Sankara Nethralaya’s Manual of Medical Laboratory Techniques

Sankara Nethralaya’s

Manual of Medical Laboratory Techniques

Sankara Nethralaya’s


Editors S Ramakrishnan MA PhD FAMS Professor Emeritus Department of Biochemistry & Cell Biology Vision Research Foundation Sankara Nethralaya, Chennai, India

KN Sulochana PhD Director Department of Biochemistry & Cell Biology Vision Research Foundation Sankara Nethralaya, Chennai, India

Foreword SS Badrinath

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Website: www.jaypeebrothers.com Website: www.jaypeedigital.com © 2012, Jaypee Brothers Medical Publishers All rights reserved. No part of this book may be reproduced in any form or by any means without the prior permission of the publisher. Inquiries for bulk sales may be solicited at: [email protected] This book has been published in good faith that the contents provided by the contributors contained herein are original, and is intended for educational purposes only. While every effort is made to ensure accuracy of information, the publisher and the editors specifically disclaim any damage, liability, or loss incurred, directly or indirectly, from the use or application of any of the contents of this work. If not specifically stated, all figures and tables are courtesy of the editors. Where appropriate, the readers should consult with a specialist or contact the manufacturer of the drug or device. Manual of Medical Laboratory Techniques First Edition: 2012 ISBN 978-93-5025-634-3 Printed at

To His Holiness Kanchi Maha Perriyaval with Our Sincere Pranams

CONTRIBUTORS

Angayarkanni N MSc MPhil PhD Reader & Head Department of Biochemistry and Cell Biology Vision Research Foundation Sankara Nethralaya Chennai, India Doreen Gracias MBBS DCP PhD Consultant Lecturer Hematology and Clinical Pathology Medical Research Foundation Sankara Nethralaya Chennai, India Jyotirmay Biswas MS FNAMS FIC (Path) Director of Uveitis & Ocular Pathology Department Sankara Nethralaya Chennai, India Krishnakumar S MD Deputy Director of Research Director Nanobiotechnology Department Head L & T Department of Ocular Pathology Vision Research Foundation Sankara Nethralaya Chennai, India Kumaramanickavel G MD Former Professor SN ONGC Department of Genetics and Molecular Biology Vision Research Foundation Sankara Nethralaya Chennai, India Lily Therese K PhD Professor & Head L & T Microbiology Research Centre Vision Research Foundation Chennai, India

Madhavan HN MD PhD FAMS FIC (Path) President Vision Research Foundation Director & Professor of Microbiology Director Vidyasagar Institute of Biomedical Technology & Science Sankara Nethralaya L & T Microbiology Research Centre Chennai, India Madhavan Jagadeesan MBBS DO PhD Former Head of the Department SN ONGC Department of Genetics and Molecular Biology Vision Research Foundation Sankara Nethralaya Chennai, India Mahalakshmi B PhD Lecturer L&T Microbiology Research Centre Medical Research Foundation Sankara Nethralaya Chennai, India Malathi J PhD Reader L & T Microbiology Research Centre Vision Research Foundation Chennai, India Punitham R BSc DMLT Lab Manager Department of Biochemistry and Cell Biology Medical Research Foundation Sankara Nethralaya Chennai, India Ramakrishnan S MA PhD FAMS Professor Emeritus Department of Biochemistry & Cell Biology Vision Research Foundation Sankara Nethralaya Chennai, India

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Ramprasad VL PhD Former Lecturer SN ONGC Department of Genetics and Molecular Biology Vision Research Foundation Sankara Nethralaya Chennai, India Soumittra N PhD Lecturer SN ONGC Department of Genetics and Molecular Biology Vision Research Foundation Sankara Nethralaya Chennai, India Sripriya S PhD Lecturer SN ONGC Department of Genetics and Molecular Biology

Vision Research Foundation Sankara Nethralaya Chennai, India Vasanthi SB MBBS Diplomate of American Board of Pediatrics

Director Sri Nathella Sampathu Chetty Clinical Laboratory Services Medical Research Foundation Sankara Nethralaya Chennai, India Vinita Kumari MS MLT Junior Scientist SN ONGC Department of Genetics and Molecular Biology Vision Research Foundation Sankara Nethralaya Chennai, India

FOREWORD

I do not indulge in any language of exaggeration when I say that the book entitled Manual of Medical Laboratory Techniques released by the Medical and Vision Research Foundation and Vidyasagar Institute of Biomedical Technology and Science, Sankara Nethralaya is “All-in-one” in two respects—it covers all the relevant subjects, namely Clinical Biochemistry, Medical Microbiology, Hematology, Genetics and Molecular Biology and all the necessary diagnostic tests in each specialty. The analytical procedures form a large spectrum—from the common tests like blood glucose estimation and simple microscopy to highly-sophisticated techniques like High Performance Liquid Chromatography. There are no two opinions in the fact that application of medical laboratory techniques goes a long way in the diagnosis and differential diagnosis of diseases. It is common knowledge that correct diagnosis is a ’MUST’ to institute proper therapy and thereby alleviation of human suffering. As such, this book will help the medical fraternity to achieve the goal of ‘EFFECTIVE PATIENT CARE’. I have no doubt that the technical staffs involved in analytical work, scientists, doctors and researchers would find the information given in the book very useful. In addition, votaries of microbiology and biochemistry in science colleges would also be benefited, as such books are few and far between. It is worth having the book as a priced, lifetime possession of the concerned staffs and the students, and in the libraries of medical institutions, hospitals and science colleges. The untiring efforts of Dr S Ramakrishnan in education and training to younger generation imparted in our institution, are most laudable. The book is jointly authored by highly qualified and experienced teachers who are also researchers and technologists of Medical and Vision Research Foundation and Vidyasagar Institute of Biomedical Technology and are experts in the field. Hence the “last word” has been given by them in the performance of each test efficiently and with precision. I could imagine the hard work they would have put in preparing the text, in spite of their heavy academic and professional schedule. I hasten to congratulate them for their valuable contributions to the discipline of medical technology and thereby service to the patients. I thank Jaypee Brothers Medical Publishers (P) Ltd., New Delhi, for having readily agreed to publish the book. I know they are second to themselves in the publication of a series of Medical books of value.

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As Chairman-Emeritus, I am happy to record that the ushering-in of the Manual of Medical Laboratory Techniques has got one more feather to adorn the crown of Sankara Nethralaya, the mother-organization of Medical Research Foundation and Vision Research Foundation and Vidyasagar Institute of Biomedical Technology and Science. SS Badrinath FRCS(C) FRCS (Edin) DSc FAMS

Chairman-Emeritus, Sankara Nethralaya Chennai, India

PREFACE

“There is no wealth above the wealth of health.” (Proverbs and wise sayings, Paul Vithayathil, 7th Edn, Vithayathil Publications, Cochin, India). One can afford to lose wealth but not health which is a ‘must’ for happiness in life. No wonder ‘health for all’ is our goal . Strictly speaking, it should be the international goal too. For achieving this, diseases have to be erased from the Globe. It is impossible, as the cheapest commodity available at present appears to be only diseases! Hence, all attempts should be made to control and cure the diseases, to make the people happy—thanks to the medical and the technological professionals who strive and serve, day-in and day-out, devoting themselves to the care of the patients and alleviation of their sufferings. Therapy and cure of a disease will be a success only if the diagnosis is correctly made. Application of Medical Laboratory Techniques is the sheet anchor for proper and precise diagnosis. Medical laboratory technologists belong to various specialties like Medical Microbiology, Hematology, Histopathology, Clinical Biochemistry, Human Genetics and Molecular Biology. Though the disciplines are different, there is unity in diversity, as the one and only mission of their votaries is to use, and, if possible, improve and innovate unfailing and sophisticated techniques in their respective fields in diagnosis, differential diagnosis, monitoring and if possible, cure of diseases of bewildering complexity—be they infectious or immunological, hematological or pathological, metabolic or genetic or just common. The book Manual of Medical Laboratory Techniques is the essence of the subject jointly authored by highly qualified and experienced teachers, researchers and technologists of Medical and Vision Research Foundations, Vidyasagar Institute of Biomedical Technology and Science, Sankara Nethralaya, India, and is a presentation of diagnostic tests in their own specialties. Principles, methodologies, results, norms, interpretations, diseases concerned and bibliography have been given for the tests. Attractive illustrative diagrams are also given. Equal coverage has also been made for instrumentation. In short, it is all-in-one in medical laboratory techniques. A fact which deserves special mention is that a laboratory scientist can perform these tests without external guidance. The authors are confident that medical and technical institutions, hospitals, clinical laboratories, teachers, clinicians, technicians and students will find the book very useful and worthy of life-time preservation.

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The authors do not find suitable words to express their sincere thanks to Dr SS Badrinath, Chairman Emeritus, and Dr Bhaskaran, Chairman of Medical Research Foundation for their constant encouragement and Jaypee Brothers Medical Publishers (P) Ltd., New Delhi, for publishing the book. Thanks are also due to many colleagues who have helped in the manuscript preparation of the book, correction of proof and secretarial work. Our special thanks are due to Smt K Parvathy Devi of the Biochemistry Research Department for sustained coordination and giving the final shape to the book. S Ramakrishnan KN Sulochana

CONTENTS

Unit 1: Biochemistry Part I: Organic .................................................................................. 2 • • • • • • • • • • • • • • • • • • • • • • • • • •

Glucose ................................................................................................................. 2 Glucose Tolerance Test ...................................................................................... 4 Urea ...................................................................................................................... 5 Creatinine ............................................................................................................ 7 Bilirubin ............................................................................................................... 9 Cholesterol ........................................................................................................ 12 Low-density Lipoprotein Cholesterol (LDL) ............................................... 15 High-density Lipoprotein – Cholesterol (HDL) .......................................... 15 Triacylglycerols (Triglycerides) ..................................................................... 18 Total Proteins .................................................................................................... 20 Albumin ............................................................................................................. 22 Ceruloplasmin .................................................................................................. 25 Cerebrospinal Fluid (CSF) Proteins ............................................................... 27 Uric Acid ............................................................................................................ 28 Homocysteine ................................................................................................... 30 Lactate ................................................................................................................ 34 Pyruvate ............................................................................................................. 36 Thiobarbituric Acid Reactive Substances (TBARS) ..................................... 37 Glutathione ........................................................................................................ 39 Sodium, Potassium and Chloride .................................................................. 41 Calcium .............................................................................................................. 44 Phosphorus ........................................................................................................ 46 Iron and Iron-binding Capacity(IBC) ............................................................ 48 Vitamin A .......................................................................................................... 51 Vitamin E ........................................................................................................... 53 Vitamin C .......................................................................................................... 54

Part II: Enzymes ............................................................................. 58 • • • • • • • • • •

Aspartate Transaminase (AST) Serum Glutamic-Oxaloacetic Transaminase (SGOT) ...................................................................................................... 58 Alanine Aminotransferase (ALT)/Serum Glutamic-Pyruvic Transaminase (SGPT) ................................................................................................................ 60 Alkaline Phosphatase (Bone Forming Enzyme) .......................................... 63 Acid Phosphatase ............................................................................................. 65 Acid Phosphatase (Tartrate-Labile) for Prostatic Cancer ........................... 67 Angiotensin Converting Enzyme (ACE) ...................................................... 68 Lactate Dehydrogenase (LDH) ....................................................................... 70 Glucose-6-Phosphate-Dehydrogenase .......................................................... 73 Amylase ............................................................................................................. 75 Ornithine Aminotransferase (OAT) .............................................................. 77


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Glutathione Peroxidase (GPx) ........................................................................ 79 Superoxide Dismutase (SOD) ......................................................................... 82

Part III: Electrophoresis ................................................................. 85 • •

Electrophoresis of Serum Proteins ................................................................. 85 Electrophoresis of Serum Lipoproteins ........................................................ 86

Part IV: Chromatography ............................................................... 89 • • • •

Sugars ................................................................................................................. 89 Amino Acids ..................................................................................................... 91 2D Chromatography ........................................................................................ 92 HPLC Analysis of Amino Acids .................................................................... 95

Part V: Inborn Errors of Metabolism ............................................. 99 • • • • • • •

Carbohydrates .................................................................................................. 99 Glucose and Other Reducing Sugars .......................................................... 100 Maple Syrup Urine Disease .......................................................................... 101 Cystine ............................................................................................................. 102 Tyrosine ........................................................................................................... 103 Homocystinuria .............................................................................................. 104 Phenylketonuria ............................................................................................. 105

Part VI: Collection of Test Sample .............................................. 106 • • • • • •

Procedure for Blood Collection .................................................................... 106 Collection of Urine Sample ........................................................................... 106 Collection of Stool Sample ............................................................................ 107 Fully Automated Chemistry Analyzers ...................................................... 108 Disposal of Materials ..................................................................................... 110 Quality Control ............................................................................................... 111

Unit 2: Genetics • • • • •

Introduction .................................................................................................... 114 Cytogenetic Methods ..................................................................................... 116 DNA Based Methods ..................................................................................... 138 Quantification of Gene Expression .............................................................. 162 Sterilization Procedures ................................................................................ 164

Unit 3: Hematology and Clinical Pathology Part I: Hematology ....................................................................... 170 • • • • • • • •

Complete Blood Count .................................................................................. 170 Total Erythrocyte (RBC) Count .................................................................... 173 Total WBC Count .......................................................................................... 176 Differential Count .......................................................................................... 178 Peripheral Smear Study ................................................................................. 182 Erythrocyte Sedimentation Rate .................................................................. 186 Platelet Count ................................................................................................. 188 Absolute Eosinophil Count ........................................................................... 190

Contents • • • • • • • • • • • • • • • • • • •

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Reticulocyte Count ......................................................................................... 193 Red Blood Cell Indices .................................................................................. 195 Packed Cell Volume ....................................................................................... 196 Hemoglobin Estimation (Cyanmethemoglobin Method) ........................ 197 Hemoglobin Electrophoresis ........................................................................ 199 Blood Grouping and Rh Typing .................................................................. 201 Direct and Indirect Coombs’ Test ................................................................ 206 Activated Partial Thromboplastin Time ..................................................... 208 Prothrombin Time .......................................................................................... 211 Bleeding Time (Ivy Method) ......................................................................... 213 Clotting Time .................................................................................................. 215 Clot Retraction ................................................................................................ 217 Fibrinogen Assay (Clot Weight Method) .................................................... 218 Euglobulin Lysis Time Test .......................................................................... 220 Factor XIII Assay ............................................................................................ 222 Examination of Malarial Parasite ................................................................. 223 Examination of Microfilaria .......................................................................... 227 LE Cell Preparation ........................................................................................ 229 Sickle Cell Preparation .................................................................................. 231

Part II: Clinical Pathology ............................................................ 233 • • • • • • • • • • • • • •

Stool Examination .......................................................................................... 233 Detection of Occult Blood in Stool ............................................................... 236 Urine Examination—Complete Automated Method ................................ 238 Qualitative Identification of Reducing Sugar in Urine by Benedict’s Test ................................................................................................................... 245 Qualitative Identification of Protein in Urine by Heat and Acetic Acid Method ............................................................................................................. 247 Qualitative Identification of Ketone Bodies in Urine ............................... 249 Qualitative Detection of Bile Pigments by Fouchet’s Method ................. 251 Qualitative Detection of Bile Salts by Hay’s Test ...................................... 252 Qualitative Identification of Urobilinogen in Urine by Ehrlich Aldehyde Test ................................................................................................................... 254 Qualitative Detection of Free Hemoglobin by Benzidine Test ................ 256 Detection of Bence Jones Protein (BJP) ........................................................ 257 Analysis of CSF ............................................................................................... 259 Mantoux Test .................................................................................................. 263 Microscopic Examination of Urine .............................................................. 264

Unit 4: Microbiology & Serology Part I: Staining Techniques ......................................................... 268 • • • • •

Gram Stain ....................................................................................................... 268 KOH-Calcofluor White Stain ........................................................................ 271 Ziehl-Neelsen Stain ........................................................................................ 273 Concentration Method for Detection of Mycobacterium ......................... 274 Modified Ziehl-Neelsen Stain ...................................................................... 277


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Giemsa Stain ................................................................................................... 279 Nigrosin Stain ................................................................................................. 281 Lactophenol Cotton Blue Stain ..................................................................... 283 Hanging Drop Method for Motility ............................................................ 284 Immunofluorescence Staining—Chlamydia trachomatis ............................ 286 Immunofluorescence Staining—For Viruses (HSV, CMV, VZV, Adenovirus) .................................................................................................... 289

Part II: Culture Methods ............................................................... 291 • • • • • • • • • • • • • • • • • •

Bacterial and Fungal Culture—Conjunctival Swab Semi-quantitative Method ............................................................................................................. 291 Bacterial and Fungal Culture—Conjunctival Scraping ............................ 294 Bacterial and Fungal Culture—Lid Margin Swab ..................................... 297 Bacterial and Fungal Culture—Corneal Scraping ..................................... 299 Bacterial and Fungal Culture—Intraocular Specimens ............................ 302 Bacterial and Fungal Culture—Other Specimens ..................................... 305 Conventional Biochemical Tests for Identification of Bacterial Isolates 308 Antibiotic Sensitivity ..................................................................................... 318 Acid Fast Bacilli (AFB) Culture ................................................................... 320 Fungal Culture ................................................................................................ 323 Bacterial and Fungal Culture Blood Culture and Sensitivity ...................................................................... 324 Processing of Cerebrospinal Fluid for Bacterial and Fungal Culture .... 326 Bacterial Culture—Urine ............................................................................... 329 Bacterial and Fungal Culture—Throat Swab ............................................. 331 Maintenance of Cell Cultures ....................................................................... 334 Acanthamoeba Culture ................................................................................. 335 Chlamydia trachomatis Culture by Rapid Shell Vial Technique ................ 337 Virus Isolation by Conventional Tube Culture Method ........................... 339

Part III: Immunology ..................................................................... 341 • • • • • • • • • • • •

Rheumatoid Arthritis Test ............................................................................ 341 C-Reactive Protein .......................................................................................... 343 Serum Antistreptolysin ‘O’ (ASO) Titer ..................................................... 347 Serum Rapid Plasma Reagin (RPR) Test .................................................... 350 Treponema Pallidum Hemagglutination (TPHA) ..................................... 353 Widal Test ........................................................................................................ 357 Brucella Agglutination Test .......................................................................... 360 Qualitative Determination of HIV1/2 Antibody ...................................... 363 Rapid Detection of Hepatitis B Surface Antigen (HBs Ag) ...................... 367 Elisa Test for the Detection of Antibodies to HIV-1 and HIV-2 .............. 368 Western Blotting Technique—Confirmation Test for Detection of HIV–1/2 Antibodies ...................................................................................... 372 Fluorescent Antinuclear Antibody Test (FANA) ...................................... 376

Unit 5. Ophthalmic Histopathology • • •

Specimens Entry ............................................................................................. 380 Fixation of Specimens .................................................................................... 381 Grossing Technique ....................................................................................... 383

Contents • • • • • • • • •

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Decalcification ................................................................................................. 387 Tissue Processing ........................................................................................... 389 Embedding ...................................................................................................... 392 Paraffin Section Cutting ................................................................................ 394 Frozen Section ................................................................................................. 397 Staining Techniques ....................................................................................... 400 Immunohistochemistry ................................................................................. 421 Cytology Study ............................................................................................... 424 Storing Process ................................................................................................ 426

Index ........................................................................................................... 429

UNIT 1

Biochemistry

S Ramakrishnan N Angayarkanni SB Vasanthi R Punitham

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Part I: Organic GLUCOSE 1. Purpose: Quantitative estimation of glucose in human serum or plasma or Cerebrospinal fluid (CSF) or other body fluids by enzymatic method (GOD-POD). Plasma Glucose determinations are useful in the diagnosis and treatment of diabetes mellitus and in monitoring the response to treatment of diabetes mellitus with insulin or oral hypoglycemic agents. Elevated glucose levels may be associated with pituitary or thyroid dysfunction, renal failure and liver disease, whereas low glucose levels may be associated with insulinoma, hypopituitary neoplasms, or insulin induced hypoglycemia. CSF and fluids have increased glucose in diabetic condition. 2. Principle: Glucose oxidase (GOD) converts glucose to gluconic acid. Hydrogen peroxide formed in this reaction, in the presence of peroxidase (POD), oxidatively couples with 4 - aminoantipyrine (AAP) and phenol to produce red quinone-imine dye. This dye has absorbance maximum at 505 nm. The intensity of color complex is directly proportional to the concentration of glucose in specimen. Glucose

GOD

gluconic acid

H 2 O 2 + AAP + Phenol

POD

H 2O 2

Red dye

3. Performance specifications 3.1. Linearity: Up to 500 mg/dL of plasma. 3.2. Measurement range: 40–500 mg/dL 3.3. Sensitivity: The minimum detection limit by this kit is 40 mg/dL 4. Primary sample 4.1. Use only plasma as specimen for the test 4.2. Collect 2 mL of venous blood in a Fluoride—EDTA mixture tube Heparin vacutainer tube. 4.3. Do not use lysed plasma for testing as it may give very high results 4.4. Do not use contaminated/turbid samples for testing 4.5. Process the sample on the same day within 3 hours of collection. 4.6. Type of container and additive: Fluoride—EDTA mixture tube. 5. Equipment: Semi-autoanalyzer 6. Reagents: Phosphate buffer; pH 7.5; glucose oxidase; peroxidase; 4 aminoantipyrine; phenol

Biochemistry

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7. Procedure: 7.1. Switch on the machine and press “FLUSH “ button by keeping the tubing in a container with 2% detergent for 2 minutes followed by distilled water for 2 minutes. 7.2. Press “PROC”. Different test procedures will be displayed. 7.3. Select the test to be processed by entering its number and then press “ENTER” key. 7.4. Now the assay parameters of the specific test procedure will be displayed. Note down the volume of the reagent and the sample to be used. 7.5. Feed the blank with each batch and ensure the absorbance of the blank is less than 0.15. If the absorbance of the ‘blank is more than 0.15’ discard the reagent. 7.6. Then feed the test samples and record the values. 7.7. Check whether the sample is hemolyzed, icteric or lipemic before processing. If the sample is lysed, collect another sample and proceed. Assay: End point Reagent volume: 1.0 mL Wavelength: 505 nm (500–550) Sample volume: 10 μL Temperature: 37°C Zero setting with Reagent blank Incubation 5 minutes 8. Interference: Turbid, lipemic, hemolyzed samples, high levels of ascorbic acid, and plasma bilirubin will interfere. Oxalate and fluoride do not interfere. 9. Calculating results: Sample absorbance × Concentration of standard = Sample concentration Standard absorbance

10. Biological reference range: Glucose Fasting is 60–110 mg/dL. Glucose PP is 90–140 mg/dL Glucose Random is 60–130 mg/dL 11. Critical/Alert level values: Below 60 mg/dL Above 400 mg/dL 12. Laboratory interpretation: Increase of blood glucose usually in diabetes mellitus, decrease in insulinoma. Decrease of CSF sugar in infection. Increase of CSF sugar in hyperglycemia. 13. Potential sources of variability: 13.1. Do not use if the absorbance of the blank reagent is greater than 0.15 at 500 nm as it indicates deterioration of the reagent. 13.2. Check if the patient has followed the instructions regarding preparation before collecting samples for fasting/post- prandial, plasma glucose/glucose tolerance test.

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Manual of Medical Laboratory Techniques 13.3. The periodic update on the reference ranges needs to be made note of.

BIBLIOGRAPHY 1. Trinder P. Ann Clin Biochem 1969; 6: 24.

