TRUE NEGATIVE d

• Positive predictive value ‐ probability of the person having the disease when test is positive

TEST

• Negative predictive value ‐ probability of the person not having the disease when the test is negative

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Relationship between prevalence, sensitivity, specificity & positive predictive value (+PV)

Diagnostic test characteristics & definitions

Positive predictive value (+PV) = a / a + b

Sensitivity X Prevalence (+PV) = (Sensitivity X Prevalence) + (1‐specificity) X (1‐prevalence)

Negative predictive value (‐PV) = d / c + d

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Relationships continued

Relationships continued

The more specific the test is, the better will be its positive predictive value

More sensitive a test is, the better will be its negative predictive value

The more confident the clinician can be that the positive test confirms or rules in the diagnosis being sought

The more confident the clinician can be that the a negative test result rules out the disease being sought

Predictive values are influenced by prevalence

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Reading list

What did you learn?

• Burnett, D. (2002). A Practical Guide To Accreditation In Laboratory Medicine. ACB Venture Publications. London • Department Of Health (2000). An Organisation With Memory. Dept Of Health, London. • MHRA (2007). Rules & Guidance For Pharmaceutical Manufacturers & Distributors. Pharmaceutical Press. London • Vorley, G., Tickle, F. (2002). Quality Management, Tools & Technique 5th Edt Quality Management & Training (Publications) Ltd, London

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Aim


• To be familiar with areas associated with quality management in a clinical setting


Lecture Quality Control Assurance II Quality Management 58

Learning outcomes

Quality Management (QM)

• To have knowledge and understanding of quality management and related systems associated with clinical laboratory and hospital setting • To be familiar with the types of good practice associated with and required in a clinical laboratory setting

• Is any system where the management has direct control of an organization with regard to quality • Quality is a never ending cycle of events that require the support and co‐operation of all the people in the organization

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QM Approach

QM Continued

• Is an attitude or culture within an organisation • Every stage of a process, system or project is a possible source of poor quality • Great potential for poor quality are complicated systems like hospitals • “Learning organisations have systems and mechanisms & processes that enhance the services they provide

• All organisations must ensure that the quality of services they provide are maintained and continually improved

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QM Continued

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The International Standards Organisation (ISO)

• Hospitals should be able to adapt to their environments and apply the results of their to achieve better results • Hence hospitals should be continually evolving and enhancing their capabilities • Need to learn from own and others mistakes to reduce the future risks to patients • Difficult to reduce totally adverse events totally from complicated systems like hospitals

• Develops standards for international organisations to follow or adopt • Document‐ISO 9001:2008 outlines guidelines for QMS • QMS is a model that may be employed to give guidelines to the selection of appropriate QC activities

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Non‐conformity

Aim of QMS

• Non‐conformity is any deviation from that which is required • An effective QMS will try and satisfy customers or consumers by concentrating on the service • Achieve this by customer surveys and monitoring complaints • A documented process designed to achieve consistency, provides a goal for the system control and maintaining the required level of quality at each stage in the process

• To prevent non‐conformity or a non‐conformance to a set of standards

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QMS: ISO 9001:2008 – Definition (Eight Requirements) • • • • • • • •

Eight requirements

1. Customer focus 2. Leadership 3. Involvement of people 4. Process approach 5. Systems approach 6. Continual improvement 7. Actual approach to decision making 8. Mutually beneficial supplier relationships

• Forms the basis for any QMS in the clinical laboratory • All eight contribute to improving the quality of care to patients

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Customer focused QM

Leadership

• Meets the needs of the clinicians who treat the patients • Clinical laboratories achieve this through user surveys, monitoring complaints and compliments • Also by analysing incidents

• This is provided by management commitment from consultants and senior laboratory managers • If senior management are not involved in the quality process it will not work • Leadership undertaken through the provision of quality policy and health & safety statements • Also through the actions & attitudes of senior managers

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High Quality Service Provision