GLUCOSE TOLERANCE TEST 1. Purpose: To recognize milder cases of diabetes and renal glycosuria. Quantitative estimation of glucose in human plasma by enzymatic method (GOD-POD). Plasma glucose determinations are useful in the diagnosis and treatment of diabetes mellitus and in monitoring the response to treatment of diabetes mellitus with insulin or oral hypoglycemic agents. Elevated glucose levels may be associated with pituitary or thyroid dysfunction, renal failure and liver disease, whereas low glucose levels may be associated with insulinoma, hypopituitarism or insulin induced hypoglycemia. CSF and fluids have increased glucose in diabetic condition. 2. Principle: Glucose oxidase (GOD) converts glucose to gluconic acid. Hydrogen peroxide formed in this reaction in the presence of peroxidase (POD), oxidatively couples with 4-aminoantipyrine and phenol to produce red quinoneimine dye. This dye has absorbance maximum at 505 nm. The intensity of color complex is directly proportional to the concentration of glucose in specimen. 3. Performance specifications 3.1. Linearity: Up to 600 mg/dL of plasma. 3.2. Measurement range: 25–600 mg/dL. 3.3. Sensitivity: The minimum detection limit 40 mg/dL. 4. Primary sample 4.1. Use only plasma as specimen for the test. 4.2. Collect 2 mL of venous blood in a fluoride–oxalate mixture tube. 4.3. Do not use lysed plasma for testing as it may give very high results. 4.4. Do not use contaminated/turbid samples for testing. 4.5. Process the sample on the same day within 3 hours of collection. 5. Type of container and additive: Fluoride—EDTA mixture tube. 6. Reagents/Consumables: For patient use, commercially available glucose (75 g) mixed with water. 7. Instrument: Semi-autoanalyzer 8. Procedures: Instructions to be given to the patient: These instructions should be given to the patients by previous day of the investigation.

Biochemistry 8.1.

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The patient should not take any food after 9 pm the previous night till the test is performed. 8.2. The subject should have normal diet for at least 3 days prior to the test. 8.3. He/she should not have taken drugs which affect blood sugar. 8.4. In exceptional cases, when the patient has to come from a distant place, light tea without sugar may be allowed (2 hours before collection). Method: Upon arrival of the patient, the following should be done: 8.5. Body weight should be noted down. 8.6. Fasting blood sample should be collected and glucose estimation should be performed. 8.7. Specimen of fasting urine is collected and test for glucose, albumin and acetone to be done. 8.8. 75 g of glucose dissolved in 300 mL of water should be given oraly. 8.9. Blood and urine samples will be collected for every half an hour interval for 2 hours after the glucose has been taken. 8.10. It is not always possible to collect urine at every half an hour interval. In such cases urine sample can be collected for every 1 hour interval. Glucose estimation: As per the method given in this manual. Urine sugar: As per the method given in this manual, with the standard curves. Normal responses: Fasting glucose within normal limit. Maximum blood glucose is reached either half or one hour after taking the glucose. The blood glucose then returns rapidly to the normal fasting limits, which are often reached in one and a half hour and almost always at two hours. There should be no sugar in any of the urine specimens. 9. Reference: The GTT curve will be interpreted with the standard curves. 10. Critical/Alert level values: Below 40 mg/dL, above 400 mg/dL. 11. Potential sources of variability: 11.1. Do not use if the absorbance of the blank reagent is greater than 0.150 at 500 nm as it indicates deterioration of the reagent. 11.2. Check if the patient has followed the instructions regarding preparation before collecting samples. BIBLIOGRAPHY 1. Harold Varley. Practical Clinical Biochemistry, 5th ed, 1980;1:406-10.

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UREA 1. Purpose: Quantitative estimation of urea in human serum by Urease– GLDH/UV kinetic method. Determination of serum urea nitrogen is an important index of kidney function. Impaired renal function or increased tissue protein breakdown is associated with increased urea nitrogen levels, whereas liver damage or pregnancy is associated with decreased levels. 2. Principle: Urea is hydrolyzed by urease to form ammonium carbonate. In the second reaction 2-oxoglutarate reacts with ammonium ion in the presence of glutamate dehydrogenase (GLDH) and the coenzyme NADH to produce L-glutamate. In this reaction two moles of NADH are oxidized to NAD+ for each mole of urea hydrolyzed. The rate of decrease in the NADH concentration is directly proportional to the urea concentration in the specimen. It is determined by measuring the absorbance at 340 nm. Urease – > 2NH4+ + 2HCO3 Urea + 2H2O ---------------GLDH 2- Oxoglutarate + NH4+ + NADH ---------------> L-Glutamate + NAD + + H2O 3. Performance specifications: 3.1. Linearity: Up to 240 mg/dL of serum 3.2. Measurement range: 2 –240 mg/dL 3.3. Sensitivity: Lower limit of detection is 2 mg/dL 4. Primary sample: 4.1. Use plasma 4.2. Collect 2 mL of venous blood from a peripheral vein in a heparin vaccutainer tube 4.3. Do not use hemolyzed/contaminated plasma for testing 5. Type of container and additive Use heparin/plain vacutainer tubes for collecting samples; do not use hemolyzed/contaminated plasma for testing 6. Instrument: Semi-autoanalyzer 7. Reagents/Consumables: The reconstituted reagent contains the following: 7.1. TRIS pH 7.8, 2-Oxoglutarate, ADP, Urease, GLDH 7.2. NADH 7.3. Urea (50 mg/dL) 8. Procedure: 8.1. Switch on the machine and press “FLUSH” button by keeping the tubing in a container with 2% detergent for 2 minutes followed by distilled water for 2 minutes.

Biochemistry

7

8.2. 8.3.

9. 10. 11. 12.

Press “PROC”, different test procedures will be displayed. Select the test to be processed by entering its number and then press “ENTER” key. 8.4. Now the assay parameters of the specific test procedure will be displayed. Note down the volume of the reagent and the sample to be used. 8.5. Run the standard with each batch of patient sample. 8.6. Then feed the test samples and record the values. Assay: 2 – point kinetic Sample volume: 10 μL Wavelength: 340 nm Reagent volume: 400 μL Start reagent 100 μL Temperature: 37 °C Biological reference range: 15–38.5 mg/dL Alert/Critical values: Above 80.0 mg/dL Laboratory interpretation: Increase suggests impaired renal function, acute nephritis, chronic glomerulonephritis. Potential sources of variability 12.1. Use of only clear, unhemolyzed plasma separated from the erythrocytes as soon as possible. Lysed plasma specimens may give falsely elevated values 12.2. On storage, the working reagent may develop a pink color which makes the use of reagent blanks necessary with every run. 12.3. This method is recommended to perform only on mechanized equipment. It is difficult to incubate all samples and reagent blank exactly for the same intervals. 12.4. The scheme may use for adaptation purpose for instruments with no specific adaptation sheet.

BIBLIOGRAPHY 1. Kassirer JP. New Eng J Med 1971;285:385. 2. Mackay EM, Mackay LL. Clin.Invest 1927;4:295. 3. Talke HN, Schubert, GE Kin. Wschr 1965;42:174.

CREATININE 1. Purpose: Quantitative estimation of creatinine in human plasma by modified Jaffe’s method (Initial rate or fixed time method) Measurement of plasma creatinine is useful in the diagnosis, treatment and follow-up of renal diseases/renal failure. Increase of serum creatinine indicates a definite damage of reneal tissue. 2. Principle: Creatinine reacts with picric acid in alkaline medium to form an orange-yellow colored complex of creatinine picrate. This colored

8

Manual of Medical Laboratory Techniques complex absorbs light at 492 nm the rate of increase in absorbance is directly proportional to the creatinine concentration in the sample. Alkali medium Creatinine + Sodium Picrate --------------------------------> Creatinine - Picrate complex (yellow-orange) 3. Performance specifications: 3.1. Linearity: Up to 24 mg/dL in plasma. 3.2. Measurement range: 0.1–24 mg/dL of creatinine in plasma. 3.3. Sensitivity: The minimum detection limit by this kit is 0.1 mg/dL 3.4. Specificity: This method measures a number of other noncreatinine substances also other than creatinine. 4. Primary sample: 4.1. Use only plasma as specimen for the test 4.2. Collect 4 mL of venous blood in a heparin vacutainer tube. 4.3. Do not use lysed plasma for testing as it may give very high results 4.4. Do not use contaminated/turbid samples for testing 4.5. Process the sample on the same day within 3 hours of collection. 5. Type of container and additive: Use heparin vacutainer tubes for collecting blood samples. 6. Instrument: Semi-autoanalyzer 7. Reagents/Consumables: 7.1. Creatinine reagent: Picric acid 8.73 mmol/L 7.2. Buffer solution: 300 mmol/L of sodium hydroxide 7.3. 25 mmol/L of phosphate. 7.4. Creatinine standard (2 mg/dL) solution containing creatinine in hydrochloric acid with preservative. 8. Procedure: 8.1. Switch on the machine and press “FLUSH “ button by keeping the tubing in a container with 2% detergent for 2 minutes followed by distilled water for 2 minutes. 8.2. Press “PROC”. Different test procedures will be displayed. 8.3. Select the test to be processed by entering its number and then press “ENTER” key. 8.4. Now the assay parameters of the specific test procedure will be displayed. Note down the volume of the reagent and the sample to be used. 8.5. Feed the blank with each batch and ensure the absorbance of reagent blank to zero

Biochemistry

9

8.6.

Then feed the test samples and record the values. Assay: Fixed time (Initial rate) Reagent volume: 500 μL/1000 μL Wavelength: 490 nm Sample volume: 25 μL/50 μL Temperature: 37°C Zero setting with distilled water No. of readings: 2 Time: 60 sec Concentration of Std: 2 mg/dL 9. Calculating results: Sample absorbance × Concentration of standard = Sample concentration Standard absorbance 10. Biological reference Range: Male: 0.7 –1.4 mg/dL Female: 0.6 –1.2 mg/dL 11. Critical/Alert level values: More than 3.0 mg/dL. 12. Laboratory interpretation: Increase of creatinine in blood suggests kidney damage. Example: Chronic glomerulonephritis. 13. Potential sources of variability: 13.1. Lysed plasma specimens may give falsely elevated values 13.2. Creatinine remains stable in plasma for up to 2 days. 13.3. Number of substances other than creatinine interfere with the assay.

BIBLIOGRAPHY 1. Allen LC. Clin Chem 1982;28(3). 2. Haeckel R, et al. Chlin Chem 1981;27(1):179–83. 3. Tanganelli E, Prencipe L, Bassi D Cambiaghi S, Murador E. Clin Chem 1982; 28(7),1461–64.

BILIRUBIN 1. Purpose: Quantitative estimation of serum bilirubin (Total and Direct) by Jendrassik and Grof Method. Measurement of total bilirubin is useful in the diagnosis of jaundice due to any cause and is an indicator of liver function. 2. Principle: Bilirubin reacts with diazotized sulfanilic acid to form an azo dye which is red in neutral and blue in alkaline solution. Whereas the water-soluble bilirubin glucuronides react “directly” (the free bilirubin). “Indirect” bilirubin reacts only in the presence of an accelerator. The total bilirubin in serum or plasma is determined using by coupling with diazotized sulfanilic acid after the addition of caffeine,

10


sodium benzoate and sodium acetate. A blue azobilirubin is formed in alkaline Fehling solution II. This blue compound can also be determined selectively in the presence of yellow byproducts (green mixed coloration) by photometry at 578 nm. The direct bilirubin is measured as the red azo dye at 546 nm using the method of Schellong and Wende without the addition of alkali. 3. Performance specifications: 3.1. Linearity: Up to 20 mg/dL. 3.2. Measurement range: As low as 0.05 mg in serum. 3.3. Sensitivity: Lower detection limit is 0.05 mg/dL 4. Primary sample: 4.1. Use only serum as specimen for the test 4.2. Collect 4 mL of venous blood in a plain vacutainer tube 4.3. Do not expose samples for serum bilirubin estimation to tube light/sunlight. 4.4. Do not use hemolyzed, contaminated or lipemic sera. 4.5. Separate serum as soon as possible; Store the serum at –10°C until required, for a maximum up to one month. 5. Type of container and additive: Use plain vacutainer tubes for collecting samples. 6. Reagents/Consumables: 6.1. Sulfanilic acid 6.2. Accelerator: Caffeine, sodium benzoate, sodium acetate 6.3. Sodium nitrite 6.4. Fehling solution II: 930 mmol/L Potassium sodium tartrate, 1.9 mol/L sodium hydroxide solution. 7. Instrument: Semi-autoanalyzer. 8. Procedure: 8.1. Switch on the machine and press “FLUSH “ button by keeping the tubing in distilled water for 2 minutes. 8.2. Press “PROC”. Different test procedures will be displayed. 8.3. Select the test to be processed by entering its number and then press “ENTER” key. 8.4. Now the assay parameters of the specific test procedure will be displayed. Note down the volume of the reagent and the sample to be used. 8.5. Feed the reagent blank with each batch of patient samples 8.6. Then feed the standard followed by test samples and record the values 8.7. Assay: End point assay Wavelength: 546 nm Temperature 30°C Reagent volume 500 μL

Biochemistry Sample volume: 50 μL No. of readings: 3

11

Incubation time: 15 minutes Times: 20.20 sec

Total Bilirubin Reagent

Blank

Test

Diazo (Sodium nitrite) Sulfanilic acid Sample Accelerator Fehling’s solution

— 50 μL 50 μL 250 μL Incubate 15 minutes at RT 250 μL Incubate 10 minutes at RT

50 μL — 50 μL 250 μL 250 μL

Measure the absorbance of the sample against distilled water or if necessary against the blank. Direct Bilirubin Reagent

Blank

Test

Diazo (Sodium nitrite) Sulfanilic acid Sample Normal saline

— 50 μL 50 μL 500 μL incubate 5 minutes at RT

50 μL — 50 μL 500 μL

Measure the absorbance of the sample against distilled water or if necessary against the blank. 9. Interferences: Turbid lipemic and lysed sera. In patients taking heavy dose of B complex, riboflavin and the folate in it may interfere by giving yellow color to the blood and urine. 10. Calculating results: For measurements against a blank: Total bilirubin concentration = A × 10.5 mg/dL. 11. Biological Reference Range: 11.1. Total Bilirubin – up to 1.0 mg/dL 11.2. Indirect or unconjugated bilirubin – 0.1 to 0.6 mg/dL 11.3. Direct or conjugated bilirubin up to 0.3 mg/dL 12. Critical/Alert level values: 3.0 mg/dL 13. Laboratory interpretation: Increase of bilirubin suggests jaundice; increase of both total and direct bilirubin suggests obstructive/hepatocellular jaundice. Increase of total bilirubin alone with normal direct bilirubin suggests hemolytic jaundice. A level of 0.4 mg/dL or more of direct bilirubin suggests liver involvement.

12


14. Potential sources of variability: 14.1. Lysed serum specimens may give falsely elevated values 14.2. Dilute the specimen if the bilirubin value is > 10 mg/dL suitable dilution can be done with normal saline. In such a case, the results obtained should be multiplied by dilution factor to be obtained correct bilirubin value. BIBLIOGRAPHY 1. Jendrassik, Grof P. Biochem Z. 1938;81:297. 2. Schellong G, Wende U. Arch Kinderheik 1960; 162:126.

CHOLESTEROL 1. Purpose: Quantitative estimation of total cholesterol in human serum by CHOD-PAP method (enzymatic photometric method) measurement of serum cholesterol is useful in the screening of the lipid status of the individual to detect atherosclerotic risks and in monitoring the response to lipid lowering measures and also in the diagnosis and classification of hyperlipidemias. Other conditions such as hepatic and thyroid diseases also influence cholesterol levels. 2. Principle: Cholesterol esters are hydrolyzed by cholesterol esterase to produce free cholesterol and fatty acids. Hydrogen peroxide is then produced from the oxidation of cholesterol by cholesterol oxidase. In a coupled reaction catalyzed by peroxidase (POD), red quinoneimine dye red is formed from 4-aminoantipyrine, phenol and hydrogen peroxide. The absorption at 500 nm of the solution of this dye is proportional to the concentration of cholesterol in the sample. (Trinder’s reaction) Chol. esterase Cholesterol ester -------------------------------> Cholesterol + Fatty acids Chol. oxidase Cholesterol -------------------------------> 2 H2O2 + Cholesten- 4 –en 3-one POD 2 H2O2 + 4-Aminoantipyrine + Phenol--------------> Red quinoneimine + H2O (Red dye) 3. Performance specifications: 3.1. Linearity: Up to 1000 mg/dL of serum 3.2. Measurement range: 1 – 1000 mg/dL of cholesterol in serum 3.3. Sensitivity: The minimum detection limit by this kit is 1 mg/dL

Biochemistry 3.4.

4.

5. 6. 7.

8.

13

Specificity: Cholesterol oxidase is not totally specific for cholesterol. Other analogs of cholesterol (dihydrocholesterol, 7- dehydrocholesterol, 20 hydroxycholesterol, etc.) are also oxidized. However, these analogs do not normally occur in any appreciable amounts in serum. Primary sample: 4.1. Use only plasma as specimen for the test 4.2. Collect 4 mL of venous blood in a heparin vacutainer tube. 4.3. Centrifugation at 2500 rpm for 10 minutes 4.4. Do not use lysed plasma for testing as it may give very high results 4.5. Do not use contaminated/turbid samples for testing 4.6. Process the sample on the same day within 3 hours of collection. 4.7. If analysis is not done on the same day/within 3 hours of collection. Type of container and additive: Use plain/heparin vacutainer tubes for collecting samples. Instrument: Semi-autoanalyzer Reagents/Consumables: 7.1. Cholesterol reagent: 4 Aminoantipyrine 7.2. Phenol 7.3. Cholesterol esterase 7.4. Cholesterol oxidase buffer pH 6.8. 7.5. Cholesterol standard: 200 mg/dL cholesterol in alcohol. Procedure: 8.1. Switch on the machine and press “FLUSH” button by keeping the tubing in a container with 2% detergent for 2 minutes followed by distilled water for 2 minutes. 8.2. Press “PROC”. Different test procedures will be displayed. 8.3. Select the test to be processed by entering its number and then press “ENTER” key. 8.4. Now the assay parameters of the specific test procedure will be displayed. Note down the volume of the reagent and the sample to be used. 8.5. Feed the reagent blank with each batch of patient samples and ensure the absorbance of the blank is less than 0.300. If the absorbance of the ‘blank is more than 0.300’ discard the reagent 8.6. Then feed the test samples and record the values. 8.7. Check whether the sample is hemolyzed, icteric before processing. If the sample is lysed, collect another sample and

14


proceed. If it is icteric or lipemic dilute the sample 1 in 10 with distilled water and proceed. Multiply the result displayed by dilution factor 10. Assay: End point Reagent volume: 1000 μL Wavelength: 510 nm Sample volume: 10 μL Temperature: 37°C Zero setting with distilled water Incubation time 5 minutes Conc. of standard: 200 mg/dL 9. Interference: Check whether the sample is hemolyzed or icteric before processing. If the sample is lysed collect another sample and proceed. Do not shake vigorously. Over the time the reagent may develop a light pink color. This is expected but it does not affect the reagent performance. Discard the reagent if the absorbance of the same exceeds 0.3 OD against distilled water at 510 nm. 10. Calculation of results: Sample absorbance × Concentration of standard = Sample concentration Standard absorbance

11. Biological reference range Serum: 135–220 mg/dL Risk classification total cholesterol in blood (mg/dL) Desirable < 200 mg/dL Borderline higher risk 200–240 mg/dL. High-risk > 240 mg/dL. 12. Critical/Alert level values: More than 300 mg/dL 13. Laboratory interpretation: Hypercholesterolemia in hypothyroidism (Myxedema), nephrotic syndrome, atherosclerosis, arteriosclerosis, uncontrolled diabetes and obstructive jaundice. Hypocholesterolemia in hyperthyroidism and acanthocytosis. 14. Potential sources of variability: 14.1. Lysed plasma specimens may give falsely elevated values 14.2. Cholesterol in plasma remains stable for up to 7 days at room temperature and at –20º C for up to 6 months. 14.3. Do not use if the reagent is turbid as it indicates contamination of the reagent. BIBLIOGRAPHY 1. 2. 3. 4. 5.

Richmond W. Clin Chem 1973;19:1350. Tarbutton PN, Gunter CR. Clin Chem 1974;20:724. Allain CC, et al. Clin Chem 1974;20:470. Richmond W Scan. J Clin Lab Invest 1972;29(suppl.26), Abst.3.25. Young DS, et al. 1975;21D.

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LOW-DENSITY LIPOPROTEIN (LDL) CHOLESTEROL 1. Purpose: The estimation of LDL cholesterol in human serum is done by calculation using a formula. Measurement of serum LDL cholesterol is useful in the screening of the lipid status of the individual to detect atherosclerotic risks and in monitoring the response to lipid lowering measures and also in the diagnosis and classification of hyperlipidemias. Relationship exists between serum LDL cholesterol and the risk of coronary heart disease. LDL cholesterol value above 130 mg/dL is considered as a risk factor for coronary and cerebral vascular disease, it is also useful for lipoprotein phenotyping. 2. Principle: By calculation 3. Procedure: Calculation of results: Total Cholesterol– (Triglycerides/5 + HDL) 4. Reference range: Desirable level : < 130 mg/dL Border line elevation : 130–159 mg/dL Elevated : > 160 mg/dL Calculation carried out up to the range 400 mg/dL TGL

HIGH-DENSITY LIPOPROTEIN – (HDL) CHOLESTEROL 1. Purpose: Quantitative estimation of HDL cholesterol in human serum by precipitation method—Precipitation of VLDL and LDL (by Magnesium ions and Phosphotungstic acid) followed by estimation of HDL cholesterol by cholesterol esterase oxidase method. Measurement of serum HDL cholesterol is useful in the screening of the lipid status of the individual to detect atherosclerotic risks and in monitoring the response to lipid lowering measures and also in the diagnosis and classification of hyperlipidemias. An inverse relationship exists between serum HDL cholesterol and the risk of coronary heart disease. An HDL cholesterol value below 30 mg/dL is considered as a risk factor for coronary and cerebral vascular disease it is also useful for lipoprotein phenotyping. 2. Principle: Phosphotungstate/Mg2+ precipitate all VLDL, LDL and chylomicron (CM) fractions in serum. The HDL fraction remains

16


unaffected in the supernatant. Centrifugation leaves the HDL cholesterol in the supernatant. The supernatant is then treated as a sample for cholesterol assay. The cholesterol content in the supernatant HDL is determined enzymatically by cholesterol esterase – cholesterol oxidase method: Phosphotungstate ---------------------> - HDL fraction (in supernatant) + Serum/Plasma -----------------Mg2+ (LDL+ VLDL + CM in the precipitate) 3. Performance specifications: 3.1. Linearity: Up to 400 mg/dL of serum 3.2. Measurement range: 1– 400 mg/dL of HDL cholesterol in serum 3.3. Sensitivity: The minimum detection limit is 1 mg/dL 3.4. Specificity: Cholesterol oxidase is not totally specific for cholesterol. Other analogs of cholesterol (dihydrocholesterol, 7-dehydrocholesterol, 20 hydroxycholesterol, etc.) are also oxidized. However, these analogs do not normally occur in any appreciable amounts in serum. 4. Primary sample: 4.1. Use only fasting serum as specimen for the test 4.2. Collect 4 mL of venous blood in a heparin vacutainer tube. 4.3. Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500-3000 rpm for 5-10 minutes 4.4. Do not use icteric/lysed plasma for testing as it may give very high results 4.5. Do not use contaminated/turbid samples for testing 4.6. Process the sample on the same day within 3 hours of collection. 4.7. If analysis is not done on the same day/within 3 hours of collection, separate the serum and store it at 2 – 8° C for up to 7 days 5. Type of container and additive: Heparin vacutainer. No additive/ Preservative is needed to be added. 6. Reagents/Consumables: 6.1. Precipitating reagent phosphotungstic acid (2.4 mM) and magnesium chloride (40 mM) 6.2. Cholesterol reagent: 4-Aminophenazone 6.3. Phenol 6.4. Cholesterol esterase 6.5. Cholesterol oxidase 6.6. Horseradish peroxidase, buffer pH 6.8, non-reactive stabilizers, and fillers. 6.7. HDL –cholesterol standard: 50 mg/dL 7. Instrument: Semi-autoanalyzer

Biochemistry

17

8. Procedure: 8.1. Bring the reagents to room temperature before use. Add 500 μL of serum and 500 μL of HDL precipitating reagent. Mix well and centrifuge at 4000 rpm for 10 minutes to obtain a clear supernatant 8.2. Assay the supernatant for HDL cholesterol using cholesterol reagent (as for total cholesterol) 8.3. Switch on the machine and press “FLUSH “button by keeping the tubing in a container with 2% detergent for 2 minutes followed by distilled water for 2 minutes. 8.4. Press “PROC”. Different test procedures will be displayed. 8.5. Select ‘Absorbance’ mode 8.6. Select the test to be processed by entering its number and then press “ENTER” key. 8.7. Now the assay parameters of the specific test procedure will be displayed. Note down the volume of the reagent and the sample to be used. 8.8. Feed the reagent blank with each batch of patient samples and ensure the absorbance of the blank is less than 0.100. If the absorbance of the blank is more than 0.100’, discard the reagent 8.9. Then feed the test samples and record the values 8.10. Check whether the sample is hemolyzed, icteric before processing. If the sample is lysed, collect another sample and proceed. If it is icteric or lipemic, dilute the sample 1 in 10 with distilled water and proceed. Multiply the result displayed by dilution factor 10. Assay: End point Reagent volume: 1000 μL Wavelength: 510 nm Sample volume: 50 μL. Temperature: 37°C Zero setting with reagent blank Incubation time 10 minutes Conc. of standard: 50 mg/dL 9. Interferences: Blood collection in fed state to be avoided, separate plasma immediately. Samples kept above 2 – 8°C and aged 1 day or more should not be used. 10. Calculating results: Sample absorbance × Concentration of standard = Sample concentration Standard absorbance 11. Biological reference range: 30 – 60 mg/dL 12. Alert/Critical values: Below 30 mg/dL 13. Laboratory interpretation: HDL cholesterol/Total cholesterol ratio less than 0.2 indicates a risk factor for coronary heart disease: If it is Total cholesterol/HDL cholesterol the ratio is 5.

18


14. Potential sources of variability: 14.1. Lysed serum specimens may give falsely elevated values 14.2. Do not use if the reagent is turbid as it indicates contamination of the reagent or if the absorbance of the blank reagent is more than 0.100. BIBLIOGRAPHY 1. Castelli WP. Circulation 1977;55;767. 2. Castelli WP. Metabolic Therapy 1977;6:1. 3. Gordon T, et al. Am J Med 1977;62:707.

TRIACYLGLYCEROLS (TRIGLYCERIDES) 1. Purpose: Quantitative estimation of triacylglycerols in human serum by enzymatic method using Glycerol -3 Phosphate Oxidase (GPO) Measurement of triglycerides in conjunction with other lipid assays is used in screening the lipid status of an individual to detect atherosclerotic risks and in monitoring the response to lipid lowering measures triglyceride determinations when performed are useful in the diagnosis of primary and secondary hyperlipoproteinemia. They are also of interest in following the course of diabetes mellitus, nephrotic syndrome, biliary obstruction and various metabolic abnormalities due to endocrine disturbances. 2. Principle: The procedure involves hydrolysis of triglycerides by lipoprotein lipase. The glycerol concentration is then determined by enzymatic assay coupled with Trinder reaction that terminates in the formation of a quinoneimine dye which is generated from 4-aminoantipyrine and 4-chlorophenol by hydrogen peroxide under the catalytic action of peroxidase. The amount of the dye formed, determined by its absorption at 500 nm, is directly proportional to the concentration of triglycerides in the sample. Lipase Triglycerides + H2O ---------------> Glycerol + Fatty acids Glycerol kinase Glycerol + ATP ----------------------------------> Glycerol-3-phosphate + ADP GPO Glycerol- 3-phosphate + O2 ---------------> DAP + H2O2 Peroxidase 2 H2O2 + 4-AA + Cholorophenol-----------------------------> Red quinone dye + 4H2O

Biochemistry

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3. Performance specifications: 3.1. Linearity: Up to 1000 mg/dL of serum 3.2. Measurement range: 1–1000 mg/dL of cholesterol in serum 3.3. Sensitivity: The minimum detection limit by this kit is 1 mg/dL. 4. Primary sample: 4.1. Use only fasting serum as specimen 4.2. Collect blood sample after an overnight fast of 12–14 hours when testing is a part of lipid profile 4.3. Collect 4 mL of venous blood in a plain vacutainer tube. 4.4. Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500–3000 rpm for 5–10 minutes 4.5. Do not use lysed serum for testing as it may give very high results 4.6. Do not use contaminated/turbid samples for testing 4.7. Process the sample on the same day within 3 hours of collection. 4.8. If analysis is not done on the same day/within 3 hours of collection, separate the serum and store it at 20–25 °C for up to 2 days or at 4–8 °C for up to 7 days 5. Type of container and additive: Use plain vacutainer tubes for collecting samples. No additive/preservative is needed to be added. 6. Reagents/Consumables: Lipoprotein lipase, magnesium acetate, 4 aminoantipyrine, glycerol-3-phosphate oxidase, glycerol kinase, peroxidase, triglyceride standard 200 mg/dL triglycerides as triolein. 7. Instrument: Semi-autoanalyzer 8. Procedure: 8.1. Switch on the machine and press “FLUSH “button by keeping the tubing in a container with 2% detergent for 2 minutes followed by distilled water for 2 minutes. 8.2. Press “PROC”. Different test procedures will be displayed. 8.3. Select the test to be processed by entering its number and then press “ENTER” key. 8.4. Now the assay parameters of the specific test procedure will be displayed. Note down the volume of the reagent and the sample to be used. 8.5. Feed the reagent blank with each batch of patient samples and ensure the absorbance of the blank is less than 0.300 at 520 nm if the absorbance of the ‘blank is more than 0.300, discard the reagent. 8.6. Then feed the test samples and record the values. 8.7. Check whether the sample is hemolyzed, icteric before processing. If the sample is lysed, collect another sample and proceed. If it is icteric or highly lipemic, dilute the sample 1 in 10 with distilled water and proceed. Multiply the result displayed by dilution factor 10.

20


Assay: End point Wavelength: 546 nm Temperature: 30°C Incubation time: 5 minutes 9. Calculating results:

Reagent volume: 1000 μL Sample volume: 10 μL Zero setting with distilled water Conc. of standard: 200 mg/dL

Sample absorbance × Concentration of standard = Sample concentration Sample concentration

10. Biological reference range: Male: 60–165 mg/dL Female: 40–140 mg/dL 11. Critical/Alert level values: 400 mg/dL 12. Laboratory interpretation: Triacylglycerolemia is a risk factor for myocardial infarction. TGA is phenomenally increased in an eye disease lipemic retinalis. 13. Potential sources of variability: 14.1. Lysed serum specimens may give falsely elevated values 14.2. Do not use if the reagent is turbid as it indicates contamination of the reagent and if the absorbance of the blank reagent is more than 0.300. BIBLIOGRAPHY 1. Annoni G, Bottasso BM. Ciaci D, Donato MF Tripoli A, Lab JJ. Res Lab Med 1982;9:115. 2. Buccolo G, David M. Clin. Chem 1973;19:476. 3. Werner M, Gabrielson DG, Estman G. Clin Chem 1981;21:268.

TOTAL PROTEINS 1. Purpose: Estimation of total protein in serum/body fluids by Biuret method. Low protein levels are observed in malnutrition, acute or chronic liver diseases, nephrotic syndrome, water intoxication, salt retention syndromes, and massive intravenous infusions. Elevated protein levels are observed in dehydration due to vomiting, diarrhea, Addison’s disease and diabetic ketoacidosis. High protein levels of over 2 g/dL in body fluids are suggestive of inflammation or malignancy and are called exudates. 2. Principle: Peptide bonds of proteins in serum react with cupric ions in alkaline solutions to form a blue colored complex, the absorbance of which is measured at 578 nm. The intensity of the blue color is

Biochemistry

3.

4.

5. 6.

7. 8.

21

proportional to the amount of protein present. The reaction sequence employed in the assay of total proteins is as follows: Alkaline pH > Cu-Protein complex (Blue color complex) Protein + Cu2+ ------------------------Performance specifications. 3.1. Linearity: Up to 12 g/dL 3.2. Measurement range: This method has a measurement range of 5.3–8.4 g/dL of total protein in serum and body fluids. 3.3. Sensitivity: The minimum detection limit by the kit is 5.3 g/dL. Primary sample: 4.1. Use serum/body fluids (Pleural, Pericardial, Ascitic fluid) as specimen for the test. 4.2. Collect blood sample in a red color vacutainer tube, separate serum within 30 minutes of collection. 4.3. Process the sample on the same day within 1 hour of collection. If analysis is done on the next day, separate the serum and store it at 2–8°C for up to 30 days. Type of container and additive: Use plain vacutainer tubes for collecting samples. No additive/Preservative is needed to be added Reagents/Consumables: 6.1. Biuret reagent 6.2. Total protein standard Instrument: Semi-autoanalyzer Procedure: 8.1 Switch on the machine and press “FLUSH “button by keeping the tubing in a container with 2% detergent for 2 minutes followed by distilled water for 2 minutes. 8.2. Press “PROC”. Different test procedures will be displayed. 8.3. Select the test to be processed by entering its number and then press “ENTER” key. 8.4. Now the assay parameters of the specific test procedure will be displayed. Note down the volume of the reagent and the sample to be used. 8.5. Feed the reagent blank with each batch of patient samples and ensure the absorbance of the blank is less than 0.150, if the absorbance of the ‘blank is more than 0.150’ discard the reagent at 546 nm. 8.6. Then feed the test samples and record the values. 8.7. Check whether the sample is hemolyzed or icteric before processing. If the sample is lysed, collect another sample and proceed. If it is icteric or lipemic, dilute the sample 1 in 10 with distilled water and proceed. Multiply the result displayed by dilution factor 10. Assay type: End point, Wavelength: 546 nm

22


Sample volume: 10 μL, Reagent volume 1000 μL Incubation time: 20 min at RT, Temperature: 37oC 9. Interferences: 9.1. Feed the reagent blank with each batch of patient samples and ensure the absorbance of the blank is less than 0.150, If the absorbance of the ‘blank is more than 0.150’ discard the reagent at 546 nm. 9.2. Keep the reconstituted reagent at 2–8ºC. Discard the same if it develops precipitate. 9.3. Highly hemolytic or icteric samples, prepare sample blank by adding 1 mL of 0–9% saline to 10 microliter samples. The value of the blank is subtracted from the corresponding sample value. 10. Calculating results: Sample absorbance × Concentration of standard = Sample concentration Standard absorbance 11. Biological reference range: Adults: 6.6–8.4 g/dL 12. Critical/Alert values: Below 5.0 g/dL and above 9.0 g/dL 13. Laboratory interpretation: Increase of proteins in dehydration, multiple myeloma and chronic infections (gammopathy); decrease in malnutrition, liver diseases, nephrotic syndrome. 14. Potential sources of variability: 14.1. The reagent is linear to 12.0 g/dL. Samples with values above 10 g/dL should be diluted 1:1 with 0.9% saline, re-run, and the result multiplied by two (2) 14.2 The biuret procedure is not sensitive at low ranges (< 1 g/dL). Do not use for urine or spinal fluid.

BIBLIOGRAPHY 1. Henry J, Winkelman JW. Clinical Chemistry Principles and Technique. Harper and Row, 2nd edn 1974. 2. Stricklad RD, Freeman ML, Gurule FF. Copper Binding by proteins in alkaline solution. Anal Chem 1961;33.

ALBUMIN 1. Purpose: Quantitative estimation of albumin in human serum by photometric method using bromocresol green (BCG) dye binding. Elevated serum albumin levels are associated with possible dehydration. Low serum albumin levels are indicative of potential malnutrition, liver diseases, kidney disorders chiefly nephrotic syndrome, and rheumatoid arthritis.

Biochemistry

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2. Principle: Albumin acts as a cation at a pH of 3.8 and selectively binds to the anionic dye, bromocresol green forming a green colored complex. The colored complex absorbs light at 630 nm. The increase in absorbance is directly proportional to the concentration of albumin in the sample. 3. Performance specifications: 3.1. Linearity: Up to 6.0 g/dL in serum 3.2. Measurement range: 0.5–6.0 g/dL of albumin in serum 3.3. Sensitivity: The minimum detection limit is 0.5 g/dL 3.4. Specificity: Ampicillin and other medications interfere with the dye-binding properties of albumin. As the dye-binding properties of albumin from various species have been found to differ widely, only standards and controls containing human albumin should be employed with this procedure as standards. Controls and standards from other species will interfere with the results. 4. Primary sample: 4.1. Use only serum, as specimen for the test and fasting specimen is advisable as lipemia interferes with the assay 4.2. Avoid venostasis during sample collection to avoid hemoconcentration which will increase albumin concentration 4.3. Collect 4 mL of venous blood in a plain red vacutainer tube. Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500 rpm for 10 minutes 4.4. Do not use lysed serum for testing as it may give very high results 4.5 Do not use contaminated/turbid samples for testing 4.6. Process the sample on the same day within 3 hours of collection. 4.7. If analysis is not done on the same day/within 3 hours of collection, separate the serum and store it at 2–8 °C for up to 30 days. 5. Type of container and additive: Use plain vacutainer tube for collecting samples. No additive/preservatives needed to be added 6. Reagents/Consumables: 6.1. Albumin Reagent: Bromocresol green (BCG), buffer pH 3.68 6.2. Standard: Bovine albumin fraction V with stabilizer (5 g/dL) 7. Instrument: RA 50 or any semi autoanalyzer 8. Procedure: 8.1. Switch on the machine and press “FLUSH “ button by keeping the tubing by distilled water for 2 minutes. 8.1. Press “PROC”. Different test procedures will be displayed. 8.2. Select the test to be processed by entering its number and then press “ENTER” key.


24 8.3.

Now the assay parameters of the specific test procedure will be displayed. Note down the volume of the reagent and the sample to be used. 8.4. Feed the reagent blank with each batch of patient samples 8.5. Then feed the standard followed by test samples and record the values. 8.6. Check whether the sample is hemolyzed, lipemic before processing. If the sample is lysed, collect another sample and proceed. If it is icteric or lipemic, dilute the sample 1 in 10 with distilled water and proceed. Multiply the result displayed by dilution factor 10. Assay: End point Reagent volume: 500 μL/1000 μL Wavelength: 628 nm Sample volume: 10 μL Temperature: 37°C Zero setting with distilled water Incubation time: 10 minutes Conc. of Standard: 5.0 g/dL Path length: 1 cm 9. Interferences: Highly hemolytic or icteric samples prefer sample blank by adding 1 mL of 0-9% saline to 10 microliter samples. The values of the blank is subtracted from the corresponding sample value. 10. Calculating results: Sample absorbance × Concentration of standard = Sample concentration Standard absorbance 11. Biological reference range: Adults: 3.5–5.0 g/dL 12. Critical/Alert level values: 2.0 g/dL 13. Laboratory interpretation: Hypoalbuminemia in liver diseases, nephrotic syndrome, malnutrition, chronic diseases, severe hemorrhage and pregnancy; lower albumin is reflected in lowered A/G ratio. Increase of albumin in dehydration. 14. Potential sources of variability: 14.1. Lysed serum specimens may give falsely elevated values. 14.2. Albumin reagent should be a clear, yellow-green solution. If turbidity or precipitation has occurred, discard the reagent. 14.3. Ampicillin and other medications interfere with the dye-binding properties of albumin. As the dye-binding properties of albumin from various species have been found to differ widely only standards and controls containing human albumin be employed with this procedure, as standards and controls from other species will interfere with results.

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25

BIBLIOGRAPHY 1. Doumas BT, Waston WA, Biggs HG. Clin Chem Acta. 1971;31:87. 2. Gustafasson JEC. Clin Chem 1976;22:676. 3. Webster D. Clin Chem 1976;22: 676.

CERULOPLASMIN 1. Purpose: To measure the amount of ceruloplasmin in serum. Lower levels have been reported in Wilson’s disease and in cases of nephrotic syndrome. It has been found to be very useful in differentiating chronic liver diseases from Wilson’s disease. 2. Principle: Ceruloplasmin, ferro-oxidase, catalyzes the oxidation of some polyamines and its action on p-phenylene diamine is measured as the amount present in serum. 3. Performance specifications: 3.1. Linearity: Up to 60 mg/dL of serum 3.2. Measurement range: 16–60 mg/dL ceruloplasmin activity in serum 3.3. Sensitivity: The minimum detection limit by this method is 16 mg/dL. 4. Primary sample: 4.1. Use only serum as specimen for the test. 4.2. Collect 4 mL of venous blood in a plain red color vacutainer tube. Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500 rpm for 10 minutes. 4.3. Do not use lysed serum for testing as it may give very high results 4.4. Do not use contaminated/turbid samples for testing. 4.5. Process the sample on the same day within 3 hours of collection. 4.6. If analysis is not done on the same day/within 3 hours of collection, separate the serum and store it at –20 °C for up to 7 days. 5. Type of container and additive: Use plain vacutainer tubes for collecting samples. No additive/preservative is need to be added. 6. Reagents/Consumables: 6.1. p-Phenylenediamine hydrochloride, 5 g in 1 liter solution in water. To purify dissolve p-phenylene diamine dihydrochloride in a minimum volume of hot distilled water, decolorize with charcoal, filter and allow to crystallize, keep the dried crystals over calcium chloride. This can be kept in sealed vials for several weeks.


26 6.2.

Acetic Acid (1 M): Make up 60 mL glacial acetic acid to 1 liter with water.

6.3.

Sodium acetate (1 M): 136 g per liter of distilled water.

6.4.

Acetate Buffer, 400 mM pH 5.5: Add approximately 1.2 mL of the acetic acid to 20 mL of sodium acetate to bring pH 5.5 and dilute to 50 mL store at 4°C.

6.5.

Sodium azide: Five gram per liter or sodium fluoride 20 g per liter can be used.

7. Instrument: Spectrophotometer. 8. Procedure: Water blank Serum Sodium azide Acetate buffer Phenylenediamine

Reag blank

Test blank

Test

— 1 mL 8 mL 1 mL

0.1 mL 1 mL 8 mL 1 mL

0.1 mL — 8 mL 1 mL

Incubate the tubes at 37°C for 1 hr Sodium azide

—

—

1 mL

Cool for 30 minutes at 4°C and read at 530 nm. Calculation: TOD – COD × 60* ×

100 mg/dL 0.1

* A conversion factor of 60 (0.06 × 1000) of King (1965) is used to express ceruloplasmin values as mg/dL. 9. Reference range value: 20–40 mg/dL. 10. Critical/Alert value: Not applicable. 11. Potential sources of variability: 11.1. Lysed serum specimens may give falsely elevated values. 11.2. If analysis is not done on the same day/within 3 hours of collection, separate the serum and store it at –20 °C for up to 7 days. BIBLIOGRAPHY 1. Harold Varley. Practical Clinical Biochemistry, 5th edn, 1980;946–47. 2. King J. Ceruloplasmin. In: Practical Clinical Enzymology, Van Nostrand, London, UK. 1965;108-10.

Biochemistry

27

CEREBROSPINAL FLUID (CSF) PROTEINS 1. Purpose: Estimation of CSF protein is done by Lowry method. Levels of proteins in CSF are useful in the diagnosis of purulent (pyrogenic) meningitis and tubercular meningitis. In respect of other body fluids like pleural or ascitic fluids it helps to know whether it is a transudate or exudate (exudate is due to inflammation or malignancy). 2. Principle: The amino acids (Tyrosine and Tryptophan) in the proteins react with the Folin’s reagent in the presence of alkaline copper reagent to form a blue-colored complex that can be read spectrophotometrically at 660 nm. 3. Performance specifications: 3.1. Linearity: Up to 5 mg/mL 3.2. Measurement range: 0.05–5 mg/mL 3.3. Sensitivity: The minimum detection limit is 0.05 mg/mL. 4. Primary sample: 4.1. Use CSF/body fluids (Pleural, Pericardial, Ascitic fluid) as specimen for the test. 4.2. Collect sample and send within 30 minutes of collection to the respective labs. 4.3. Process the sample on the same day, if analysis is not done on the next day, store it at –20°C for up to 1 day. 5. Type of container and additive: Use plain tubes for collecting samples. No additive/Preservative is needed to be added. 6. Reagents/Consumables: 6.1. Standard BSA - 0.1 g/dL 6.2. Na2CO3 - 2% in 0.2 N NaOH 6.3. CuSO4 - 0.5% in 1% Trisodium citrate 6.4. Alkaline copper reagent - 49 mL of 6.2 + 1 mL of 6.3 6.5. Folin’s Ciocalteu reagent - 1:1 dilution 7. Instrument: Spectrophotometer. 8. Procedures: Std: BSA (μL) Sample (μL) Alkaline copper reagent (mL)

Blank

Standard

Sample

— — 5.0

50 — 5.0

— 50 5.0

Room temperature, 10 minutes Folin’s reagent (mL)

0.5

Room temperature; 20 minutes; read at 660 nm.

0.5

0.5

28


9. Calculation: From the standard graph, the test absorbance will be plotted and the value will be calculated for 100 mL. 10. Interferences: Cells. 11. Biological reference range: CSF Proteins – 15–45 mg/dL 12. Critical/Alert values: Above 1 g/dL 13. Laboratory interpretation: Increase to about 2 g/dL and more shows purulent meningitis, about 1 g/dL may suggest tuberculous meningitis, mild increase in encephalitis. 14. Potential sources of variability: The reagent is linear up to 5 mg/mL. Samples with values above 3 mg/mL should be diluted 1:2 with 0.9% saline, re-run, and the result multiplied by 2. BIBLIOGRAPHY 1. Lowry OH, Rosenborough NJ, Farr AL, Randall RJ. Protein measurement with folin phenol reagent. J Biol Chem 1951;193:265–75.

URIC ACID 1. Purpose: Quantitative estimation of uric acid in human serum by enzymatic uricase method. The quantitation of uric acid is an aid in the diagnosis of gout, decreased renal function, and myeloproliferative disorders. The quantitation of uric acid is also an aid in the diagnosis of hyperuricemia due to any cause. Serum uric acid levels will increase with urea and creatinine under conditions of elevation of NPN substances in serum. 2. Principle: Uric acid is converted by uricase into allantoin and hydrogen peroxides. The hydrogen peroxide initiates the coupling of 4-aminoantipyrine to 3, 5-dichloro-2-hydroxybenzene sulfonic acid (DCHBS) to form the chromogen which is measured at 520 nm and which is proportional to the amount of hydrogen peroxide generated from uric acid. Uricase Uric acid +H2O + O2 ---------------> Allantoin +H 2O 2 + CO 2 Peroxidase 2H2O2 + 4-AAP + DCHBS -------------------------------> Red colored complex HCl + H2O 3. Performance specifications: 3.1. Linearity: 25 mg/dL of serum 3.2. Measurement range: 1–25 mg/dL 3.3. Sensitivity: The minimum detection limit by this kit is 1 mg/dL

Biochemistry

29

4. Primary sample: 4.1. Use serum/plasma 4.2. Collect 4 mL of venous blood from a peripheral vein in a plain red-topped vacutainer tube. 4.3. Do not use hemolyzed/contaminated serum for testing 5. Type of container and additive: Use plain vacutainer tubes for collecting samples, do not use hemolyzed/contaminated serum for testing 6. Reagents/Consumables: The reconstituted reagent contains the following: 6.1. Uric acid reagent: 4-Aminoantipyrine, 3, 5 dichloro-2 hydroxybenzenesulfonate, stabilizer and surfactant, uricase, peroxidase (horseradish), buffer pH 7.5 6.2. Uric acid Standard 6 mg/dL. 7. Instrument: Semi–autoanalyzer 8. Procedure: 8.1. Switch on the machine and press “FLUSH” button by keeping the tubing in a container with 2% detergent for 2 minutes followed by distilled water for 2 minutes. 8.2. Press “PROC”, different test procedures will be displayed. 8.3. Select the test to be processed by entering its number and then press “ENTER” key. 8.4. Now the assay parameters of the specific test procedure will be displayed. Note down the volume of the reagent and the sample to be used. 8.5. Feed the blank with each batch and ensure the absorbance of the blank is less than 0.4 if the absorbance of the ‘blank is more than 0.4’, discard the reagent. 8.6. Run the standard with each batch of patient sample. 8.7. Then feed the test samples and record the values. 8.8. Check whether the sample is hemolyzed, icteric or lipemic before processing. If the sample is lysed, collect another sample and proceed. If it is icteric or lipemic, dilute the sample 1 in 10 with distilled water and proceed. Multiply the result displayed by dilution factor 10. Assay: End point Sample volume: 50 μL Wavelength: 520 nm Reagent volume: 500 μL Temperature: 37°C Incubation time: 5 minutes Concentration of standard: 6 mg/100 mL 9. Calculation of results: Sample absorbance × Concentration of standard = Sample concentration Standard absorbance

30


10. Biological reference range: Male: 3.4 –7.0 mg/dL Female: 2.4–5.7 mg/dL 11. Critical/Alert level values: Above 7 mg/dL. 12. Laboratory interpretations: Specific for gout in which there is increase of blood level. Increase is also found in leukemia, polycythemia, renal dysfunctions, lactic acidosis alcoholics, atherosclerosis, uncontrolled diabetes, hypothyroidism and glycogen storage diseases. There is decrease in Wilson’s disease. 13. Potential sources of variability 13.1. Use of only clear, unhemolyzed serum, separated from the erythrocytes as soon as possible. Lysed serum specimens may give falsely elevated values. 13.2. On storage, the working reagent may develop a pink color, which makes the use of reagent blanks necessary with every run. 13.3. Do not use if the absorbance of the blank reagent is greater than 0.4 as it indicates detoriation of the reagent. 13.4. Uric acid remains stable in serum for up to 7 days if the serum specimen is stored at 2- 8° C. Hence if analysis is not done on the same day within 3 hours of collection, separate the serum and store it at 2-8° C. 14. Interference: Icteric, hemolyzed and turbid sera should not be used. BIBLIOGRAPHY 1. Barham D, Trinder P. Analyst 1972;97:142. 2. Fossati P, Prenciple L, Berti G. Clin Chem 1980;26(2):227.