Leadership‐ Pathology Lab

• Only arises from commitment of everyone in the organisation • It needs everyone to be involved and aware of the system in place • Often the case that people performing specific tasks have a better understanding of problems than the manager • Need a well motivated work force who feel valued & take pride in their work • Excellence in the service provided is the responsibility of everyone concerned

The senior team may consist of: • Clinical director • Pathology service manager • Head biomedical scientist • Or clinical scientist for each department

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A process approach

Continual efforts

• Individual areas must not be looked at in isolation • The whole process must be examined • Changing something in one area will have a knock‐on‐effect in other areas • Therefore likely to require collaboration of a different services such as pathology, radiology & pharmacy

• Must be made to improve the clinical laboratory service and to improve the accuracy and timelines of results as an aid to the diagnostic process • Various tools require including audit, lean thinking, cause & effect, & process redesign • Usually no single improvement tool is likely to provide all the answers to improve any one situation • Essential to select the tool that best fits the one in hand

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Factual approach to decision making

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Mutual beneficial supplier relationship

• Means making decisions based on facts and the analysis of data • This should allow the clinical laboratory to match the needs of the service users with business planning • Example: purchase equipment for present that will also meet future needs‐increased workloads • Make the right decision using – audit, incident reporting, statistical analysis to predict future trends

• Improves services in a number of ways • Thus, if the laboratory & the suppliers of laboratory equipment & its maintenance & service contracts, reagents and consumables work together • This will improve the quality of the laboratory provision

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Clinical governance

Personnel

• Is a form of QM brought in to improve the quality of care for patients • Safe guard high standards of care • Create an environment that allows clinical excellence to flourish • Requires the creation of a culture, as well as systems & methods of working to ensure opportunities for quality improvement are identified • Need to provide a better experience for patients & staff

• QM requires to be led by senior management • Employ personnel to implement and maintain QMs & the clinical governance framework • Quality manager or clinical governance manager • Need to work with the eight requirements

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Effective process

Improving the quality of medical laboratories

• Once established the focus should be on the continual improvement of the operation of the laboratory • This could mean‐establishing more effective ways of working, reducing costs or improving training opportunities • To improve – must learn from mistakes, from analysis of data & from others • Process of continual improvement – a never ending cycle

• Adverse healthcare events • Such events in hospitals and clinical laboratories can lead to serious harm to a person or even cost lives • When dealing with hundreds of samples per day it is it possible for a mistake to be made • Laboratories have efficient processes in place that strive to deliver the correct test result to the right patient in a timely & efficient manner • This ensures appropriate diagnosis & effective treatment can be given

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Documentation in the clinical laboratory

Documentation – standards & requirements

• Possible hierarchy of documentation in a quality management system

• ISO 15189:2007 Medical Laboratories – Particular Requirements For Quality And Competence • ISO 22870:2006 – Point Of Care Testing Requirements For Quality And Competence • These Outline A Set Of Criteria That Need To Be Fulfilled By Clinical Laboratories • These Standards Also Include Requirements For A QMs As Outlined In ISO 9001‐quality Management Systems – Requirements • Documentation Must Be Provided To Confirm That Laboratories Are Meeting These Standards

• Top: quality policy & quality manual • Middle: policies • Bottom: Standard Operating Procedures (SOPs)

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Minimum requirements

Quality policies (QPs)

• Need to meet requirements specified in standards • QMs in a laboratory require quality policy (QP), quality manual & standard operating procedures (SOPs) for the processes in place • There are also procedures to control the documents and to control changes made to any processes

• Are short one page document that outlines the scope of the service the laboratory intends to provide • Contains details of laboratory’s commitment to its users and staff & outlines the organization’s quality objectives • Designed by a senior manager – clinical director • Communicated & available to all laboratory staff • Displayed on the notice board

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Quality manual

Standard Operating Procedures (SOPs)

• Documentation that outlines how the objectives stated in the op are to be achieved • Sections are linked to or referenced to standards with which the organisation is trying to comply • It also contains organisational‐specific information Including: organisational structure, processes and procedures & resources needed to implement quality to meet to objectives described in SOPs

• SOPs are written instructions that describe in a clear & concise manner how procedures and methods are to be performed • How are individual tasks to be carried out • That is what is to be done • By whom and when

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SOPs continued

What did you learn?