HOMOCYSTEINE 1. Purpose: Analysis of homocysteine in the blood by ELISA method using S-adenosyl-L-homocysteine (SAH). Levels of homocysteine are needed for diagnosis and therapy wherever necessary. Increased homocysteine in blood has been found in many eye diseases like ectopia lentis, Eale’s disease, uveitis, age-related macular degeneration (ARMD), central retinal vein occlusion (CRVO), etc. and in heart diseases. It could cause oxidative stress also. 2. Principle: Homocysteine Microplate Enzyme Immunoassay is an enzyme immunoassay for the determination of Homocysteine in blood. Protein bound Homocysteine is reduced to free Homocysteine and enzymatically converted to S-adenosyl-L-homocysteine (SAH) in a separate procedure prior to the immunoasssay. This enzyme is specific for the L-form of homocysteine, which is the only form present in the blood.

Biochemistry

3.

4.

5. 6.

31

Reduction: Homocysteine (Hcy), mixed disulfide of homocystine and protein bound forms of Homocysteine in the sample are reduced to free Homocysteine by use of dithiothreitol (DTT). Prot‐SS‐Hcy *R ‐ SS ‐ Hcy DTT Homocysteine Hcy‐SS‐Hcy *R is any thiol residue Enzymatic conversion: Homocysteine in the test sample is converted to S -adenosyl-L-homocysteine by the use of SAH hydrolase and excess adenosine. Homocysteine + Adenosine ---------------> SAH + H2O The solid-phase enzyme immunoassay is based on competition between SAH in the sample and immobilized SAH bound to the walls of the microtiter plate for binding sites on a monoclonal anti-SAH antibody. After removal of anti-SAH antibody, secondary rabbit antimouse labeled with the enzyme horseradish peroxidase (HRP) is added. The peroxidase activity is measured spectrophotometrically after the addition of substrate and the absorbance is inversely related to the concentration of the Total Hcy in the sample. Performance specifications: 3.1. Correlation coefficient: 0.99 3.2. Measurement range: This method has a measurement range of 2–50 μmol in plasma. 3.3. Sensitivity: The minimum detection limit by this kit is 1 μmol/L. Primary sample: 4.1. Use only EDTA plasma as specimen for the test 4.2. Collect 2 mL of venous blood in an EDTA vacutainer tube. 4.3. Separate the plasma by centrifugation at 2500 rpm for 15 min. Plasma should be separated within 30 min. 4.4. Do not use contaminated/lysed plasma for testing as it may give very high results 4.5. Process the sample on the same day or store at –20° C till analysis. Type of container and additive: Use ethylenediaminetetra-acetic acid (EDTA) vacutainer tube for collecting samples. Reagent/Consumables:

Component

Color code

Reference

Description

Volume

Reagent A Assay buffer

Brown

194–5376

Phosphate buffer, 0.1% sodium azide

54 mL

Reagent B Adenosine/DTT

White

194–5377

Adenosine/dithiothreitol citric acid.

3.5 mL Contd...

32


Contd... Reagent C SAH- hydrolase

White

Reagent D enzyme inhibitor

194 –5378

Bovine S-adenosyl- L homocysteine hydrolase, tris buffer glycerol, methylparaben.

3.5 mL

Orange 194–5379

0.2% thimerosal phosphate buffer

55 mL

Reagent E Adenosine deaminase

Red

194–5380

Adenosine deaminase, phosphate buffer, 0.1% Sodium azide, BSA, phenol-red dye.

55 mL

Reagent F a-SAH antibody

Green

194–5381

Monoclonal mouse—antiS-adenosyl- L homocysteine antiobody, BSA thimerosal

Reagent G Enzyme conjugate

Blue

194–5382

Rabbit anti-mouse antibody 15 mL enzyme conjugate, BSA, horseradish peroxidase (HRP) blue eye.

Reagent H Substrate

Violet

194–5383

<10% N-methyl-2-pyrrolidone, 15 mL propylene carbonate.

Reagent S Stop solution

Yellow

194–5384

0.8 M sulfuric acid.

20 mL

Wash concentrate

Black

194–5369

10x phosphate buffer thimerosal Tween 20, BSA

60 mL

Cal –1 to 6

White

194–5388

25 mL

7. Instrument: ELISA Reader 8. Procedure: Make sure all solutions and microtiter strips are equilibrated to room temperature before use. It is recommend to run a set of calibrators every time when a new kit is opened and to run 1 or 2 calibrators during each run. 8. 1. Sample pre-treatment solution, SPS has to be made within 1 hour prior to the start of the assay. Volume needed per 10 samples: (No dead volume calculated) 4.5 mL Reagent A (brown); 0.25 mL Reagent B (white); 0.25 mL Reagent C (white) Mix 8.2. Dilute calibrators and samples/controls in plastic or glass tubes as follows: 25 μL calibrator/sample/control + 500 μL SPS Mix well. Incubate 30 minutes at 37°C. Cap the tubes or cover with parafilm during incubation. Note: Proceed with step 3 before the samples have cooled. 8.3. Add 500 μL Reagent D (orange) Mix well. Incubate 15 minutes at 18–25°C

Biochemistry 8.4.

9. 10. 11. 12.

13.

33

Add 500 μL Reagent E (red) Mix well. Incubate 5 minutes at 18–25°C 8.5. Microtiter plate procedure: 8.6. Pipette 25 μL diluted calibrator/sample/control from step 4 into the wells of the SAH-coated microtiter strips. 8.7. Add 200 mL reagent F (green) to each well. 8.8. Incubate 30 min at 18–25ºC Use the enclosed lid during all incubations. 8.9. Wash with diluted wash buffer, 3 times. 3 x 400 mL 8.10. Add 100 μL reagent G (blue) to each well. Incubate 20 min at 18–25ºC 8.11. Wash with diluted. Wash buffer, 3 times. 3 x 400 mL 8.12. Add 100 μL reagent H (violet) to each well. 8.13. Incubate 10 min at 18–25ºC 8.14. Add 100 μL reagent S (yellow) to each well. 8.15. Shake and read at 450 nm within 15 min using ELISA reader. Automatic plate shaker is preferred to ensure proper mixing. 8.16. Plot the log graph and read the results. Interferences: Blood should be collected, the EDTA tubes should be kept in ice immediately after drawing. a. Food consumptions can affect homocysteine levels protein-rich meals give high level so avoid late in the day before sampling. b. Specimens from patient who are in drug therapy involving S-adenosyl methionine. c. Specimens from patient who have received preparations of mouse monoclonal antibodies for diagnosis or therapy. d. Specimens from patients taking methotrexate, carbamaze, phenyltoin, methoxide, anticonvulsants or 6 azauridine triacetate. Calculating results: Plot the calibrator values in log graph and read the results directly. Biological reference range: Plasma: 0–15 μM/L Critical/Alert values: More than 30 μM/L Laboratory interpretation: Homocysteinemia is a risk factor for atherothrombosis and is found in several eye diseases namely ectopia lentis, secondary glaucoma, cataract, optic atrophy, Eale’s disease, uveitis, ARMD, and CRVO. B12 and folate therapy is recommended. Potential sources of variability: 13.1. Lysed plasma specimens may give falsely elevated values. 13.2. Bilirubin, hemoglobin, lipids, red blood cells, protein and sodium fluoride were spiked into plasma samples for interference it was shown to be less than 10%.

34

Manual of Medical Laboratory Techniques 13.3. Do not use if the absorbance of the blank reagent is greater than 1.000 as it indicates deterioration of the reagent and also if the color of the reconstituted ALP reagent has turned yellow.

BIBLIOGRAPHY 1. Ueland PM, Refsum H, Stabler SP, Mailnow R, Andersson A, Allen RH. Total homocysteine in plasma or serum: Methods and clinical applications. Clin Chem 1993;39:1764-79.

LACTATE 1. Purpose: To estimate lactic acid in blood. The blood lactate concentration is dependent on the extent of metabolism in the liver and muscle and excretion by the kidneys. The purpose of determination of lactate in blood is to diagnose lactic acidosis which is associated with shock, hypovolemia, diabetes mellitus, liver disease, etc. 2. Principle: Lactic acid is converted quantitatively to acetaldehyde on heated with concentrated sulfuric acid. This acetaldehyde reacts with p-hydroxydiphenyl in the presence of copper sulfate forms violet colored compound, which can be estimated spectrophotometrically. 3. Performance specifications: 3.1. Linearity: Up to 5 mg. 3.2. Measurement range: 0.5–5.0 mg. 3.3. Sensitivity: 0.5 mg. 4. Primary sample: 4.1. 10% TCA precipitated blood sample. 4.2. To 2 mL of ice cold TCA add equal amounts of blood, which is collected without using tourniquet. Mix properly. Centrifuge the sample at 2500 rpm for 10 min. Use the supernatant for analysis. 4.3. Transport the sample in ice. 4.4. Process the sample on the same day. If analysis is not done on the same day, separate the supernatant and store at –20°C for up to 7 days. 4.5 The blood should be collected by vein puncture without using tourniquet. 5. Type of container and additive: 15 mL falcon tube is used for collecting sample, containing 2 mL of 10% cold TCA 6. Reagents/Consumables 6.1. 10% TCA: 10 g in 100 mL 6.2. 4% CuSO4.5H2O: 4 g in 100 mL

Biochemistry

35

6.3. 6.4.

Concentrated sulfuric acid. p-hydroxy diphenyl reagent: Dissolve 1.5 g of p-hydroxy diphenyl in 95% ethanol. 6.5. Stock standard: Lithium lactate—21.3 mg of lithium lactate in 100 mL. 6.6. Working standard: Dilute the stock standard with water to get a solution containing 10 μg/mL. 6.7. Sample preparation: To 2.0 mL of 10% cold TCA added 2.0 mL of blood sample, which was collected without using tourniquet. Mixed and centrifuged, the supernatant was used for the estimation. 7. Instrument: Spectrophotometer. 8. Procedure: S.No.

Reagents (mL)

Test

Control Blank

1.

Supernatant

0.05

0.05

—

2.

Distilled water

0.45

0.45

0.5

3.

Conc. sulfuric acid

3

3

3

Keep the tubes in 95–100º for 10 minutes. Cool it in running water. After cooling add 4.

4% CuSO4 (drop by drop)

0.05

0.05

0.05

p-hydroxy diphenyl reagent (drop by drop)

0.1

0.1

0.1

Keep the tubes at room temperature for 30 min. Read at 570 nm against water as blank using spectrophotometer.

Standardization S. No.

Reagents (mL)

B

S1

S2

S3

S4

S5

1.

Std. lithium lactate

—

0.1

0.2

0.3

0.4

0.5

2.

Concentration (mg)

—

1

2

3

4

5

3.

Distilled water

0.5 0.4

0.3

0.2

0.1

—

4.

Conc. H2SO4

—————— 3.0 ——————

Keep the tube at 95–100º for 10 min. Cool it in running water. After cooling add 5.

CuSO4 (drop by drop)

————— 0.05 ———————

6.

p-hydroxy diphenyl reagent (drop by drop)

——— 0.1 ————

Keep the tubes at room temperature for 30 minutes. Read at 570 nm against water as blank using spectrophotometer.

Calculation: Concentration from graph = X mg in 0.05 mL supernatant =

X ×2 0.05

36


where 90 M met of locate to convert to mM; Multiplied by 2 – 1; 2 dilution 1000 – to convert to liter 9. Reference range: 0.5–1.3 mM 10. Potential sources of variability: 10.1. After the blood collection, it should be immediately precipitated with cold TCA. 10.2. If the supernatant obtained is turbid, it should not be used for the analysis. 10.3. Sample to be transported in ice. BIBLIOGRAPHY 1. Barker SB, Summerson WH, J Biol Chem 1941;138:535.

PYRUVATE 1. Purpose: To estimate pyruvic acid in blood. It is useful in diagnosing diseases like congestive heart disease, diarrhea, other digestive disturbances, liver damage, acute infections, beriberi and some neurological diseases. 2. Principle: Pyruvate reacts with 2,4-dinitrophenylhydrazine forms 2,4-dinitrophenylhydrazone, which reacts with strong alkali to form a reddish compound, which can be estimated spectrophotometrically. 3. Performance specifications: 3.1. Linearity: Up to 50 μg 3.2. Measurement range: 70 – 210 μg 3.3. Sensitivity: The minimum detection limit 5 μg 4. Primary sample: 4.1. 10% TCA precipitated venous blood sample. 4.2. To 2 mL of ice cold TCA add equal amounts of venous blood, which is collected without using tourniquet. Mix properly. Centrifuge the sample at 2500 rpm for 10 min. Use the supernatant for analysis. 4.3. Process the sample on the same day. If analysis is not done on the same day, separate the supernatant and keep at – 20°C at the maximum of 7 days. 4.4. Sample to be transported in ice. 5. Type of container and additive: 15 mL falcon tube for collecting blood sample. Equal volume of 10% cold TCA should be added in whole blood. 6. Reagents/Consumables: 6.1. 200 mg of DNPH in hot 1000 mL of 1 NHCl

Biochemistry

37

6.2. NaOH: 16 g/liter. 6.3. Standard: Pyruvic acid 1 mg/mL. 7. Instrument: Spectrophotometer 8. Procedure: S. No.

Reagents

Blank

Test

S1

S2

S3

S4

1.

Standard (mL)

—

—

5

10

15

20

2.

Concentration (μg)

5

10

15

20

3.

H2O (mL)

0.5

—

0.495

0.490

0.485

0.80

4.

Supernatant (mL)

—

0.5

—

—

—

—

5.

DNPH (mL)

1.0

1.0

1.0

1.0

1.0

1.0

Keep at 37ºC for 30 minutes NaOH (mL)

10 mL

10 mL

10 mL 10 mL 10 mL 10 mL

Read at 440 nm against blank.

Calculation: Concentration from graph = X μg in 0.5 mL supernatant Calculate for the total volume of the supernatant = 2.0 mL blood Calculate for 1000 mL blood say = Y μg/L Y × 1000 × 2 = Z mM 110 where molecular weight of pyruvate = 110 1:2 dilution with TCA = 2 9. Reference range: 90–200 μM/L 10. Critical/Alert level values: Not applicable. 11. Potential sources of variability: 11.1. After the blood collection it should be immediately precipitated with cold TCA. 11.2. If the supernatant obtained is turbid, it should not be used for the analysis. 11.3. Sample to be transported in ice.

=

BIBLIOGRAPHY 1. Varley H. Practical Clinical Biochemistry 1960;612-5.

THIOBARBITURIC ACID REACTIVE SUBSTANCES (TBARS) 1. Purpose: To determine the oxidant status and lipid peroxidation. 2. Principle: Malondialdehyde (MDA) is produced during peroxidation of unsaturated fatty acids of lipids. It reacts with thiobarbituric acid (TBA) to generate a colored product, which absorbs light at 532 nm.

38


3. Performance specifications: 3.1. Linearity: Up to 60 nM/g Hb 3.2. Measurement range: 10–60 nM/gHb 3.3. Sensitivity: The minimum detection limit is 5 nM/g Hb. 4. Primary sample: 4.1. Collect 2 mL of venous blood in an EDTA vacutainer tube. 4.2. Do not use lysed blood sample for the analysis, which gives false positive values. 4.3. Process the sample on the same day of collection if not stored at 4°C for 24 hours. 5. Type of container and additive: Use EDTA vacutainer tubes for collecting samples 6. Reagents/Consumables 6.1. 10% TCA: 10 gm in 100 mL distilled water. 6.2. Thiobarbituric acid (TBA): 500 mg TBA is dissolved in 6 mL of 1 M NaOH and to this solution 69 mL distilled water was added. 6.3. Stock Standard MDA: 0.05 mL stock solution of 1,1',3,3'tetraethoxy propane bis (diethylacetate) (TEP) is made to 1 mL with 0.9% NaCl and 0.03 mL of 6 N HCl is added and made to 100 mL in distilled water. 6.4. Working Standard: 1 mL of the stock is diluted to 50 mL with distilled water to obtain a concentration of 50 nm/mL. 6.5. Saline: 0.9% NaCl. 7. Instrument: Spectrophotometer 8. Procedure: 8.1. Wash the erythrocytes thrice with saline after removal of plasma. 8.2. The buffy coats along with the upper layer are discarded with each saline wash to remove leukocytes. 8.3. Add to the packed cells 1.5 mL of 10% TCA and allow to stand for 15 min at room temperature. Centrifuge the tube and to the supernatant add 1.5 mL of TBA solution and heat a boiling water bath for 15 minutes. Measure the absorbance of the chromophore at 532 nM. Construct a standard curve using TEP hydrolyzed MDA containing 5–25 nM. B

S1

S2

S3

S4

S5

T1

Working standard (μL)

—

100

200

300

400

400

—

Concentration in nM

—

5

10

15

20

25

—

Washed erythrocytes (mL) —

—

—

—

—

—

1

Water (μL)

500

400

300

200

100

—

—

TCA (mL)

1.5

1.5

1.5

1.5

1.5

1.5

1.5 Contd...

Biochemistry

39

Contd... Incubate at room temperature for 15 minutes TBA (mL)

1.5

1.5

1.5

1.5

1.5

1.5

1.5

The contents are heated in a boiling water bath for 15 minutes and cooled. Absorbance is read at 532 nm.

Calculation:

(value from graph ) × 100 Hb

9. Reference range: 18–46 nM MDA/g Hb. 10. Critical/Alert values: Not applicable. BIBLIOGRAPHY 1. Ledwozyw A, Michalak J, Stepien A, Kadziolka A. The relationship between plasma triglycerides, cholesterol, total lipid and lipid peroxidation products during human atherosclerosis. Clin Chim Acta 1986;155:275-84.

GLUTATHIONE 1. Purpose: Glutathione is the specific donor of hydrogen for glutathione peroxidase for the reduction of H2O2, lipid and non-lipid hydroperoxides. During oxidative stress conditions, they react with the oxidant species and reduce their effect. Glutathione is used to know the antioxidant status of the patient. 2. Principle: Virtually all the non-protein sulfhydryl groups of red cells are in the form of glutathione (GSH). 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) is a disulfide compound that is readily reduced by sulfhydryl compounds, forming a highly colored yellow anion. The optical density of this yellow substance is measured at 412 nm. 3. Performance specifications 3.1. Linearity: Up to 5 mg/g Hb. 3.2. Measurement range: 0.5–5 mg/g Hb. 3.3. Sensitivity: The minimum detection limit is 0.5 mg/g Hb. 4 Primary sample: 4.1. Use only EDTA red blood cells as specimen for the test. 4.2. Collect 2.0 mL of venous blood in an EDTA vacutainer tube. 4.3. Do not use hemolyzed sample. 4.4. Process the sample immediately and if not done, store the RBC at 4°C until processed, for a maximum of 24 hours. 5. Type of container and additive: 5.1. Collect 2 mL of EDTA vacutainer tube for blood collection. 6. Reagents/Consumables: 6.1. Precipitating solution: 1.67 g—glacial metaphosphoric acid 0.2 g—EDTA


40 6.2.

Phosphate solution: (0.3 M)

6.3.

DTNB reagent:

6.4.

Standard glutathione:

30 g—sodium chloride 100 mL distilled water. 5.34 g disodium hydrogen phosphate 100 mL distilled water Crystals developed during storage at 4°C were dissolved by heating 20 mg of 5,5'-dithiobis (2-nitrobenzoic acid) 100 mL—phosphate solution This reagent is stable for 12 weeks at 4°C. 20 mg/dL.

7. Instrument: Spectrophotometer 8. Procedure: 8.1. Take 1.0 mL of EDTA blood and wash the erythrocytes thrice with saline after removal of plasma. 8.2. Discard the buffy coat along with the upper layer of erythrocytes with each saline wash to remove leukocytes. 8.3. Remove the supernatant and add 1.0 mL of distilled water. Mix well. 8.4. Mix 2 mL of the precipitating solution to 100 mL of hemolysate. 8.5. After standing for 5 minutes, filter the mixture through a medium grade filter paper or centrifuge for 2500 rpm. 8.6. Add 2 mL of the filtrate to 2.0 mL DTNB solution. Read the color intensity at 412 nm. 8.7. Glutathione content is expressed as mg/dL of whole blood/Hb. Standard: Reagents

Blank

S1

S2

S3

S4

Standard (μL)

—

25

50

75

100

D. Water (μL)

—

75

50

25

—

Precipitating solution (mL)

2.0

2.0

2.0

2.0

2.0

2.0

2.0

2.0

Concentration (μg)

Kept at RT for 10 min DTNB (mL)

2.0

2.0

Read OD at 412 nm

Calculation: The test OD was plotted from the graph = Z Z × 2000 = mg/g Hb Hb (gm)

Biochemistry

41

9. Reference range: 2–5 mg/g of Hb. 10. Critical/Alert values: Not applicable. 11. Potential sources of variability: 11.1. Lysed RBC samples may give falsely decreased values as the enzymes are released from the erythrocytes. 11.2. Hemolysate has to be preserved in –20o C if the assay is not being performed immediately. BIBLIOGRAPHY 1. Beutler E, Duran O, Belly BM. Improved method for the determination of blood glutathione. J Lab Clin Med 1963;61:882-8.

SODIUM, POTASSIUM AND CHLORIDE 1. Purpose: Quantitative estimation of sodium, potassium and chloride in human plasma by using ion sensing electrodes in electrolyte analyzer. The levels are useful is assessment of water and electrolyte balance in diseases with acidosis or alkalosis and endocrine diseases. Knowledge of hyperkalemia is useful for prompt therapy to save the heart. 2. Principle: Its methodology is based on the selective electrode measurement (SLE) principle to precisely determine measurement values. Three different electrodes used in the AVL 9180 electrolyte analyzer, sodium, potassium, chloride, and a reference electrode. Each electrode has an ion selective membrane that undergoes a specific reaction with the corresponding ions contained in the sample being analyzed. The membrane is an ion exchanger, relating to the electrical charge of the ion causing a change in the membrane potential or measuring voltage, which is built up in the film, between the sample and the membrane. 3. Performance specifications 3.1. Linearity: 51–196 mEq/L, potassium: 2.0 –12.6 mEq/L, Chloride: 56–194 mEq/L. 3.2. Measurement range: This method has a measurement range of sodium 116–150 mEq/L, potassium 2.0–7.0 mEq/L, chloride 94 –115 mEq/L. 3.3. Sensitivity: The minimum detection limit by the kit, Sodium: 51 mEq/L, potassium: 1.5 mEq/L, chloride: 56 mEq/L. 4. Primary sample: 4.1. Use plasma/body fluids as specimen for the test. 4.2. Collect blood sample in 4 mL heparin vacutainer tube. 4.3. Separate plasma within 30 minutes of collection.


42 4.4.

Process the sample on the same day within 1 hour of collection. If analysis is done on the same day, keep it 2–4 °C. 5. Type of container and additive: Use 4 mL heparin vacutainer tubes for collecting samples. 6. Reagents/Consumables: 6.1. ISE SNAP PAKTM consists of Std A solution (350 mL), Std B solution (85 mL), Std C solution (85 mL) Reference solution: A salt bridge for calibration and measurement in the AVL 9180 electrolyte analysis. 6.2. Separately packed reagents: Cleaning solution A: For cleaning the measuring system. Conditioning solution B: For daily conditioning of the stdelectrode and sample sensor in the AVL 9180 6.3. Electrolyte analyzer: 100 mL 7. Instrument: AVL 9180 electrolyte analyzer Analyzer components: The AVL electrolyte analyzer is a fully automatic microprocessor-controlled medical instrument that measures sodium, potassium and chloride. The analyzer consists of several major components that are important for us to communicate with the analyzer through a keypad with Yes/No keys. With these keys, we can perform all analyzer functions, including sample measurement data input, programming and quality control testing. The measuring chamber consists of the movable left locking device that holds the electrode in place, the electrodes, the right electrode holder, with sample sensor connector and the measuring chamber bare. Electrodes are labeled: Reference electrolyte, sodium (Na+), potassium (K+), chloride (Cl–) Operation: Running a sample with the AVL electrolyte analyzer: The AVL electrolyte analyzer provides fast easy operation. Whenever READY appears on the display, the unit is prepared to conduct sampling measurement. To analyze a std. sample, press No. To get QC/STD/DIALYSATE/URINE SAMPLE? appears press Yes. To analyzer a sample lift the sample door. The promptly, “Introduce sample”, will be displayed and the pump will begin to aspirate. Introduce the sample to the probe. Hold the sample, under the probe will wipe probe close sample door is displayed. Use a lint-free tissue to clean the probe, and then close the sample door when prompted. The analyzer will display Thank You! and a brief countdown will begin. Upon completion of analysis, the test results will be displayed and printed.

Biochemistry

8.

9. 10. 11.

12.

43

Note: Values that is higher or lower than the programmed normal range will be indicated by an arrow pointing up or down. The process involves cleaning and conditioning the sampling path, including the probe and electrodes. You will need to have ready the bottles of cleaning solution. A and electrolyte conditioning solution B and a package of lint-free tissues to use in drying sample probe. Check the bottles to ensure that the expiration date has not been reached. Standby Mode: The AVL electrolyte analyzer is designed to calibrate automatically every four hours during normal operation. If sampling will be displayed for an extended period of time, such as evening and weekends, you may place the analyzer into standby mode to suspend automatic calibration. Important points to be noted when operating the electrolyte analyzer: It is very important that the main door is closed during sampling, since it provides shielding from sources of electromagnetic interference. Procedure: 8.1. Sample size: 95 mL 8.2. Sample types: whole blood, serum, plasma 8.3. Sample container: Capillary, AVL microsampler, syringe, collection tube, sample cup. 8.4. Ambient temperature: +15 to +32º C (60–90º F) 8.5. Relative humidity: 5 to 85% (non-condensing) 8.6. Type of measurement: Direct potentiometry. 8.7. Calibration: The analyzer contains software, which permits one of three configurations. (Na+, K+, Cl–) Each of the configurations is done with the same calibration solutions. 8.8. A two-point calibration is performed automatically every 4 hours. 8.9. In ready mode and a one-point calibration is automatically performed with every measurement. 8.10. Automatic calibration procedure is also performed shortly after power on or reset. A calibration cycle can also be initiated manually at times when no sample measurements are performed. Interference: Lysed sample should not be used. Calculating results: Automatic calculation done by the machine. Biological reference range: Sodium 136–145 mEq/L Potassium 3.5–5.1 mEq/L Chloride 97–111 mEq/L Critical/Alert values: Plasma sodium < 120–150 mEq/L

44


Plasma potassium < 2.0–6.0 mEq/L Plasma chloride < 97–120 mEq/L 13. Laboratory interpretation: Hyponatremia and hyperkalemia show abnormal cardiac functions. Hyponatremia in Addison’s disease and salt-losing nephrites and hyper natremia its Cushing’s syndrome and primary aldosteronism. BIBLIOGRAPHY 1. AVL, 9180 Electrolyte Analyzer Operator’s Manual, AVL Scientifici Corporatation, Georgia, USA, 1st edn Oct 1996, 3-118. 2. Tietz, Norbert W. Clinical Guide to Laboratory Tests, 2nd edn, Philadelpia: WB Saunders, Co, 1994, 1362.

CALCIUM 1. Purpose: Quantitative estimation of Calcium in human serum/by Direct Photometric Method using Arzenazo III. Hypercalcemia (increased serum calcium) is observed in hyperparathyroidism, hypervitaminosis D, sarcoidosis, multiple myeloma, and certain cancers of the bone. Hypocalcemia can cause osteoporosis and tetany. Monitoring of serum calcium level is useful during calcium supplementation in the treatment of osteoporosis 2. Principle: Calcium reacts with Arzenazo III at neutral pH to form blue colored complex that absorbs at 650 nm. The intensity of the color is directly proportional to the calcium concentration in specimen. Interference by magnesium is eliminated by the addition of 8-hydroxy quinoline 5 sulfonic acid. Neutral pH Calcium + Arzenazo III ---------------------------> Blue colored complex 3. Performance specifications: 3.1. Linearity: This method is linear for calcium concentrations up to 16 mg/dL of serum 3.2. Measurement range: This method has a measurement range of 5– 16 mg/dL of calcium in serum 3.3. Sensitivity: The minimum detection limit by this kit is 5 mg/dL. 4. Primary sample: 4.1. Use only serum as specimen 4.2. Collect 2 mL of venous blood in a plain vacutainer tube. Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500 rpm for 10 minutes 4.3. Do not use lysed serum for testing as it may give very high results 4.4. Do not use contaminated/turbid samples for testing

Biochemistry

45

4.5. 4.6.

5.

6.

7. 8.

9.

10.

Do not collect blood in EDTA as calcium is chelated by EDTA Do not use torniquet while collecting venous blood sample as it may lead to falsely elevated levels of calcium in the sample 4.7. Process the sample on the same day within 3 hours of collection. 4.8. If analysis is not done on the same day/within 3 hours of collection, separate the serum and store it at 20–25° C for up to 7 days or at 4–8º C for up to 21 days. Type of container and additive: For serum calcium, use a plain vacutainer tube for collecting venous sample. No additive/ preservative is needed to be added. Reagents/Consumables: 6.1. The reconstituted reagent contains Arzenazo III 6.2. Calcium Standard: 10 mg/dL Instrument: Spectrophotometer. Procedure: Assay: End point Reagent volume: 1000 μL Wavelength: 650 nm Sample volume: 10 μL Temperature: 37°C Zero setting with distilled water Incubation time 5 minutes Conc. of standard: 10 mg/dL Interferences: Turbid, lypemic, incteric and lysed samples should not be used. Tourniquet should not be used, because tissue fluids will affect the value. Calculating Results:

Sample absorbance × Concentration of standard = Sample concentration Standard absorbance 11. Biological reference range: Serum: 8.8–10. 2 mg/dL 12. Critical/Alert level values: < 8 mg/dL >12.0 mg/dL 13. Laboratory interpretation: Hypercalcemia is found in sarcoidosis, hyperparathyroidism and multiple myeloma; hypocalcemia is found in hypoparathyroidism. It can cause tetany and irritability. 14. Potential sources of variability: 14.1. Lysed serum specimens may give falsely elevated values. 14.2. Do not use torniquet while collecting venous sample as it may result in falsely elevated values 14.3. If plasma is used as specimen use heparinized plasma only. If EDTA is used for collecting blood samples it may give very low values due to chelation of calcium by EDTA 14.4. As calcium is an ubiquitous ion, to prevent accidental contamination, all glassware should be rinsed in diluted hydrochloric acid and water before use. Even water and glassware containing calcium will react with the reagent.

46


BIBLIOGRAPHY 1. Baginski ES, Marrie SS, Clarke WL, Zak. Clin Chem Acta 1973;46:49. 2. Henry RJ, Dryer RL. Standard Methods of Clinical Chemistry, Acord Press, New York, 1963,205. 3. Young SD, Pestaner LC, Gibberman V. Clin Chem 1975;21(5).

PHOSPHORUS 1. Purpose: Quantitative estimation of phosphorus in serum UV End Point Method. Measurement of Serum Phosphorus is useful in the diagnosis of bone disorder. Increased serum phosphorus levels are seen in hypervitaminosis D, hyperparathyroidism, and renal failure. Reduced serum phosphorus levels are seen in rickets (vitamin D deficiency) hypoparathyroidism, and Fanconi syndrome. 2. Principle: Inorganic phosphorus reacts with ammonium molybdate in an acid medium to form a phosphomolybdate complex, which absorbs light at 340 nm. The absorbance at this wavelength is directly proportional to the amount of inorganic phosphorus present in the sample. Acid pH Phosphorus + Ammonium molybdate -----------------------> Phosphomolybdate complex 3. Performance specifications: 3.1. Linearity: Up to 20 mg/dL in serum 3.2. Measurement range: 1–20 mg/dL in serum 3.3 Sensitivity: The minimum detection limit is 1 mg/dL 4. Primary sample: 4.1. Use only serum as specimen for the test 4.2. Collect 4 mL of venous blood from a peripheral vein in a plain vaccutainer tube 4.3. Do not use hemolyzed/contaminated serum for testing 4.4. Process the sample on the same day within 3 hours of collection. 4.5. If analysis is not done on the same day/within 3 hours of collection, separate the serum and store it at 2–8° C for up to 7 days. 5. Type of container and additive: Use plain vacutainer tubes for collecting. No additive/Preservative is needed to be added 6. Instrument: Semi-autoanalyzer 7. Reagents/Consumables: 7.1. Inorganic phosphorus reagent: Ammonium molybdate 0.3 mM, sulfuric acid 1% with surfactant. 7.2. Inorganic phosphorus standard: 5.0 mg/dL

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8. Procedure: 8.1. Switch on the machine and press “FLUSH” button by keeping the tubing in a container with 2% detergent for 2 minutes followed by distilled water for 2 minutes. 8.2. Press “PROC”. Different test procedures will be displayed. 8.3. Select the test to be processed by entering its number and then press “ENTER” key. 8.4. Now the assay parameters of the specific test procedure will be displayed. Note down the volume of the reagent and the sample to be used. 8.5. Feed the blank with each batch and ensure the absorbance of the blank is less than 0.3 if the absorbance of the ‘blank is more than 0.300’ discard the reagent. 8.6 Run the phosphorus standard with each batch of patient sample 8.7. Then feed the test samples and record the values. 8.8. Check whether the sample is hemolyzed, icteric or lipemic before processing. If the sample is lysed, collect another sample and proceed. If it is icteric or lipemic, dilute the sample 1 in 10 with distilled water and proceed. Multiply the result displayed by dilution factor 10. Assay: UV- End point Reagent volume: 1000 μL Wavelength: 340 nm Sample volume: 10 μL Temperature: 37°C Conc. of Standard: 5 mg/dL Incubation time: 5 min Zero setting with: Distilled water No. of readings: 1 Time: 30 sec 9. Interferences: Turbid, lipemic, icteric and lysed samples should not be used. Feed the blank with each batch and ensure the absorbance of the blank is less than 0.300 if the absorbance of the ‘blank is more than 0.300’ discard the reagent. 10. Calculating results: Sample absorbance × Concentration of standard = Sample concentration Standard absorbance

11. Biological reference range Male : 2.1–5.6 mg/dL Female : 1.5–6.8 mg/dL 10 days-24 months : 4.5 –6.7 mg/dL 24 months-11 years : 4.5 –5.5 mg/dL 12. Critical/Alert level values: 6.0 mg/dL 13. Laboratory interpretation: Lowered phosphorus in hyperparathyroidism and vice-versa.

48


In rickets, osteomalacia, renal rickets and Fanconi syndrome there is lowered phosphorus. Increase of phosphorus could cause tetany. 14. Potential sources of variability: 14.1. Detergents used in glassware washing and disposable wipes used in the laboratory contain phosphates, and the use of improperly rinsed glassware may result in elevated inorganic phosphorus values. 14.2. Use only clear, unhemolyzed serum, separated from the erythrocytes as soon as possible. Lysed serum specimens may give falsely elevated values, as erythrocytes contain organic phosphates that can hydrolyze on standing or can be enzymatically cleaved by phosphatases. Inorganic phosphates can then leak through the cell walls, increasing the concentration. 14.3. Phosphorus remains stable in serum for up to 7 days if the serum specimen is stored at 2–8°C hence if analysis is not done on the same day within 3 hours of collection, separate the serum and store it at 2–8°C. 14.4. Do not use if the absorbance of the blank reagent is greater than 0.300 as it indicates detrioration of the reagent. BIBLIOGRAPHY 1. Amador E. Urban J Clin Chem 1977;18:60. 2. Daly JA. Clin Chem 1972;18:263. 3. Gamst O, Try K, Scand. J Clin Lab Invest 1980;14.

IRON AND IRON-BINDING CAPACITY (IBC) 1. Purpose: Iron found in blood is mainly present in the hemoglobin of the RBCs. Its role in the body is mainly in the transport of oxygen and cellular oxidation. Iron is absorbed in the small intenstines and bound to a globulin in the plasma, called transferrin and transported to bone marrow for the formation of hemoglobin. Increased serum levels are found in hemolytic anemias, hepatitis, and lead and iron poisoning. Decreased serum levels are found in anemias caused by iron deficiency due to insufficient intake or absorption of iron, chronic blood loss, late pregnancy and cancer. Increase in total iron-binding capacity (TIBC) is found in iron deficient anemias and pregnancy. Decrease in TIBC is found in hypoproteinemia, hemolytic/pernicious/sickle cell anemias, inflammatory diseases and cirrhosis. 2. Principle: Iron, bound to transferrin, is released in and acidic medium and the ferric ions are reduced to ferrous ions. The Fe (II) ions react

Biochemistry

49

with ferrozine to form a violet colored complex. Intensity of the complex formed is directly proportional to the amount of iron present in the sample. For TIBC, the serum is treated with excess of Fe (II) to saturate the iron binding sites of transferrin. The excess Fe (II) is adsorbed and precipitated and the iron content in the supernatant is measured to give the TIBC. Acid medium

Fe (III) ---------------------------------------------------> Fe (II) Fe (II) + Ferrozine -----------------------------> violet colored complex 3. Performance specifications: 3.1. Linearity: Up to 1000 μg/dL. If the value exceeds this limit, dilute the serum with distilled water and repeat the assay. Calculate the value using the proper dilution factor. 3.2. Measurement range: This method has a measurement range 60–200 μg/dL of iron in serum. 3.3. Sensitivity: The minimum detection limit by this kit is 40 μg/dL. 4. Primary sample: 4.1. Use only serum as specimen 4.2. Collect 4 mL of venous blood in a plain vacutainer tube. 4.3. Allow the tube to stand for 30 min and separate the serum by centrifugation at 2500 rpm for 10 min. 4.4. Do not use lysed serum for testing as it may give very high results. 4.5. Do not use contaminated/turbid samples for testing. 4.6. Process the sample on the same day within 3 hours of collection. 4.7. If analysis is not done on the same day/within 3 hours of collection, separate the serum and store it at 4–8º C for up to 7 days or at –20 to 25° C for up to 21 days. 4.8. Sample should be collected before 11.00 am. 5. Reagents/Consumables: 5.1. L1: Iron buffer reagent 35 mL 5.2. L2: Iron color reagent 35 mL 5.3. S: Iron standard (100 μg/dL) 2 mL 5.4. Storage/Stability: Contents are stable at 2–8°C till the expiry. 5.5. The reconstituted reagent contains Arzenazo III, 8-Hydroxyquinoline 5-sulfonic acid, Phosphate buffer: Non-reactive ingredients, and stabilizers. 6. Instrument: Semi-autoanalyzer/Spectrophotometer 7. Procedure: 7.1. Wavelength/filter : 570 nm (Hg 578 nm)/Yellow Temperature : RT Light path : 1 cm


50 7.2.

Iron assay: Pipette into clean dry test tubes labeled as Blank (B), Standard (S), Sample Blank (SB) and Test (T)

Addition Sequence

B (mL)

S (mL)

SB (mL)

T (mL)

Iron buffer reagent (L1)

1.0

1.0

1.05

1.0

Milli Q H2O

0.2

-

-

-

Iron standard (S)

-

0.2

-

-

Sample

-

-

0.2

0.2

Iron color reagent (L2)

0.05

0.05

—

0.05

Mix well and incubate at RT for 5 min. Measure the absorbance of the blank (Abs.B), standard (Abs. S), Sample Blank (Abs. SB) and Test sample (Abs.T) against DW.

7.3.

TIBC assay: Pipette into a clean dry test tube Serum 0.5 mL TIBC saturating reagent (L1) 1.0 mL Mix well and allow to stand at RT for 10 min and add TIBC precipitating reagent (L2) Approx. 50 mg Mix well and allow to stand at R-T for 10 min. Centrifuge at 2500 rpm for 10 min to obtain a clear supernatant. Determine the iron content in the supernatant as above mentioned iron assay. Calculation Iron in μg/dL =

Abs.T – (Abs. SB + Abs.B) × 100 Abs.S – Abs. B

Abs.T – (Abs. SB + Abs.B) × 300 Abs.S – Abs. B UBIC in μg/dL = TIBC in μg/dL – iron in μg/dL Switch on the machine and press “FLUSH “ button by keeping the tubing in a container with 2% detergent for 2 minutes followed by distilled water for 2 minutes. Press “PROC”. Different test procedures will be displayed. Select the test to be processed by entering its number and then press “ENTER” key. Now the assay parameters of the specific test procedure will be displayed. Note down the volume of the reagent and the sample to be used. Zero with distilled water. Feed the reagent blank with each batch of patient samples and ensure the absorbance of the blank is less than 0.300 at 650 nm if the absorbance of the ‘blank is more than 0.300’, discard the reagent. TIBC in μg/dL =

7.4.

7.5. 7.6. 7.7.

7.8. 7.9.

Biochemistry

8. 9.

10. 11. 12.

13.

51

7.10. Then feed the standard and ensure the value of the standard is 100 μg/dL. Then feed the test samples and record the values. 7.11. Before processing patient samples, check whether the sample is hemolyzed, icteric before processing. If the sample is lysed collect another sample and proceed. Interferences: Turbid, lipemic, lysed and icteric samples interfere with the absorbance. Reference range: Serum Iron: Male 80–140 μg/dL Female 80–155 μg/dL Newborns 12–67 μg/dL Children up to 10 yrs 30–150 μg/dL Serum IBC: 250–400 μg/dL UBIC: 160–360 μg/dL Reportable interval of examination: Within 4 hours. Critical/Alert level values: Below 50 μg/dL Laboratory interpretation: Decreased iron and increased TIBC and UBIC suggest iron-deficiency anemia. Increase of iron suggests hemolytic anemia, hemosiderosis and hemochromatosis. Potential sources of variability: 13.1. Lysed serum specimens may give falsely elevated values. 13.2. As iron is a ubiquitous ion, to prevent accidental contamination, all glassware should be rinsed in dilute hydrochloric acid and water before use. Even water and glassware containing iron will react with the reagent.

BIBLIOGRAPHY 1. Duffy JR, Gaudin J. Clin Biochem 1977;10:122. 2. Higgins T. Clin Chem 1981;27:1619. 3. Kalpan LA, Peasce AJ. Clinical Chemistry. Mosby Ed 1989.

VITAMIN A 1. Purpose: Quantitative estimation of vitamin A in human serum or plasma by high performance liquid chromatography (HPLC) at 280 nm. Vitamin A estimation is useful in diseases with possible weak antioxidant defenses like atherosclerosis, cancer, Eale’s disease, uveitis, etc. HPLC analysis provides a quantitative and sensitive detection of vitamin A in serum samples. The advantages include small sample size requirement, nondestructive nature and speed of analysis and highly accurate and reproducible separation.

52


2. Principle: The serum proteins are precipitated by ethanol and the vitamin A is extracted with hexane, the organic solvent is evaporated under the nitrogen gas atmosphere and re-dissolved in the ethanol and injected in the HP-HPLC and detected at 280 nm. 3. Performance specifications: 3.1. Linearity: Up to 250 μg/dL in human serum or plasma 3.2. Measurement range: 20 ng to 250 μg for vitamin A in serum 3.3. Sensitivity: Detection limit of vitamin A is 20 ng. 4. Primary sample: 4.1. Use serum as specimen for the test 4.2. Collect 4 mL of venous blood in a plain vacutainer tube. 4.3. Do not use hemolyzed sample. 5. Type of container and additive: 5.1. Use plain vacutainer tubes for collecting samples 5.2. No additive/preservative is needed to be added 6. Reagents/Consumables: 6.1. Vitamin A stock standard –Retinol (1 mg/mL) 6.2. Working standard (20–100 ng) 6.3. Solvent – 100% methanol 6.4. Hexane 6.5. Ethanol 7. Instrument: Reverse-phase high performance liquid chromatography (RP-HPLC) 8. Procedure: 8.1. Take 100 μL of the serum sample and to it add equal volume of ethanol. 8.2. Vortex the mixture well for 2 minutes. 8.3. Add 400 μL of hexane and again vortex for 2 minutes and centrifuge at 2500 rpm for 10 minutes. 8.4. Remove 300 μL of the clear supernatant. 8.5. Pass nitrogen gas until completely gets dried. 8.6. Add 100 μL of ethanol to the dry tube and vortex for 1 minute. 8.7. Load 50 μL into HPLC and the values are calculated against the standard area. 8.8. Prior to the analysis degas the methanol solvent and keep the column for equilibrium/Flow rate/min. 9. Reference Range: 30–120 μg/dL 10. Critical/Alert level values: Not applicable. 11. Potential sources of variability: 11.1. Lysed serum samples may give falsely increased values. 11.2. Freeze thaw of the sample may give false values.

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BIBLIOGRAPHY 1. Kenneth W, Miller, Nancy A Lorr, Chung S. Yang. Simultaneous determination of plasma retinal, α-Tocopherol, Lycopene, α-Carotene, and β-Carotene by High Performance Liquid Chromatography. Anal Biochem 1984; 138:340-45.

VITAMIN E 1. Purpose: Quantitative estimation of Vitamin E in human serum or plasma by HPLC at 280 nm. Vitamin E has got protective effects as biological antioxidant against environmental and drug toxicity as well as carcinogenesis. Its estimation is useful in diseases with possible weak antioxidant defenses like atherosclerosis, cancer, Eale’s disease, uveitis, etc. HPLC analysis provides a quantitative and sensitive detection of vitamin E in serum samples. The advantages include small sample size requirement, nondestructive nature and speed of analysis and highly accurate and reproducible separation. 2. Principle: The serum proteins are precipitated by ethanol and the vitamin E is extracted with hexane, the organic solvent is evaporated under the nitrogen gas atmosphere and re-dissolved in the ethanol and injected in the HP-HPLC and detected at 280 nm. 3. Performance specifications: 3.1. Linearity: Up to 30 mg/L in human serum or plasma 3.2. Measurement range: 1–30 mg/L for vitamin E in serum 3.3. Sensitivity: The minimum detection limit is 1 mg/L. 4. Primary Sample: 4.1. Use serum as specimen for the test 4.2. Collect 4 mL of venous blood in a plain vacutainer tube. 4.3. Do not use hemolyzed sample. 5. Type of container and additive: 5.1. Use plain vacutainer tubes for collecting samples 5.2. No additive/Preservative is needed to be added 6. Reagents/Consumables: 6.1. Vitamin E stock standard (1 mg/mL) 6.2. Working standard (200–1800 ng) 6.3. Solvent – 100% methanol 6.4. Hexane 6.5. Ethanol 7. Instrument: Reverse-phase high performance liquid chromatography (RP-HPLC) 8. Procedure: 8.1. Take 100 μL of the serum sample and to it add equal volume of ethanol.