• Accurately describe the process & methods that are to be used in the laboratory • Contain essential information such as health & safety instructions • Correct use of equipment • Correct use of controls • Always need up‐to‐date SOPs • Correctly describe what is to be done to ensure no step is missed & that the quality of results are not compromised

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88


Reading list • Burnett, D. (2002). A Practical Guide To Accreditation In Laboratory Medicine. ACB Venture Publications. London • Department Of Health (2000). An Organisation With Memory. Dept. Of Health, London. • MHRA (2007). Rules & Guidance For Pharmaceutical Manufacturers & Distributors. Pharmaceutical Press. London • Vorley, G., Tickle, F. (2002). Quality Management, Tools & Technique 5th Edt Quality Management & Training (Publications) Ltd, London

Lecture Quality Control Assurance III Standards & Regulations 89

460

Aims

Learning outcomes

• To be familiar with standards & regulatory requirements associated with a clinical laboratory

• To have a knowledge and understanding of the key standards and regulatory requirements associated with a clinical laboratory setting • To familiar with the types of good practice associated with and required in a clinical laboratory setting

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Accreditation

Standards & regulatory requirements in the medical laboratory

• Accreditation is the process by which an organisation gains recognition that its activities & products have attained a certain level of compliance against a set of defined standards

• Clinical laboratories subject to rigorous inspections by various assessment & accreditation bodies

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Standards

UKAS

• Are codes of best practice that improve safety & efficiency • ISO is the world's largest developer & publisher of international standards (ISO 9001) • The Clinical Pathology Accreditation Ltd (CPA UK Ltd) which is part of UKAS– sets standards related to ISO 15189 & 9001 • The Human Tissue Authority (HTA) • The Medicines & Healthcare Products Regulatory Agency (MHRA) – inspect clinical labs

• This body assess against national & international standards as published by the British Standards Institute (BSI) & the International Organisation for Standardization (ISO)

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CPA

Clinical Pathology Accreditation (CPA) Standards • Organisations that declare adherence to a defined standard of practice & have their ability to attain these standards independently confirmed that they are ‘accredited’ can reassure their users of the quality of service they provide

• Based on ISO 15189 medical laboratories‐particular requirements for quality & competence • This an international standard that incorporates some elements of ISO 9001

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Standards – cover eight major areas • • • • • • • •

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Organisation & QMs

1. Organisation & quality management system (a standards) 2. Personnel (b standards) 3. Premises & environment (c standards) 4. Equipment, information systems & materials (d standards) 5. Pre‐examination process (e standards) 6. Examination process (f standard 7. Post‐examination process (g standard) 8. Evaluation & quality assurance (h standard)

• • • • •

Needs and requirements of users QP documentation Quality objectives and plans for the laboratory The quality manual Document control procedures, and control process and quality record

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Premises & Environment

Personnel • • • •

100

Personnel management Staff induction Records and meetings Staff training & education

Encompass: • The work areas • Staff & patient facilities • Health & safety in the laboratory

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Pre‐examination Process

Equipment, Information Systems & Materials

Encompasses: • Information for users • Specimen collection & handling • Requirements for specimen request forms • Transport of specimens

• Covers procurement & management of equipment , materials, data & information

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Pre‐examination process

Post‐examination Process

• Sets standards relating to the selection and validation of the examination and the quality of the examination that is performed on the specimens

• Covers areas such as final report & how the results are reported

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CPA Assessment process

Evaluation & QA

Laboratory enrol with CPA Inspected every 4 years by CPA 1‐2 day external audit by trained assessors Team consists of a regional assessor and at least two peer assessors • Regional are employed full‐time by UKAS and coordinate the visit • Peer assessors drawn from consultant pathologists, clinical scientists & biochemical scientist & specific areas (immunology etc) *There are other bodies • • • •

• Investigates how the laboratory assess user satisfaction, the laboratory audit process & quality improvement processes in place in the laboratory

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Blood Safety & Quality Regulations (BSQR)