54


8.2. Vortex the mixture well for 2 minutes. 8.3 . Add 400 μL of hexane and again vortex for 2 min and centrifuge at 2500 rpm for 10 minutes. 8.4. Remove 300 μL of the clear supernatant. 8.5. Pass nitrogen gas until completely gets dried. 8.6. Add 100 μL of ethanol to the dry tube and vortex for 1 minute. 8.7. Load 50 μL into HPLC and the values are calculated against the standard area. 8.8. Prior to the analysis degas the methanol solvent and keep the column for equilibrium/Flow rate/min. 9. Reference range: 5–15 mg/L. 10. Critical/Alert level values: Not applicable. 11. Potential sources of variability: 11.1. Lysed serum samples may give falsely increased values. 11.2. Serum sample unused can be stored in at –20ºC with occasional freeze-thaw cycles for 3 to 5 weeks. BIBLIOGRAPHY 1. Kenneth W, Miller, Nancy A Lorr, Chung S Yang. Simultaneous determination of plasma retinal, α-Tocopherol, Lycopene, α-Carotene, and β-Carotene by High Performance Liquid Chromatography. Anal Biochem 1984; 138:340-5.

VITAMIN C 1. Purpose: To estimate the vitamin C in plasma. It is an effective antioxidant and is estimated in oxidative stress conditions like Eales disease. It is decreased in scurvy. 2. Principle: Ascorbic acid in plasma is oxidized by Cu (II) to form dehydroascorbic acid, which reacts with acidic 2, 4-dinitrophenylhydrazine to form a red bis-hydrazine, which is measured at 520 nm. 3. Performance specifications: 3.1. Linearity: This method is linear up to 15 mg/L 3.2. Measurement range: This method has a measurement range of 5–15 mg/L 3.3. Sensitivity: The minimum detection limit by this method is 5 mg/L. 4. Primary sample: 4.1. Use only heparinized plasma as specimen for the test 4.2. Do not use lysed plasma for testing as it may give very high results

Biochemistry 4.3. 4.4. 4.5.

55

Do not use contaminated/turbid samples for testing Process the sample immediately on the same day. As soon as received, the plasma is treated with 6% metaphosphoric acid and processed immediately, if not the supernatant is stored at –20o C. 5. Type of container and additive: Collect 4 mL of blood in heparin vacutainer tube. 6. Reagents/Consumables: 6.1. 6% Metaphosphoric acid solution. Dissolve 30.0 g of metaphosphoric acid (HPO3) in distilled water and bring to a final volume of 500 mL. Prepare immediately before use. 6.2. 4.5 mol/L sulfuric acid. Add slowly 250 mL of concentrated sulfuric acid, reagent grade, to 500 mL of cold water in a 1 L flask and fill to mark with distilled water. Caution: Since significant heat is generated when concentrated sulfuric acid is diluted; the flask should be placed in an ice bath. The concentrated acid should be added slowly to water in small quantities at a time and the resulting solution mixed constantly. 6.3. 12 mol/L Sulfuric acid: Add 650 mL of concentrated sulfuric acid to 300 mL of cold water in a 1 L flask, cool, and fill to mark with distilled water. The concentrated acid should be added slowly to water in small quantities at a time and the resulting solution mixed constantly and refrigerate. 6.4. 2% 2,4-dinitrophenylhydrazine (DNPH) reagent: 4.5 mol/L sulfuric acid. Dissolve 2 g (DNPH) in 100 mL 4.5 mol/L sulfuric acid. Let it stand in the refrigerator overnight, and then filter. 6.5. 5% Thiourea solution: Dissolve 5 g of thiourea in distilled water and dilute to a final volume of 100 mL. This reagent is stable for 1 month at 4° C. 6.6. 6% Copper sulfate solution. Dissolve 0.6 g of anhydrous copper sulfate in distilled water and dilute to a final volume of 100 mL. 6.7. Dinitrophenylhydrazine-thiourea-copper sulfate (DTCS) reagent: Mix 5 mL of the thiourea solution, 5 mL of the copper sulfate solution, and 100 mL of the 2, 4-dinitrophenylhydrazine reagent. Store in a bottle at 4°C for a maximum of 1 week. 6.8. Calibrators: All ascorbic acid calibrators should be prepared daily. 6.9. 50 mg/dL Ascorbic acid stock calibrator. Dissolve 50 mg of ascorbic acid in metaphosphoric acid (6.0 g/dL) and bring to a final volume of 100 mL with metaphosphoric acid.

56


6.10. 5 mg/dL Intermediate ascorbic acid calibrator. Pipette 10.0 mL of stock calibrator into a 100 mL colorimetric flask and dilute to mark 6% with metaphosphoric acid. 6.11. Working calibrators: In a series of 25 mL volumetric flasks, pipette the following amounts of intermediate calibrator: 0.5, 2.0, 4.0, 6.0, 10.0, 15.0 and 20.0 mL. Bring to a final volume of 25 mL with 6% metaphosphoric acid to yield working calibrators of 0.10, 0.40, 0.80, 1.20, 2.00, 3.00 and 4.00 mg/dL. 7. Instrument: Spectrophotometer. 8. Procedure: 8.1. Add 0.5 mL of heparinized plasma to 2.0 mL of freshly prepared metaphosphoric acid in a 13 × 10 mm test tube, and mix well in a vortex mixer. Centrifuge the plasma-metaphosphoric acid mixture for 10 min at 2500 rpm. Pipette 1.2 mL of the clear supernatant into a 13 × 100 mm Teflon-lined, screw-cap test tube. 8.2. Add 1.2 mL of each concentration of working calibrator into 13 × 100 mm screw-cap test tubes. Prepare calibrators in duplicate. Add 1.2 mL of metaphosphoric acid to two tubes for use as blank. 8.3. Add 0.4 mL of DTCS reagent to all tubes. Cap the tubes, mix the contents, and incubate the tubes in a water bath at 37°C for 3 hrs. 8.4. Remove the tubes from the water bath and cool for 10 min in an ice bath. While mixing, slowly add to all tubes 2.0 mL of cold sulfuric acid, 12 mol/L, cap, and mix in a vortex mixer (The temperature of the mixture must not exceed room temperature). 8.5. Adjust the spectrophotometer with the blank to read zero at 520 nm, and read the calibrators and unknowns. Plot the concentration of each working calibrator versus absorbance values. The calibration curve obeys Beer’s law up to an ascorbic acid concentration of 2.0 mg/dL. Calculation: From the standard graph the test absorbance will be plotted and the value will be calculated for 100 mL. Standardization protocol Reagents (mL)

Blank

Intermediate calibrators

S1

S2

S3

S4

S5

0.5

2.0

4.0

6.0

10.0

6% Metaphosphoric acid

1.2

24.5

23.0

21.0

19.0

15.0

Working calibrators

—

1.2

1.2

1.2

1.2

1.2

Vit C conc in mg/dL

—

0.1

0.4

0.8

1.2

2.0 Contd...

Biochemistry

57

Contd...

DTCS

0.4

0.4

0.4

0.4

0.4

0.4

2.0

2.0

Mix and Incubate at 37 °C for 3 hrs, Then cool the tubes in ice bath for 10 min 12 M H2SO4

2.0

2.0

2.0

2.0

OD at 520 nm

9. Reference range: 5–15 mg/L 10. Potential sources of variability: 10.1. Do not use lysed plasma samples it may give false elevated values. 10.2. Immediately add metaphosphoric acid to the collected sample as it may get oxidized. BIBLIOGRAPHY 1. Carl A, Burtis Edward R Ashwood. Tietz Textbook of Clinical Chemistry – 2nd edn. 1994;1313-14.

58


Part II: Enzymes ASPARTATE TRANSAMINASE (AST) SERUM GLUTAMIC-OXALOACETIC TRANSAMINASE (SGOT) 1. Purpose: Quantitative estimation of AST activity in human serum by optimized UV kinetic (Modified IFCC Method). Measurement of AST (SGOT) is useful in diagnosis and treatment of heart and hepatobiliary diseases such as cirrhosis, metastatic carcinoma, and viral hepatitis. Increased AST (SGOT) levels indicate damage to heart/skeletal muscle. Injury to these tissues results in the release of the AST (SGOT) enzyme to general circulation. Following myocardial infarction, serum levels of AST (SGOT) are elevated and reach a peak in 48 to 60 hours after onset. 2. Principle: AST catalyzes the transfer of an amino group between L-aspartate and 2-oxoglutarate. The oxalocetate formed in the first reaction reacts with NADH in the presence of malate dehydrogenase (MDH) to form NAD+. AST activity is determined by measuring the rate of oxidation of NADH at 340 nm. Lactate dehydrogenase is included in the reagent to convert endogenous pyruvate in the sample to lactate during the lag phase prior to measurement. Addition of pyridoxal 5-phosphate stabilizes the transaminase and avoids falsely low values in samples containing insufficient endogenous pyridoxal phosphate. The enzymatic reaction-sequence employed in the assay of aspartate aminotransferase is as follows: GOT L-aspartate + α-ketoglutarate ------------------> Oxaloacetate + L-Glutamate MDH Oxaloacetate + NADH + H+ --------------------> L- Malate + NAD+ 3. Performance specifications: 3.1. Linearity: Up to 500 IU/L of serum 3.2. Measurement range: 2 – 500 IU/L of AST activity in serum 3.3. Sensitivity: The minimum detection limit by this kit is 2 IU/L of serum 4. Primary sample: 4.1. Use only serum as specimen for the test 4.2. Collect 4 mL of venous blood in a plain vacutainer tube. 4.3. Allow the tube to stand for 30–45 min and separate the serum by centrifugation at 2500 rpm for 10 min. 4.4.. Do not use lysed serum for testing as it may give very high results 4.5. Do not use contaminated/turbid samples for testing. 4.6. Process the sample on the same day within 3 hours of collection.

Biochemistry 4.7.

5. 6.

7. 8.

9.

59

If analysis is not done on the same day/within 3 hours of collection, separate the serum and store it at 2–8° C for up to 7 days. Type of container and additive: Use plain vacutainer tubes for collecting samples. No additive/preservative is needed to be added. Reagents/Consumables: 6.1. Reagent I : NADH, α-ketoglutarate MDH, LDH 6.2. Reagent II: Tris buffer and L-aspartate Discard the reagent if the initial absorbance, read against water at 340 nm, is below 0.800. Instrument: Semi-autoanalyzer Procedure: 8.1. Switch on the machine and press “ FLUSH “ button by keeping the tubing in a container with 2% detergent for 2 minutes followed by distilled water for 2 minutes. 8.2. Press “PROC”. Different test procedures will be displayed. 8.3. Select the test to be processed by entering its number/name of the test and then press “ENTER” key. 8.4. Now the assay parameters of the specific test procedure will be displayed. Note down the volume of the reagent and the sample to be used. 8.5. Feed the blank with each batch of patient samples and ensure the absorbance of the blank is less than 0.800. If the absorbance of the ‘blank is more than 0.800’, discard the reagent 8.6. Then feed the test samples and record the values. 8.7. Check whether the sample is hemolyzed, icteric or lipemic before processing. If the sample is lysed collect another sample and proceed. If it is icteric or lipemic dilute the sample 1 in 10 with distilled water and proceed. Multiply the result displayed by dilution factor 10. Assay: Kinetic assay Reagent volume: 500 μL Wavelength: 340 nm Sample volume: 50 μL Temperature: 37°C Zero setting with distilled water Number of readings: 4 Time: 60 sec Interferences: Samples with a very high SGOT activity cause an excessive consumption of NADH, resulting in very low initial absorbance and/or nonlinear reaction. When this occurs, the assay should be repeated with a diluted sample. Pyridoxal phosphate can elevate AST values by activating the apoenzyme of the transaminase. Pyridoxal phosphate may be found in diluent water, contaminated with microbial growth. High levels of pyruvate may also interfere with assay performance.

60


10. Calculating results: ΔA/min × factor given - (Factor 1746 given in the kit). 11. Biological references: Up to 46 IU/L at 37°C. 12. Critical/Alert level values: Above 100 IU/L Laboratory interpretation: Damage of the cells of heart, liver and skeletal muscle. Peak values of AST on the second day of myocardial infarction. Increase also in pancreatitis and mononucleosis. 13. Potential Sources of Variability: Lysed serum specimens may give falsely elevated values as erythrocytes contain fifteen times the AST activity in serum. AST activity remains stable in serum for up to 7 days if the serum specimen is stored at 2–8°C hence if analysis is not done on the same day within 3 hours of collection, separate the serum and store it at 2–8°C. Do not use if the absorbance of the blank reagent is greater than 0.800 as it indicates deterioration of the reagent. Pyridoxal phosphate can elevate AST values by activating the apoenzyme form of the transaminase. Pyridoxal phosphate may be found in diluent’s water contaminated with microbial growth. High levels of pyruvate may also interfere with assay performance. BIBLIOGRAPHY 1. Expert panel of the IFCC on enzymes. Clin Chem Acta 1976;F19:70.

ALANINE AMINOTRANSFERASE (ALT)/SERUM GLUTAMIC-PYRUVIC TRANSAMINASE (SGPT) 1. Purpose: Quantitative estimation of ALT in human serum by optimized UV kinetic (Modified IFCC Method). Measurement of ALT (SGPT) is useful in diagnosis, treatment of hepatobiliary diseases such as cirrhosis, metastatic carcinoma, and viral hepatitis. Increased ALT levels also indicate damage to heart/skeletal muscle. Injury to these tissues results in the release of the ALT enzyme to general circulation. Following a myocardial infarction, serum levels of ALT are elevated and reach a peak 48 to 60 hours after onset. 2. Principle: ALT catalyzes the transfer of an amino group between 2-Oxoglutarate and L-alanine. The pyruvate formed in the first reaction reacts with NADH in the presence of lactate dehydrogenase (LDH) to form NAD+. ALT activity is determined by measuring the rate of oxidation of NADH at 340 nm. Lactate dehydrogenase is included in the reagent to convert endogenous pyruvate in the sample to lactate during the lag phase prior to measurement. The enzymatic reaction

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61

sequence employed in the assay of alanine aminotransferase is as follows: ALT 2-oxoglutarate + L-alanine ---------------> L-glutamate + Pyruvate 3.

4.

5. 6.

7. 8.

LDH Pyruvate + NADH + H+ ---------------> Lactate + NAD+ Performance specifications 3.1. Linearity: This method is linear for ALT concentrations up to 240 IU/L of serum 3.2. Measurement range: This method has a measurement range of 2– 440 IU/L of ALT activity in serum 3.3. Sensitivity: The minimum detection limit by this kit is 4 IU/L 3.4. Precision CV: Within run 4.7–5.8 between RUN 4.4 –10.1 Primary sample: 4.1. Use only serum as specimen for the test 4.2. Collect 4 mL of venous blood in a plain vacutainer tube. 4.3. Allow the tube to stand for 30 min and separate the serum by centrifugation at 2500 rpm for 10 min. 4.4. Do not use lysed serum for testing as it may give very high results 4.5. Do not use contaminated/turbid samples for testing 4.6. Process the sample on the same day within 3 hours of collection. 4.7. If analysis is not done on the same day/within 3 hours of collection, separate the serum and store it at 2-8°C for up to 7 days. Type of container and additive: Use plain vacutainer tubes for collecting samples. No additive/preservative is need to be added. Reagents/Consumables: Reagent A: Composition in the test: Tris buffer 100 mM pH 7.15, L-alanine 500 mM, B: 2-oxoglutarate 15 mM, NADH 0.18 mM, LDH 1.2 kU/L store all components in 2–8°C. Instrument: Semi-autoanalyzer. Procedure: 8.1. Switch on the machine and press “FLUSH “button by keeping the tubing in a distilled water for 2 minutes. 8.2. Press “PROC”. Different test procedures will be displayed. 8.3. Select the test to be processed by entering its number and then press “ENTER” key. 8.4. Now the assay parameters of the specific test procedure will be displayed. Note down the volume of the reagent and the sample to be used. 8.5. Feed the blank with each batch of and ensure the absorbance of the blank is less than 0.8. If the absorbance of the ‘blank is more than 0. 8’, discard the reagent.


62 8.6. 8.7.

9.

10. 11.

12. 13. 14.

Then feed the test samples and record the values Check whether the sample is hemolyzed, icteric or lipemic before processing. If the sample is lysed collect another sample and proceed. If it is icteric or lipemic, dilute the sample 1 in 10 with distilled water and proceed. Multiply the result displayed by dilution factor 10. Assay: Kinetic Reagent volume: Reagent A 400 μL Reagent B 100 μL Wavelength: 340 nm Sample volume: 50 μL Temperature: 37° C Zero setting with distilled water Number of readings: 3 Time: 60 sec Interferences: High levels of ascorbic acid (above 40 mg/dL) hemoglobulin above 400 mg/dL and lipemia above 2000 mg/dL triacyl glycerol. Calculating of results: Calculation: U/L = ΔA/min × factor given. Biological reference range: Male: 41 IU/L Female: 31 IU/L. Alert level values: More than 200 IU/L Laboratory interpretation: Increase of ALT suggests necrosis of liver, say in hepatocellular jaundice and in myocardial infarction. Potential sources of variability: 14.1. Lysed serum specimens may give falsely elevated values as erythrocytes contain fifteen times the ALT activity in serum 14.2. ALT activity remains stable in serum for up to 7 days if the serum specimen is stored at 2–8°C hence if analysis is not done on the same day within 3 hours of collection, separate the serum and store it at 2–8°C. 14.3. Do not use if the absorbance of the blank reagent is greater than 0.800 as it indicates deterioration of the reagent 14.4. Pyridoxal phosphate can elevate ALT values by activating the apoenzyme form of the transaminase. Pyridoxal phosphate may be found in diluent water contaminated with microbial growth High levels of pyruvate may also interfere with assay performance.

BIBLIOGRAPHY 1. Clin Chem lab Med 2002;40:718-24. 2. TH-books Verlags Gesel lschaft; 1st ed. Frankfurt 1998;55-65. 3. Tietz Textbook of Clinical Chemistry, 3rd ed, Philadelphia: WB Saunders Company.

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63

ALKALINE PHOSPHATASE 1. Purpose: Quantitative estimation of alkaline phosphatase in human serum by PNPP-DEA kinetic method. Measurement of ALP is useful in the diagnosis and treatment of hepatobiliary diseases of obstructive origin both intrahepatic and extrahepatic and bone diseases such as rickets, osteomalacia and Paget’s disease and healing fractures. Elevations also occur in the third trimester of pregnancy and levels are elevated during periods of active bone growth. Marked elevations of serum ALP in the absence of jaundice in the presence of primary source indicates metastasis. 2. Principle: At pH 9.8 alkaline phosphatase catylyses the hydrolysis of colorless 4-nitrophenyl phosphate to yellow colored 4-nitrophenol and phosphate. 4-nitrophenol absorbs light at 405 nm the rate of increase in absorbance at 405 nm is directly proportional to the enzyme activity. The enzymatic sequence employed in the assay of alkaline phosphatase is follows: ALP > Phosphate + 4-nitrophenol. 4-nitrophenyl phosphate + H2 O ----------------pH 9.8 3. Performance specifications: 3.1.

Linearity: This method is linear for A concentrations up to 2800 IU/L of serum

3.2.

Measurement range: This method has a measurement range of 1– 2800 IU/L of ALP activity in serum

3.3.

Sensitivity: The minimum detection limit by this kit is 1 IU/L

4. Primary sample: 4.1. Use only serum as specimen for the test. 4.2. Collect 2 mL of venous blood in a plain vacutainer tube. Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500 rpm for 10 minutes. 4.3. Do not use lysed serum for testing as it may give very high results 4.4. Do not use contaminated/turbid samples for testing. 4.5. Process the sample on the same day within 3 hours of collection. 4.6. If analysis is not done on the same day/within 3 hours of collection, separate the serum and store it at 2–8 °C for up to 24 hours only. 5. Type of container and additive: Use plain vacutainer tubes for collecting samples. 6. Reagents/Consumables: 6.1. Reagent A, Reagent B.


64 6.2.

7. 8.

9.

10. 11.

Diethanolamine Buffer (pH 9.8) 1M, Magnesium chloride (as acetate) 0.5 mM, 4-nitrophenylphosphate 10 mM. 6.3. Stabilizer to arrest autohydrolysis. 6.4. Discard the reagent if the initial absorbance, read against water at 405 nm, is above 1.000. The absorbance of working ALP reagent increases slowly on storage 6.5. Do not use the constituted reagent if it has turned yellow. Instrument: Semi-autoanalyzer Procedure: 8.1. Switch on the machine and press “FLUSH “button by keeping the tubing in a container with 2% detergent for 2 minutes followed by distilled water for 2 minutes. 8.2. Press “PROC”. Different test procedures will be displayed. 8.3. Select the test to be processed by entering its number and then press “ENTER” key. 8.4. Now the assay parameters of the specific test procedure will be displayed. Note down the volume of the reagent and the sample to be used. 8.5. Feed the blank with each batch and ensure the absorbance of the blank is less than 1.000. If the absorbance of the ‘blank is more than 1.000’ or if the reagent has turned yellow, discard the reagent 8.6. Then feed the test samples and record the values. 8.7. Check whether the sample is hemolyzed, icteric or lipemic before processing. If the sample is lysed, collect another sample and proceed. If it is icteric or lipemic dilute the sample 1 in 10 with distilled water and proceed. Multiply the result displayed by dilution factor 10. Assay: Kinetic Reagent volume: 500 μL Wavelength: 405 nm Sample volume: 10 μL Temperature: 37°C Zero setting with distilled water Number of readings: 3 Time: 60 sec Light path: 1 cm Interferences: Fluoride, oxalate, citrate and EDTA inhibit alkaline phosphate activity and should not be used as anticoagulants. Hemolysis interferes due to the high concentration of alkaline phosphatase in red cells. Calculation of results: ΔA/min x 3300 = U/L ALP Biological various reference range: Children up to 1 – 14 years: <480 U/L at 30°C Adult: 73–207 U/L at 30°C

Biochemistry

65

12. Critical/Alert level values: 250 U/L 13. Laboratory interpretations: High values in obstructive jaundice (above 213–249 U/L) and moderately high in hepatocellular jaundice and, an increase in rickets, osteomalacia, hyperparathyroidism and Paget’s disease. 14. Potential sources of variability: 14.1. Lysed serum specimens may give falsely elevated values 14.2. ALP activity remains stable in serum for up to 24 hours only if the serum specimen is stored at 2–8°C. Hence if analysis is not done on the same day within 3 hours of collection, separate the serum and store it at 2–8°C 14.3. Do not use if the absorbance of the blank reagent is greater than 1.000 as it indicates deterioration of the reagent and also if the color of the reconstituted ALP reagent has turned yellow. BIBLIOGRAPHY 1. 2. 3. 4.

Burtis A, et al. Tietz Textbook of Clinical Chemistry, 3rd ed AACC 1999. Rosalki S, et al. Clin Chem 1993;39(4);648-52. Tietz N W, et al. Clinical Guide to Laboratory Tests, 3rd ed AACC 1995. Wenger C, et al. Alkaline Phosphatase, Kaplan A, et al. Clin Chem. The CV Mosby Co, St. Louis Toronto. Princeton 1984; 1094-8. 5. Young DS. Effect of Drugs on Clinical Lab Tests, 4th ed, AACC press, 1995. 6. Young DS. Effects of Disease on Clinical Lab Tests, 4th ed AACC 2001.

ACID PHOSPHATASE 1. Purpose: Quantitative estimation of acid phosphatase in serum by testing for the released phenol from the substrate disodium phenyl phosphate in acid pH. The levels are helpful in diagnosis and therapy of certain types of cancers especially metastasizing prostatic cancer. 2. Principle: Disodium phenyl phosphate in hydrolyzed by acid phosphatase at pH 5.0 with liberation of phenol and formation of sodium phosphate. The phenol thus produced reacts with 4 amino antipyrine and gives red or purple color, which could be measured spectrophotometrically. 3. Performance specifications: 3.1. Linearity: This method is linear for acid phosphatase up to 10 KA/dL of serum 3.2. Measurement range: This method has a measurement range of 1-15 KA/dL of acid phosphatase in serum.

66


4. Primary sample: 4.1. Use only serum as specimen 4.2. Collect 4 mL of venous blood in a plain red color vacutainer tube. 4.3. Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500 rpm for 10 minutes 4.4. Do not use lysed serum for testing as it may give very high results 4.5. Do not use contaminated/turbid samples for testing 4.6. Process the sample on the same day within 3 hours of collection. 4.7. If analysis is not done on the same day/within 3 hours of collection, separate the serum and store it at 2–8°C for up to 1 day. 5. Type of container and additive: For serum use a plain vacutainer tube for collecting venous sample. 6. Reagents/Consumables 6.1. Buffer: Citric acid and sodium citrate buffer. 6.2. Dissolve 4.2 g of citric acid in water add 7.6 mL of 1 N NaOH and made up to 100 mL with distilled water pH 4.9 (use sodium hydroxide for adjusting the pH) 6.3. Substrate: Dissolve 0.218 g of disodium phenyl phosphate in 100 mL – the distilled water. Bring the solution quickly to boiling to sterilize, cool and add a little chloroform. 6.4. 0.5 N sodium hydroxide: 2 g of sodium hydroxide in 100 mL of distilled water. 6.5. 0.5 M sodium bicarbonate: 42 g of sodium bicarbonate in 1 liter. Store in amber colored bottle. 6.6. 4-aminoantipyrine (AAP): 0.6% in water 6.7. Potassium ferricyanide: 2.4 g in 100 mL water 7. Instrument: Spectrophotometric 8. Procedure: Reagent Buffer (mL) Substrate (mL) Distilled water (mL)

Reagent Blank

Test Blank

3.0 3.0 — 3.0 3.0 — Incubate for 5 minutes at 37°C Serum (mL) — — Incubate for 60 minutes at 37°C 0.5 N NaOH (mL) 0.8 0.8 0.5 M NaHCO3 (mL) 1.2 1.2 4-aminoantipyrine (mL) 1.0 1.0 Serum (mL) — 0.1 Potassium ferricyanide (mL) 1.0 1.0

Read at 510 nm.

Test 3.0 3.0 — 0.1 0.8 1.2 1.0 — 1.0

Biochemistry

67

Standardization: Stock standard: 100 mg phenol in 100 mL of 0.1 N HCl Working standard: 2 mL of stock is diluted to 100 mL. 1 mL contains 20 mg. Reagents

B

S1

S2

S3

Working standard (mL) — 0.125 0.25 0.375 Distilled water (mL) 1.0 0.875 0.75 0.625 Buffer (mL) 1.1 1.1 1.1 1.1 NaOH (mL) 0.8 0.8 0.8 0.8 1.2 1.2 1.2 1.2 NaHCO3 (mL) 4AAP (mL) 1.0 1.0 1.0 1.0 Potassium ferricyanide (mL)1.0 1.0 1.0 1.0

S4

S5

0.5 0.5 1.1 0.8 1.2 1.0 1.0

0.625 0.375 1.1 0.8 1.2 1.0 1.0

S6

S7

S8

0.75 0.875 1.0 0.25 0.125 — 1.1 1.1 1.1 0.8 0.8 0.8 1.2 1.2 1.2 1.0 1.0 1.0 1.0 1.0 1.0

Read at 510 nm against blank.

9. Reference range: 1 to 5 KA units/dL. 10. Critical/Alert level values: Above 5 KA units/dL. 11. Potential sources of variability: Lysed serum specimens may give falsely elevated values. BIBLIOGRAPHY 1. Harold Varley. Practical Clinical Biochemistry, 4th ed, 1969;458-64.

ACID PHOSPHATASE (TARTRATE-LABILE) FOR PROSTATIC CANCER 1. Purpose: Qualitative estimation of acid phosphatase in serum by testing for the released phenol from the substrate disodium phenyl phosphate in acid pH. The levels are helpful in diagnosis and therapy of certain types of cancers especially metastasizing prostatic cancer in serum. In the presence of tartrate, testing at acid pH and by difference, the levels give the tartrate-labile acid phosphatase which is specific for prostate and is helpful in the diagnosis of metastasizing prostatic cancer. 2. Principle: Prostatic acid phosphatase is strongly inhibited by L (+) tartrate, which has no action on red cell acid phosphatase. 3. Primary sample: 3.1. Use only serum as specimen 3.2. Collect 4 mL of venous blood in a plain vacutainer tube. 3.3. Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500 rpm for 10 minutes 3.4. Do not use lysed serum for testing as it may give very high results. 3.5. Do not use contaminated/turbid samples for testing.


68

4. Type of container and additive: Use plain vacutainers tubes for collection blood samples. 5. Reagents/Consumables: 5.1. Reagents same as for the acid phosphatase except the buffer, which is as follows. 5.2. Tartrate buffer: Dissolve 15 g of L (+) tartaric acid in about 70 mL of water add 12.5 mL of normal sodium hydroxide and adjust pH to 4.9 makes up to 100 mL. 6. Techniques: In addition to the two tubes for test and blank used in process no. 25 (Acid Phosphatase), a third tube containing 2 mL of buffer substrate, 1 drop of the tartrate solution and 0.1 mL of serum is also incubated and put though in exactly the same way as for acid phosphatase. Calculation:

Reading of the test – Reading of test containing tartrate × 10 Reading of standard – Reading of standard blank

7. Critical/Alert level values: Below and above the reference range. 8. Reference range: 0–0.8 KA/dL 9. Potential sources of variability: Lysed serum specimens may give falsely elevated values. BIBLIOGRAPHY 1. Harold Varley. Practical Clinical Biochemistry 4th edn, 1969;458-64.

ANGIOTENSIN CONVERTING ENZYME (ACE) 1. Purpose: To measure the activity of angiotensin converting enzyme in serum. Elevated levels of ACE activity occur in serum of patients with active sarcoidosis, occasionally in premature infants with respiratory distress syndrome; in adults with tuberculosis, Gaucher’s disease, leprosy, and in many lung and liver diseases. 2. Principle: The following reaction is catalyzed by ACE: FAPGG ---------------> FAP + Glycylglycine FAPGG is hydrolyzed to furylacryloylphenylalanine (FAP) and glycylglycine. Hydrolysis of FAPGG results in a decrease in absorbance at 340 nm. The rate of decrease in absorbance is directly proportional to ACE activity in the sample. 3. Performance specifications: 3.1. Linearity: This method is linear up to 250 U/L of serum 3.2. This method has a measurement range of 8–250 IU/L 3.3. Sensitivity: The minimum detection limit by this kit is 8 IU/L

Biochemistry

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4. Primary sample: 4.1. Use only serum as specimen for the test 4.2. Collect 2 mL of venous blood in a plain vacutainer tube. 4.3. Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500 rpm for 10 min 4.4. Do not use lysed serum for testing as it may give very high results 4.5. Do not use contaminated/turbid samples for testing 4.6. Process the sample on the same day within 3 hours of collection. 4.7. If analysis is not done on the same day/within 3 hours of collection, separate the serum and store it at –20°C for up to 24 hours only. 5. Type of container and additive: Use plain tubes for collecting samples No additive/preservative is needed to be added 6. Reagents/Consumables: 6.1. Reagent I : Furyl acryloyl phenylalanyl glycyl glycine 28 mmol/L 6.2. Reagent 2: Hepes 40 mmol/L pH 8.4 Sodium chloride 0.185 mol/L Stabilizers and preservatives Stable at 2–8°C up to the expiration date on the label. Reconstitution of working reagent: Reconstitute a vial of reagent 1 with exactly 4.2 mL of reagent 2. Mix until complete solubilization. 7 Instrument: Semi-autoanalyzer—Spectrophotometer (to take absorbance at 37°C) 8. Procedure: 8.1. The temperature of the reaction mixture should be maintained at 37°C. 8.2. ACE reagent is reconstituted. 8.3. ACE reagent is reconstituted with 4.2 mL 8.4. 1.0 mL ACE reagent is pipetted into test tubes and is brought to temperature of 37°C. 8.5. 0.1 mL of serum sample is added to the test tube labeled test. 8.6. Reaction mixture is aspirated. After 1 minute read A1 the initial absorbance read at 340 nm and after exactly 5 minutes from the first reading read A2 absorbance at 340 nm. 8.7. The absorbance (A) of test and calibrator at 340 nm vs. water as blank. These are initial A1 read and exactly 5 minutes later the absorbance is read A2 absorbance. 8.8. Calculations: ACE activity (in U/L) = (A1 – A2) × 2200 or as per kit. 8.9. Final value will be calculated and displayed by the analyzer.

70

9. 10. 11.

12.

Manual of Medical Laboratory Techniques One unit of ACE activity is defined as that amount of enzyme that will catalyze formation of one micromole of FAP per minute under the conditions of assay. Interferences: ACE is a metal protein so does not use chelate in sample preparation. Turbid lipemic icteric and lysed sera will interfere. Biological reference range: 67 – 113 U/L (37°C) Critical/Alert level values: Below and above values of reference range. Laboratory interpretations: Sarcoidosis, asbestosis, silicosis, leprosy and Gaucher’s disease will have increased values. Reduced levels are found in lung cancer, tuberculosis and cystic fibrosis. Potential sources of variability: 12.1. Lysed serum specimens may give falsely elevated values 12.2. ACE activity remains stable in serum for up to one week only if the serum specimen is stored at – 20 °C hence if analysis is not done on the same day within 3 hours of collection, separate the serum and store it at – 20 °C. 12.3. Discard the vial if dry reagent exhibits caking due to possible moisture penetration, does not dissolve completely or if the solution appears turbid. 12.4. ACE activity is inhibited by EDTA and by heavy metal ions that may serve to replace the zinc ion of the enzyme. Upon administration of the angiotensin converting enzyme-inhibitory drug, captopril, currently used for treating hypertension, ACE serum activity is markedly reduced but usually returns to normal levels in about 12 hours. Administration of other such drugs may produce a similar response.

BIBLIOGRAPHY 1. Harjanne A. Clin Chem 1984;30:901.

LACTATE DEHYDROGENASE (LDH) 1. Purpose: LDH is present in almost all the tissues of the body. Its increased activity in serum reflects several pathologic states, i.e. myocardial infarction, liver disorders, pernicious anemia, megaloblastic anemia, and progressive muscular dystrophy and cancer. 2. Principle: 2.1. Lactate dehydrogenase is a hydrogen transfer enzyme that catalyzes the following reaction:

Biochemistry

71

LDH Lactic acid + NAD+ ---------------> Pyruvic acid + NADH 2.2.

3.

4.

5.

6.

The reaction is reversible but the conditions for the reverse reaction are different than those for the forward (e.g. the pH for the forward reaction is 8.8 to 9.8 and for the reverse reaction is 7.4 to 7.8). 2.3. LDH activity can be determined colorimetrically using 2,4-dinitrophenylhydrazine (2,4 DNPH) as the chromogen in alkaline medium. Pyruvic acid produced during the LDH activity, reacts with 2, 4-DNPH produces red color product which can be read at 510 nm spectrophometrically. Performance specifications: 3.1. Linearity: This method is linear up to 1000 Units/L of serum 3.2. Measurement range: This method has a measurement range of 100–200 Units/L 3.3. Sensitivity: Lower limit of detection is 100 Units/L Primary sample: 4.1. Use serum sample for the analysis. 4.2. Collect 2 mL of venous blood from a peripheral vein in a plain vaccutainer tube. 4.3. Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500 rpm for 10 minutes 4.4. Do not use hemolyzed/contaminated serum for testing Type of container and additive: 5.1. Use plain tubes for collecting samples. 5.2. Do not use hemolyzed/contaminated serum for testing Reagents/Consumables: 6.1. Preparation of substrate 6.2. Glycine buffer: Glycine–3.753 g NaCl–2.922 g Water–500 mL 6.3. 35% Sodium lactate solution: 6.4. 0.1 N NaOH 6.5. Buffered substrate: Glycine buffer – 120 mL NaOH solution – 20 mL Sodium lactate – 10 mL. Adjust the pH to 10 with 0.1 N NaOH. Store in refrigerator. 6.6. NAD+: 10 mg in 2.0 mL distilled water 6.7. Standard: 220 mg sodium pyruvate in 100 mL glycine buffer. 6.8. Working standard: Dilute 5 mL of stock to 100 mL with glycine buffer (1 μ mole pyruvate/mL). 6.9. 2,4-Dinitrophenylhydrazine: 200 mg DNPH in 1000 mL of 1 N HCl.


72

6.10. 0.4 N Sodium hydroxide 6.11. Serum: 1:5 dilution in saline. 7. Instrument: Spectrophotometer. 8. Procedure: Standardization protocol Reagents (mL)

Blank

S1

S2

S3

S4

S5

Buffered substrate

1.0

0.9

0.8

0.7

0.6

0.5

Water

0.3

0.3

0.3

0.3

0.3

0.3

Std Sodium pyruvate

—

0.1

0.2

0.3

0.4

0.5

Conc. U/L

—

333

666

999

1998

3996

1.0

1.0

1.0

1.0

10

10

10

10

Incubate at 37° C for 5 min 2,4-DNPH

1.0

1.0

Incubate at 37° C for 15 min 0.4N NaOH

10

10

Incubate at RT for 10 min OD at 510 nm Reagent

Blank

Control

Test

Buffered substrate (mL)

—

1.0 mL

1.0 mL

NAD+

—

0.2

0.2

—

0.1 1.0

(mL)

Incubate at 37 1.5 Diluted serum (mL)

oC

for 5 minutes

— Keep at 37°C for 15 minutes

DNPH (mL)

1.0

1.0

Diluted serum (mL)

—

0.1

Keep at 37°C for 15 minutes 0.4 N NaOH (mL)

10.0 10.0 Room temperature for 10 minutes

10.0

Read at 510 nm against water.

Calculation: (At – Ab) 1 1000 × std. conc. × × As 15 0.02 Note: At – Test absorbance, Ab- Blank absorbance, As- std absorbance 1/15 = per 15 minutes 1000/0.02 = conversion of aliquot of specimen taken to 1 liter 9. Reference range: Adults: 70 to 240 U/L; Children: 150 to 590 U/L 10. Potential sources of variability: 10.1. Use of only clear, unhemolyzed serum separated from the erythrocytes as soon as possible. Lysed serum specimens may give falsely elevated values

LDH activity U/L =

Biochemistry

73

10.2. On storage, the working reagent may develop a turbid, which makes the use of reagent blanks necessary with every run. 10.3. Repeated freezing and thawing is harmful for the enzyme. BIBLIOGRAPHY 1. Kanai L Mukherjee. Medical Laboratory Technology 1988; Vol III: 1051-54.

GLUCOSE-6-PHOSPHATE-DEHYDROGENASE 1. Purpose: Glucose-6-phosphate-dehydrogenase (G6PD) deficiency is one of the most common human enzyme deficiencies, in the world. During G6PD deficiency, the red cells are unable to regenerate reduced Nicotinamide adenine dinucleotide phosphate (NADPH) a reaction that is normally catalyzed by the G6PD enzyme. Since the X chromosome carries the gene for G6PD enzyme, this deficiency mostly affects the males as females are protected by the other normal X chromosome. The two major conditions associated with G6PD deficiency are hemolytic anemias and neonatal jaundice, which may result in neurological complications and death. Screening and detection of G6PD deficiency helps in reducing such episodes, through appropriate selection of treatment, patient counseling and abstinence from disease-precipitating drugs such as anti-malarials like primaquine and other agents and favism. 2. Principle: G6PD in RBCs is released by a lysing agent present in the reagent. The G6PD released catalyzes the oxidation of Glucose-6 phosphate with the reduction of NADP+ to NADPH. The rate reduction of NADP+ to NADPH is measured as an increase in absorbance, which is proportional to the G6PD activity in the sample. G6PD G6P + NADP + -------------------------> 6-Phosphogluconic acid +NADPH + H+ 3. Performance specifications: Linearity: This method is linear for up to 20 Unit/g of Hb. 4. Primary sample: 4.1. Use only whole blood as specimen 4.2. Collect 2 mL of venous blood in a vacutainer EDTA anticoagulant tube. 4.3. Do not use lysed blood for testing as it may give very high results 4.4. Do not use contaminated/turbid samples for testing. 4.5. Sample materials: Fresh whole blood sample collected in EDTA, heparin or ACD, red cell G6PD in whole blood is reported to be stable for 7 days at 2–8°C, but is unstable in hemolysates freezing is not recommended.

74


5. Type of container and additive: Add 2.0 mL of blood in an EDTA vacutainer tube and shake well for 30 sec. 6. Reagents/Consumables: L1: G6PDH reagent 5 × 1 mL L2: Starter reagent 10 mL Reagent preparation: Make up G6PD reagent (L1) with distilled water as per the volume mentioned on the label. This working reagent is stable for 6 hours at RT and at least 5 days when store at 2–8°C. The starter reagent (L2) is ready to use. Add 1.0 mL of blood to the EDTA added tube and shake well for 30 sec. 7. Instrument: Semi-autoanalyzer Chema. 8. Procedure: 8.1. Wavelength: 340 nm Temperature: 37°C Light path: 1 cm 8.2. Pipette into a clean dry test tube labeled test (T) 8.3. Addition sequence T (mL) G6PD working reagent (L1) 1.0 Whole blood 0.01 Mix well and incubate for 5–10 min at RT and add Starter reagent 2.0 8.4. Mix well and incubate for 5 min at 37°C and read the initial absorbance A0 and repeat the absorbance reading after every 1, 2 and 3 minutes. 8.5. Calculate the mean absorbance change per minute (ΔA/min) If the G6PD activity is very low the absorbance change per minute will also be very low. In such cases, read the initial absorbance A1 and read another absorbance A2 exactly 5 min later. Calculate the mean absorbance change per min. A – A1 ΔA/min = 2 5 9. Reference range: G6PD Activity (U/g Hb) : 6.4 to 18.7 at 37°C 10. Critical/Alert values: Not applicable 11. Safety precautions: 11.1 Handle all samples as potentially infectious 11.2. Handle all reagents with care and avoid contact with eye, mouth and skin 11.3. Do not perform mouth pipette 11.4. Discard used reagents and sample as per disposal procedure

Biochemistry

75

12. Potential sources of variability: Lysed blood specimens may give falsely elevated values.

AMYLASE 1. Purpose: Quantitative estimation of the activity of amylase in human serum. The clinical significance of the estimation of amylase lies almost entirely in the diagnosis of acute pancreatitis, in which the enzyme level frequently exceeds more than 10 times the normal values. Some other causes include salivary gland disorder, abdominal disturbances affecting the pancreas and intake of drugs. 2. Principle: Amylase is a hydrolytic enzyme that splits complex carbohydrates such as starch and glycogen to glucose. Amylase

Starch -----------------> glucose The iodometric method is based on the ability of iodine to form a vivid blue color in combination with starch. The byproduct of amylase action may also form colored substances with iodine but at different wavelengths from the characteristic starch-iodine complex. In this method, iodine color reagent is added to the substrate-sample mixture after an incubation period. The greater the amount of amylase activity, the lighter will be the color. 3. Performance specifications: 3.1. Linearity: This method is linear for amylase activity up to 300 Somogyi units/dL in serum. 3.2. Measurement range: This method has a measurement range of 50–300 Somogyi units/dL of amylase activity in serum. 3.3. Sensitivity: The minimum detection limit of amylase in serum by this method is 50 Somogyi units/dL. 3.4. The amylase of serum is activated by chloride ions, so the dilutions of serum must be made in physiological saline. 4. Primary sample: 4.1. Collect 4 mL of blood in a plain vacutainer tube for blood collection. 4.2. Do not use lysed serum for testing as it may give very high results 4.3. Do not use contaminated/turbid samples for testing 4.4. Process the sample on the same day within 3 hours of collection or store at –20 °C for one week. 5. Type of container and additive: Use plain vacutainer tube for blood collection.

76


6. Reagents/Consumables: 6.1. Phosphate buffer: Na2HPO4 - 1.735 g - 1.009 g (for 1 liter) KH2PO4 Mix 84 mL of A and 16 mL of B solution (pH 7.4) 6.2. Starch solution: 5 g/liter 6.3. Prepare the buffer and starch solution fresh every time a sample is processed or at intervals of not more than 2 to 3 days. 6.4. Buffered substrate: 0.4 mg/mL. Weigh 20 mg starch in 50 mL of phosphate buffer. 6.5. Iodine reagent stock: Dissolve 30 g of potassium iodide with 250 mL water, weigh 13 g iodine in a closed container and transferred quantitatively to a liter volumetric flask with the iodide solution. Shake well to dissolve and make to the mark with water. Standardize against thiosulphate. The iodine concentration should be 47.5 to 52.5 mmole/liter. Adjust if necessary. 6.6. Working iodine standard solution: Prepare from reagent 3 by diluting 1 to 10 with water. 6.7. Sodium chloride solution: 0.9 g in 100 mL water. 7. Instrument: DU- 640 Beckman spectrophotometer. 8. Procedure: Dilute the serum/plasma 1: 10 with saline

Buffered substrate (mL) Incubate 37°C for 5 minutes Serum (1:10) (mL) Incubate at 37°C for 15 minutes Iodine, working standard (mL) Water (mL)

Test

Control

1.0

1.0

0.1

—

0.4 8.5

0.4 8.6

Then measure the color at 660 nm using water as a blank.

9. Calculation: (Absorbance of control – Absorbance of test) × 800 Absorbance of control 1 Somogyi unit of amylase activity = 5 mg starch hydrolyzed under aforesaid conditions (Enzymatic reaction for 15 minutes at 37°C at pH 7.4). Amount of starch present in the reaction mixture = 0.4 mg

Amylase activity =

0.4 = amylase units 5 Enzyme activity factor (amylase activity/dL serum)

Biochemistry

77

0.4 100 × × 10* 5 0.1 *Dilution factor =

10. Reference range: 60–180 Somogyi units/dL (or) 95 –290 IU//L 11. Critical/Alert level value: Not applicable 12. Potential sources of variability: 12.1. Lysed serum specimens may give falsely elevated values 12.2. Amylase activity remains stable in serum for up to one week, on refrigeration for two months 12.3. With the exception of heparin, all common anticoagulants inhibit amylase activity because they chelate Ca (II) Citrate, EDTA, and oxalate inhibit it by 15% 12.4. The amylase of serum is activated by chloride ions, so the dilutions of serum must be made in physiological saline 12.5. Misleading increases in the activities of amylase and pancreatic amylase in the serum of a patient with macroamylasemia. BIBLIOGRAPHY 1. Kanai L Mukherjee. Medical Laboratory Technology Iodometric Method 1988;Vol III:1037-9. 2. Method of Huggins and Ruisel. Practical Clinical Biochemistry by Harold Varley 1980;1080.

ORNITHINE AMINOTRANSFERASE (OAT) 1. Purpose: To determine ornithine aminotransferase enzyme activity in cultured lymphocytes. 2. Principle: OAT is a pyridoxal phosphate requiring mitochondrial transaminase that catalyzes the reversible interconversion of ornithine and alpha ketoglutarate to pyrolline-5-carboxylate and glutamate. The pyrolline-5-carboxylate released is estimated using ortho amino benzaldehyde by the method of Katsunuma et al using ornithine as substrate. 3. Primary sample: 3.1. Use only heparinized blood to culture lymphocytes. 3.2. Collect 4 mL of venous blood in sterile heparin vacutainer tube. 3.3. The sample should be processed the same day. 4. Type of container and additives: Collect 4 mL of heparin vacutainer tube collecting sample.