Good Laboratory Practice (GLP)

• Changed two European directives in to UK law – [directives 2002/98/Ec & 2004/33/Ec]

• GLP provides the framework within which laboratory work is performed • The medicines & healthcare products regulatory agency (MHRA) • Assesses the compliance of hospital blood banks & blood establishments to the BSQR

• The UK as a duty to translate the European directives into national legislation • BSQR requires hospital blood banks & blood establishments to maintain a quality system based on the principles of Good Laboratory Practice (GLP)

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Records

MHRA • • • • •

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Laboratory submits online compliance report to MHRA MHRA assesses online report against the BSQR MHRA may decide to have an onsite assessment/visit Usually one day & single assessor Assessor checks to see that there is accurate & complete traceability of blood and blood components

• Records kept by the hospital laboratory and wards will be examined • Also check records and documents of satellite sites that receive and store blood & blood components

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Shot & Sabre

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Human Tissue Act (England 2004)

• One requirement of BSQR is that the laboratory must notify the MHRA and serious hazards of transfusion (SHOT) of any serious adverse blood reactions and events (SABRE) • Online reporting system that can be accessed through the MHRA website • The reporting of SABRES allows MHRA & SHOT teams to identify any trends in blood transfusion incidents

• Provides a legal framework for issues relating to the taking, storage & use of human tissue & organs & whole body donations

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Point of Care Testing (POCT)‐requirements for quality & competence

The Act

• POCT can be defined as testing at or near the site of patient care • Increases the likely hood that the patient well receive the result of a clinical test in a timely manner • Often used in A & E departments • Neonatal care units • GP surgeries • The patient’s home

• The act makes made consent the foundation for the lawful storage, and use of human bodies, body parts, organs and tissue & the removal tissue from the deceased, & regulates the use of tissues and cells that can be used for transplant & stem cell research from loving donors

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Standards

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POCT continued

• ISO 22870:2006 • Point of Care Testing (POCT) – Requirements for quality & competencies outlines the requirements for POCT • The clinical laboratory must ensure that POCT Is co‐ordinated effectively & efficiently in the hospital environment • Policies & procedures must be in place that outline how the equipment must be used • Staff must be trained on use of equipment & records of training maintained

• Medico‐legal considerations must be taken into account • Requirements of data protection act • Laboratory must be involved in the purchase of new equipment for use outside of the laboratory • Must be involved in training the staff how to use it • Must ensure that equipment is serviced in accordance with manufacturer’s requirements

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POCT continued

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Tools and techniques for continual improvement

• “POCT” Is not assessed or accredited in isolation but as part of the overall laboratory accreditation process

To check or assure that the processes & methods used in a clinical laboratory comply with required standards These tools include: • Audit • Root Cause Analysis (RCA)

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Audit

Audit continued

• ISO9001 Defines an audit: • Integral part of any QMs • Requirement for accreditation (CPA) • Means for continually improving the service produced by a laboratory • Identify areas for improvement by gathering information – observation/interview/sampling • Also identifies processes that are working well • Opportunity to identify good practice and transfer the details

• ‘A systematic & independent examination to determine whether quality activities & related results comply with the planned arrangements and whether these arrangements are implemented effectively and are suitable to objectives’

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Audit continued

Audit continued • Check practices against procedures & to thoroughly document any differences

• Enables evaluation of processes • Determine deficiencies • Generate a cost effective & efficient solutions to problems

• Only a sample exercise so cannot confirm that all aspects of the process are being complied with at all times

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Example process

Types of audit • • • •

• • • • • • • •

1. Vertical 2. Horizontal 3. Witness or examination 4. Self‐assessment

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Step 1. Start of process – skin lesion removed from patient Step 2. Sample transported to lab & received by sample reception# Step 3. Sample labelled, details added to computer Step 5. Sample suitably processed in lab Step 6. Slides made from sample Step 7. Slides stained, cover slips added. & Checked for problems Step 8. Slides examined by consultant & report typed Step 9. Report issued‐end of process

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Vertical & Horizontal

A Witness/Examination Audit

• Vertical audit – examines a single item from start to finish ( step 1. To step 9 previous slide)

• Examines the person undertaking a task • Step 3 – observing a person labelling samples to check that he or she have read & are following the relevant sop

• Horizontal audit – examines one element in a process that is performed on more that one item (step 6. EXAMINES MANY SLIDES FOR ONE ELEMENT; section quality)

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Internal, External & Cooperative Audits

Self‐Assessment Audit

• Internal; conducted by clinical laboratory staff themselves • Example: – 1. Biomedical scientist in haematology could conduct an internal audit in the microbiology lab – 2. An internal vertical audit could be conducted by the lab staff on a clinical sample subject to a particular process or method

• This is a careful evaluation that is usually performed by the organisations own management which results in an opinion or judgement as to its effectiveness & efficiency

• Identify any non‐compliance with standards or in QMs hence make recommendations

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External and cooperative audits

Audit plan or schedule

External: • Conducted by an outside person or body • Possible accreditation • An external horizontal can be conducted on a specific element ( staff training records) Cooperative: • Conducted between the lab & another party for mutual benefit –clinical audits/user satisfaction surveys/benchmarking activities

• Must be done before the audit can be carried out • What audits are to be undertaken, when, where & who will be performing them • Must be discussed & agreed by senior management at the beginning of the year

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Audit schedule

Stages of an audit

• Audit of laboratory equipment: • January ‐ horizontal audit on equipment such as analysers/centrifuges/microscopes used in lab • Staff records: • February ‐ horizontal audit on CPD of staff • Transport of samples: • March ‐ vertical audit on receipt of samples from general practitioners to the issuing of results of clinical tests

Four stages: • 1. Prepare the audit check list • 2. Undertake the audit • 3. Write a summary of the audit • 4. Undertake corrective action to improve the laboratory process

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The Audit Cycle

Incident Reporting

A never ending cycle of improvements • Step 1. Plan: the audit schedule & checklist • Step 2. Do: undertake the audit, report & identify non‐ compliance • Step 3. Act: put in place changes/actions to close out the non‐compliance • Step 4. Check: that actions are in place & are working • Back to step 1.

• Major tool for examining what happened when an error or problem occurred • An appropriate system is required • Must have a core of sound & representative information on which to base any analysis of errors/problems • In order to make recommendations to prevent their repetition • Value in systematic approaches to reporting & recording adverse events

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Adverse Events

Root Cause Analysis (RCA)

• All countries have organisations or bodies that record these events‐logged in central data bases • Can be caused by medical devices, lab equipment, exposure to biological samples, reagents or pharmaceutical products • Incidents assigned a specific type or level • Outcomes of investigations subject to formal review • Issue guidance of how to improve process and reduce further risk • Do not ignore healthcare near misses – report!!

• Estimated that for every major injury 29 minor ones and 300 accidents with no injury occur • A powerful tool that identifies records & visualises the possible causes of a problem, error or incident • Investigation into the error to determine its basis that is its root cause

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Example: cause & effect diagram illustrating how an equipment failure should be investigated

Basis of RCA

• Potential cause of failure: 1. Maintenance & cleaning of equipment not undertaken 2. Lack of lab staff leading to incomplete or absence of QA checks 3. Lack of staff training in recognising problems 4. Reagents passed their expiry dates

• Break down problem into smaller more manageable tasks which allows the problem to be identified and described • Assist in finding a solution or corrective action that will prevent the same problem reoccurring

1. To 4. Effect is to give inaccurate results to patients

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Reading List • Burnett, D. (2002). A Practical Guide To Accreditation In Laboratory Medicine. ACB Venture Publications. London • Department Of Health (2000). An Organisation With Memory. Dept. Of Health, London. • MHRA (2007). Rules & Guidance For Pharmaceutical Manufacturers & Distributors. Pharmaceutical Press. London • Vorley, G., Tickle, F. (2002). Quality Management, Tools & Technique 5th Edt Quality Management & Training (Publications) Ltd, London

What did you learn?

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Laboratory Trainer Theory

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