78


5. Reagents/Consumables: Reagents for lymphocyte culture: 5.1. Lymphoprep—commercially available from sigma 5.2. Phosphate buffered saline (PBS) - pH 7.2–7.4 Disodium hydrogen phosphate (Na2HPO4) - 14.8 g Potassium dihydrogen phosphate (KH2PO4) - 4.7 Sodium chloride (NaCl) - 68.0 g Distilled water - 1000 mL 5.3. F12 medium with fetal calf serum (FCS)—To get from tissue culture lab. 5.4. Phytohemagglutinin (PHA)—commercially available 5.5. Tris—HCl Buffer (pH 8.0) 121 g/L 5.6. Ornithine 20 mM (mol wt –132) 26 mg/10 mL 5.7. α-ketoglutarate (α-KG) 10 mM (mol wt – 146) 14 mg/10 mL 5.8. Pyridoxal phosphate 10 mM (mol wt – 247) 24 mg/10 mL 5.9. 10% TCA. 5.10. Ortho aminobenzaldehyde (OAB) 10 mg/1 mL methanol 5.11. Pyroll 5 carboxylic acid (mol wt – 377) 6. Instrument: Spectrophotometer. 7. Procedure: Lymphocyte separation - Perform using sterile things 7.1. Mix 4 mL of heparin blood with equal volume of working PBS. 7.2. To 10 mL of lymphoprep overlay 10 mL of the blood diluted with PBS. 7.3. Centrifuge at 3000 rpm for 30 minutes. 7.4. Carefully discard the supernatant. 7.5. Remove the lymphocyte layer and add to a sterile vial or bottle. 7.6. Add equal volume of F12 with FCS to the lymphocyte separated and add 10 μL of PHA. 7.7. Keep for 72 hours at 37°C in CO2 incubator. 7.8. After 72 hrs, centrifuge the cells and remove the supernatant. 7.9. Wash the cells in PBS twice. 7.10. Re-suspend the cells in 1.5 mL in PBS and sonicate the culture at 60-kilo cycles for 10 sec. 7.11. Perform protein estimation by Lowry method. 7.12. Use 0.5 mL for OAT assay.

Biochemistry Sl. No. Reagents 1. 2. 3. 4. 5.

Tris HCl (mL) pH 8.0 Ornithine (mL) α-KG (mL) Pyridoxal (mL) Enzyme (mL)

79

Blank

T with B6

T without B6

3.5 — — — —

1.0 1.0 1.0 10 μL 0.5

1.0 1.0 1.0 — 0.5

Incubate at 37°C for 30 minutes 6. 7.

10% TCA (mL) OAB (mL)

1.0 0.1

1.0 0.1

1.0 0.1

Keep at boiling water bath for 5 minutes cool and read at 440 nm

8. Reference range: Compared with the control and interpreted. 9. Potential sources of variability: Lysed blood specimens should not be used for the separation. 10. 3.7 mg Pyroll 5 carboxylic acid/100 ml = 100 μM from this stock plot a standard graph from 10 to 50 nM. 0.5 mL of lymphocyte is used for the assay. Therefore, for 100 mL = 100 × X/0.5 The value obtained is for 1/2 hour so multiply by 2 for 1 hour this gives the unit activity For specific gravity = Unit activity/mg of protein. BIBLIOGRAPHY 1. Berger SL. Methods in enzymology, New York, USA: Acad Press, 1979,58;48694. 2 Katsunuma N, Matsuda Y, Tomino I: Studies on ornithine-ketoacid transaminase: I purification and properties. J Biochem 1964;56:499-503.

GLUTATHIONE PEROXIDASE (GPx) 1. Purpose: Glutathione peroxidase (GPx) is a selenium dependent selenoprotein, an antioxidant enzyme present in our system. During oxidative stress conditions, this enzyme reacts with the oxidant species and reduces their effect. It is used to know the antioxidant status of the patient. 2. Principle: Glutathione peroxidase, reacts with H2O2 in the presence of GSH and converts it into H2O and thus functions as an antioxidant. GPx 2H2O2 ---------------> 2H2O +O2 GSH

GSSG

80 3.

4.

5. 6.

Manual of Medical Laboratory Techniques The enzyme activity is expressed as μ moles of glutathione utilized minutes/gm hemoglobin. Performance specifications: 3.1. Linearity: This method is linear for GPx concentrations up to 60 μ mole glutathione utilized/g Hb. 3.2. Measurement range: This method has a measurement range of 5.0-60 μ mole glutathione utilized/g Hb. 3.3. Sensitivity: The minimum detection limit by this method is 5.0 μ mole glutathione utilized/g Hb. Primary sample: 4.1. Use only EDTA red blood cells as specimen for the test 4.2. Collect 2.0 mL of venous blood in EDTA vacutainer tube. 4.3. Do not use hemolyzed sample. 4.4. Process the sample immediately and if not done, store the RBC at 4°C until processed, for a maximum of 24 hours. Type of container and additive: Collect 2 mL of EDTA vacutainer tube for blood collection. Reagents/Consumables: 6.1. Sodium phosphate buffer, 0.32 M, pH 7.0: Na2HPO4 - 4.23 g in 100 mL NaH2PO4 2H2O-4.1 g in 100 mL 6.2. EDTA (0.8 mM) : 30 mg in 100 mL. 6.3. Sodium azide (2.0 mM) : 12.2 mg in 100 mL. 6.4. Test glutathione (4 mM) : 12.2 mg in 10 mL. 6.5. Hydrogen peroxide (2.5 mM) : 1:100 dilution again 3 mL made up to 100 mL. 6.6. Trichloroacetic acid (10%) : 10 g in 100 mL. 6.7. Phosphate solution (0.3 M) : 5.34 g - disodium hydrogen phosphate 100 mL - distilled water. Crystals developed during storage at 4°C are dissolved by heating. 6.8. DTNB reagent: 20 mg 5,5'-dithiobis-(2 nitrobenzoic) acid in 100 mL of one percent sodium citrate solution. This solution is stable for 10 weeks at 4°C. Instrument: Spectrophotometer Procedure: 8.1. 1.0 mL of EDTA blood is taken and the erythrocytes were washed thrice with saline after removal of plasma.

}

7. 8.

Biochemistry 8.2.

8.3.

81

The buffy coats along with the upper layer of erythrocytes are discarded with each saline wash to remove leukocytes. Add 1.0 mL of hemolysine solution. The estimation is done as follows:

Reagents

Blank

Test

Control

S1

S2

Standards S3

S4

S5

—

—

Buffer (μL)

400

400

400

400

400

400

400

400

Sodium azide (μL)

200

200

200

200

200

200

200

200

EDTA (μL)

200

200

200

200

200

200

200

200

H2O2 (μL)

200

200

200

200

200

200

200

200

Standard (μL)

—

25

50

75

100

125

—

—

Concentration (μg)

—

5

10

15

20

25

—

—

Test GSH solution (μL) —

—

—

—

—

—

200

200

10% TCA (mL)

—

—

—

—

—

—

—

1.0

Hemolysate (mL)

—

—

—

—

—

—

0.1

0.1

Distilled water (mL)

0.7

0.675 0.65

0.575 0.7

0.7

0.625 0.600

Keep at room temperature for 10 min 10%TCA (mL)

— — — — — — Centrifuge and take the supernatant

1.0

—

Phosphate solution (mL)

8

8

8

8

8

8

8

8

DTNB (mL)

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

Read the absorbance at 412 nm

Calculation: TOD – COD × Std. Conc. × 20,000 = μ mole glutathione utilized/g Hb SOD × 307 × 10 × Hb 9. Reference range: 27.6–48.4 μ mole glutathione utilized/g Hb 10. Critical/Alert values: Not applicable 11. Safety precautions: 11.1. Handle all samples as potentially infectious 11.2. Handle all reagents with care and avoid contact with eye, mouth and skin 11.3. Ensure the reagents and specimens are at room temperature before use. 11.4. Do not perform mouth pipetting 11.5. Discard used reagents and sample as per disposal procedure. 12. Potential sources of variability: 12.1. Below and above the reference range.

82

Manual of Medical Laboratory Techniques 12.2. Lysed RBC samples may give falsely decreased values as the enzymes are released from the erythrocytes. 12.3. Hemolysate has to be preserved in –20oC if the assay is not being performed immediately.

BIBLIOGRAPHY 1. Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG. Selenium: Biochemical role as a component of glutathione peroxidase purification and assay. Science 1973;179:558-90.

SUPEROXIDE DISMUTASE (SOD) 1. Purpose: Quantitative estimation of Superoxide Dismutase. Measurement of SOD is useful in the diagnosis of antioxidant status. 2. Principle: Superoxide anion, O2– ., is an intermediate in the autoxidation of epinephrine. The ability of super oxide dismutase to inhibit the autoxidation of epinephrine at pH 9.8 provides the basis of the assay for the enzyme. SOD catalyzes the following reaction: 3.

4.

5. 6.

7.

SOD O2– · + O2– · + 2 H+ ---------------> O2 + H2O2 Performance specifications: 3.1. Linearity: This method is linear for up to 6000 U/g Hb 3.2. Measurement range: This method has a measurement range of 500–6000 U/g Hb of SOD activity in RBC. 3.3. Sensitivity: The minimum detection limit is 500 U/g Hb Primary sample: 4.1. Use only heparin vacutainer tube for blood collection 4.2. Do not use hemolyzed sample. 4.3. Process the sample immediately and if not done store the RBC at 4°C until processed, for a maximum of 24 hours. Type of container and additive: Collect 4 mL of heparin tube for blood collection. Reagents/Consumables: 6.1. 50 mM of Carbonate-bicarbonate buffer, pH 9.8 Na2 CO3-529 mg; NaHCO3 – 420 mg; EDTA-1 mg made up to 100 mL with distilled water. 6.2. Epinephrine: 1.8 mM solution, prepared freshly - 1 mg in 1 mL, dissolved by adding dilute HCl. 6.3. Absolute ethanol. 6.4. Chloroform. Instrument: Spectrophotometer (Kinetic assay mode)

Biochemistry

83

8. Procedure: 8.1. Preparation of the Enzyme: The extraction of the enzyme is carried out according to the method of Bartosz et al (1978). 8.2. Take 1 mL of heparinized blood. Wash with saline three times after removing the plasma. 8.3. The buffy coats along with the upper layer of erythrocytes are discarded with each saline wash to remove leukocytes. 8.4. Add 1 mL of cold distilled water to the remaining RBCs. Cyclomix well to get the hemolysate. 8.5. To the 0.5 mL of the hemolysate add 3.5 mL cold distilled water. 8.6. Following this, add 1.0 mL of chilled ethanol and 0.6 mL of icecold chloroform. 8.7. Shake the mixture well for a few minutes at 4°C and then centrifuge at 5000 rpm for 15 minutes 8.8. Take the supernatant for the enzyme assay as follows: 8.9. Assay: Water (mL) Carbonate buffer (mL) Sample (mL) Epinephrine (mL)

Blank

Control

Test

1.2 1.8 — —

0.8 1.8 — 0.4

0.75 1.8 0.05 0.4

8.10. As soon as the epinephrine is added, immediately the increase in absorbance at 480 nm is measured in a spectrophotometer by kinetic assay for interval of 15 seconds and total of 240 seconds. 8.11. Autoxidation of epinephrine to adrenochrome is performed in a control tube without the enzyme. One unit of enzyme activity is defined as the quantity of enzyme required to produce 50% inhibition in epinephrine autoxidation. Calculation: T – (165 seconds – 105 seconds) C – (165 seconds – 105 seconds) C–T × 45000 = Units/min/g Hb C/2 9. Reference range: 2600–4963 Units/min/g Hb. 10. Safety precautions: 10.1. Handle all samples as potentially infectious 10.2. Handle all reagents with care and avoid contact with eye, mouth and skin 10.3. Ensure the reagents and specimens are at room temperature before use 10.4. Do not perform mouth pipetting 10.5. Discard used reagents and sample as per disposal procedure

84


11. Potential sources of variability: 11.1. Lysed RBC samples may give falsely decreased values as the enzymes are released from the erythrocytes. 11.2. Do not use if the lysate has been stored in the fridge even for 24 hours. 11.3. Hemolysate has to be preserved in –20oC if the assay is not been performed. BIBLIOGRAPHY 1. Misra HP and Ferdovich IC. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 1972; 243:3170-75.

Biochemistry

85

Part III: Electrophoresis ELECTROPHORESIS OF SERUM PROTEINS 1. Purpose: By the technique of electrophoresis serum proteins are separated into individual proteins, viz. albumin, α 1 globulin, α2 globulin, β globulin and γ globulin. They are compared with the pattern of proteins of normal serum. The electrophorogram helps in diagnosis, of diseases like nephrotic syndrome, systemic lupus erythematosis, lymphogranuloma venerum, cirrhosis of liver, multiple myeloma, and hypoalbuminemia, etc. The pattern is significant in chronic infections in which A/G ratio is reversed. 2. Principle: Electrophoresis is the movement of the charged particles in an applied electric field. The migration in an electric field is influenced by the size, shape, charge and chemical composition of the molecule. The technique uses a buffer-saturated gel type matrix as a support medium. The sample to be analyzed is applied to the medium as a spot or thin band, and the proteins are separated by well-defined zones; hence the term, “zone” electrophoresis. 3. Performance specifications: Sensitivity: The minimum detection limit is 50 mg 4. Primary sample: Use only serum as specimen Collect 4 mL of venous blood in a plain vacutainer tube. Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 3000 rpm for 5 minutes. Do not use lysed serum for testing as it may give very high results. Do not use contaminated/turbid samples for testing. Process the sample on the same day within 3 hours of collection. If analysis is not done on the same day/within 3 hours of collection, separate the serum and store it at –20°C for up to 7 days or at 4–8 °C for up to 3 days. 5. Type of container and additive: Plain vacutainer tube for collecting venous sample. No additive/preservative is needed to be added. 6. Reagents/Consumables: 6.1. Glass slide 6.2. Pipettes and beakers 6.3. 1% Agarose in barbitone (barbiturate) buffer. 6.4. Barbitone buffer (pH 8.6): 10.3 g—Sodium diethyl barbiturate, 1.84 g—Barbitone Dissolved in 1000 mL distilled water. 6.5. Staining reagent: 0.25% Coomassie brilliant blue R250 in methanol, glacial acetic acid, water in the ratio of 45: 5: 50. (0.25 g in 45 mL methanol, 5 mL glacial acetic acid, 50 mL water).


86 6.6.

7. 8.

9. 10.

11.

Destaining reagent: Methanol: Glacial acetic acid: Water in the ratio of 50: 7: 43. 6.7. Serum sample. 6.8. Tracking dye: 0.1% Bromophenol blue. 6.9. Methanol. Instrument: Power pack, and electrophoresis tank Procedure: 8.1. Place the slides on an even surface. 8.2. Layer about 2.5 mL of molten agarose on the slide. 8.3. Allow to cool undisturbed, to form a flat surface. 8.4. Using the coverslip apply the sample ½ inch from the bottom of the slide. 8.5. Place the slides in the electrophoresis tank. 8.6. Fill the tank with barbitone buffer to make the connection between the electrodes using paper wicks. 8.7. Apply 200 voltage until the tracking dye reaches the other end of the slide. 8.8. Switch off the voltage and fix the slides in methanol for 30 minutes. 8.9. Then dry the slides at 60°C until the gel forms a thin film. Stain the slide and identify the bands. Interpretation of results: Compare with control serum proteins profile. Abnormal bands are interpreted in disease conditions. Safety precautions: 10.1. Handle all samples as potentially infectious. 10.2. Handle all reagents with care and avoid contact with eye, mouth and skin. 10.3. Do not perform mouth pipette. 10.4. Discard used reagents and sample as hayadarous electrophonetic waste per disposal procedure. Potential sources of variability: Lysed serum specimens may give falsely elevated bands.

BIBLIOGRAPHY 1. John D Bauer. Clinical Laboratory Methods, 9th edn, 1982;554. 2. Harold Varley. Practical Clinical Biochemistry, 4th edn, 1969;68.

ELECTROPHORESIS OF SERUM LIPOPROTEINS 1. Purpose: By the technique of electrophoresis, serum lipoproteins from patients are separated into individual lipoproteins viz. chylomicrons, β, pre β and α fractions. Lipoproteins are stained by Oil Red O.

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3.

4.

5.

6.

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The analysis helps to find out the type of lipoprotein abnormality according to Frederickson’s classification of familial hyperlipoproteinemias and in acquired diseases like alcoholism, kidney diseases, etc. Principle: Electrophoresis is the movement of the charged particles in an applied electric field. The migration in an electric field is influenced by the size, shape, charge and chemical composition of the molecule. The technique uses a buffer-saturated gel type matrix as a support medium. The sample to be analyzed is applied to the medium as a spot or thin band, and the lipoproteins are separated by well-defined bands, hence the term, “zone” electrophoresis. Performance specifications: 3.1 Linearity: This method is linear for up to 500 μg lipoprotein. 3.2 Measurement range: This method is qualitative. 3.3 Sensitivity: The minimum detection limit depends on the staining technique. Primary sample 4.1. Use only serum as specimen 4.2. Collect 2 mL of venous blood in a plain vacutainer tube. 4.3. Allow the tube to stand for 30 min and separate the serum by centrifugation at 3000 rpm for 5 min. 4.4. Do not use lysed serum for testing as it may give very high results 4.5. Do not use contaminated/turbid samples for testing 4.6. If analysis is not done on the same day/within 3 hours of collection, separate the serum and store it at – 20 °C for up to 7 days or at 4–8 °C for up to 3 days. Type of container and additive: Use a plain vacutainer tube for collecting venous sample. No additive/preservative is needed to be added Reagents/Consumables 6.1. Glass slides 6.2. Pipettes and beakers 6.3. Paper wick 6.4. Electrophoresis tank 6.5. 1% agarose in barbitone (barbiturate) buffer 6.6. Barbitone buffer (pH 8.6) 10.3 g sodium diethyl barbiturate, 1.84 g barbitone Dissolved in 1000 mL distilled water 6.7. Staining reagent: Oil Red O – 0.4 g Methanol – 70 mL, Water – 30 mL. Boil till it gets dissolved. Cool and store at 37°C. Use it without filtering. 6.8. Destaining reagent: Concentrated sodium hypochlorite 5 mL is mixed with 100 mL of 5% acetic acid.

88

7. 8.

9. 10.

11.

Manual of Medical Laboratory Techniques 6.9. Serum sample. 6.10. Tracking dye: 0.1% Bromophenol blue. 6.11. Methanol. Instrument: Power pack and electrophoresis tank. Procedure: Place the slides on an even surface. 8.1. Layer about 2.5 mL of molten agarose on the slide. 8.2. Allow cooling undisturbed to form a flat surface. 8.3. Using the coverslip apply the sample mixed with tracking dye ½ inch from the bottom of the slide. 8.4. Place the slides in the electrophoresis tank. 8.5. Fill the tank with barbitone buffer, make the connection between the electrodes using paper wicks. 8.6. Apply 200 voltage until the tracking dye reaches the other end of the slide. 8.7. Switch off the voltage and fix the slides in methanol for 30 minutes. 8.8. Then dry the slides at 60°C until the gel forms a thin film. Stain the slide and identify the bands. Reference range: Compare with normal control serum lipid profile. Altered intensities are seen in diseases. Safety precautions: 10.1. Handle all samples as potentially infectious 10.2. Handle all reagents with care and avoid contact with eye, mouth and skin 10.3. Do not perform mouth pipette 10.4. Discard used reagents and sample as per disposal procedure. Potential sources of variability: Lysed serum specimens may give falsely elevated bands.

BIBLIOGRAPHY 1. John D Bauer. Clinical Laboratory Methods, 9th edn, 1982;554.

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Part IV: Chromatography SUGARS 1. Purpose: By the technique of chromatography chemically closely similar substance can be separated using the differences in their partitioning between two partially miscible liquids like butanol and water. They will have different Rf values. Using the Rf values, they can be identified. Standards can also be used for identification. The work is useful in diagnosis of diseases like hyper phenyl alaninemia, tyrosinosis, cystinuria, galactosemia, pentosuria, gyrate atrophy of the choroid and retina, etc. Chromatography of sugars helps in diagnosis of galactosemia, fructosuria, pentosuria, lactosuria, etc. 2. Principle: Chromatography has two phases—a mobile phase and a stationary phase. The mobile phase may be gas or liquid. The sample, which contains one or more components, comes into contact with the mobile phase. The components distribute themselves between the mobile and stationary phases. The components have different partition coefficient in the organic and inorganic solvents which are partially miscible. If the components are preferentially bound by the stationary phase, they spend more time in the stationary phase and are retarded in their movement. Molecules that show weak affinity for the stationary phase spend more time with the mobile phase and move rapidly. The different affinities of the molecules for the stationary and mobile phases bring about the separation of molecules. Rf =

Distance moved by the solute Distance moved by the solvent

3. Performance specifications: Sensitivity: Lower limit of detection is in microgram. 4. Primary sample: 4.1. Collect 5.0 mL of urine specimen in case urine is specified. 4.2. Collect 2 mL of blood sample in a heparin vacutainer tube in case blood sample is specified. 4.3. If the urine specimen is not analyzed within 3 hours, it should be stored at 4–8°C and –20°C in the case of plasma samples. 5. Type of container and additive: 5.1. Use plain urine container with a pinch of sodium azide. 5.2. Add 20 μL of heparin and to that add 2.0 mL of blood sample. 6. Reagents/Consumables: 6.1. Sugar standard: 1 mg/mL Mobile phase solvent: Pyridine: Isoamyl alcohol : water (10: 10 : 7)


90 6.2.

7. 8.

9. 10.

11.

Staining reagent (Aniline Hydrogen Oxalate): Redistilled aniline – 0.95 mL 0.1 M oxalic acid – 100 mL (0.1 M oxalic acid – 1.26 g/100 mL of water.) 6.3. Whatman No 1 filter paper 6.4. Chromatography tank 6.5. Capillary tubes Instrument: Chromatography tank. Procedure: 8.1. A straight strip of Whatman no. 1 filter paper is cut and a line is drawn an inch above the lower end of the paper. A dot is marked at the center of the line and encircled for sample application. 8.2. Two to three drops of the sugar samples are placed on the circled mark with intermittent drying, using a capillary tube. 8.3. The paper is then stuck to the inner surface of the lid of the tank containing the mobile phase, such that the paper just touches the solvent. 8.4. After allowing the mobile phase to move for 12 hours, it is removed from the tank and hanged outside for drying. 8.5. After complete drying, the paper is stained with aniline hydrogen oxalate using a pipette from top to bottom, horizontally. 8.6. After staining, the paper is again dried completely and then, is kept in the hot air oven at 90°C for a few seconds, to visualize the spot, and the Rf is calculated for the given sugar sample. Interpretation of results: The spot obtained for the plasma/urine sample will be compared with the standard solutions. Safety precautions: 10.1. Handle all samples as potentially infectious 10.2. Handle all reagents with care and avoid contact with eye, mouth and skin 10.3. Do not perform mouth pipetting 10.4. Discard used reagents and sample as per disposal procedure Potential sources of variability: 11.1. Urine with bacterial contamination should not be used. 11.2. Lysed plasma should not be used.

Reference textbook on which the procedure is based 1. Harold Varley. Practical Clinical Biochemistry 1980;5(1):494-96.

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AMINO ACIDS 1. Purpose: By the technique of chromatography chemically closely similar substances can be separated using the differences in their partitioning between two partially miscible liquids like butanol and water. They will have different Rf values. Using the Rf values they can be identified, standards can also be used for identification. The work is useful in diagnosis of diseases like hyperphenylalaninemia, tyrosinosis, cystinuria, gyrate atrophy of the choroid and retina. 2. Principle: Chromatography has two phases—a mobile phase and a stationary phase. The mobile phase may be gas or liquid. The sample, which contains one or more components, comes into contact with the mobile phase. The components distribute themselves between the mobile and stationary phases according to their partition coefficients. If the components are preferentially bound by the stationary phase, they spend more time in the stationary phase and are retarded in their movement. Molecules that show weak affinity for the stationary phase spend more time with the mobile phase and move rapidly. The different affinities of the molecules for the stationary and mobile phases bring about the separation of molecules. Rf =

Distance moved by the solute Distance moved by the solvent front

3. Performance specifications: Sensitivity: Lower limit of detection is in micrograms (say 20-50 mg). 4. Primary sample: Blood should be collected in fasting condition. It should be centrifuged at 2500 rpm and the plasma sample is separated for analysis if not processed immediately stored at –20°C for 1 week. 5. Type of container and additive: The blood should be collected in tube containing acid citrate dextrose (ACD) 0.3 mL of ACD/2 mL of blood. 6. Reagents consumables: 6.1. Mobile phase solvent: Butanol : Acetic acid: Water 4:1:1 6.2. Staining reagent (Ninhydrin): 0.1% ninhydrin in acetone. 6.3. Amino acid standard: 1 mg/mL 6.4. Acid citrate dextrose (ACD): Trisodium citrate – 22 g Citric acid – 8 g Dextrose – 25 g Water – 1000 mL 300 mL of ACD for 2.0 mL blood. 6.5. Whatman No 1 filter paper 6.6. Chromatography tank 6.7. Capillary tubes

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7. Instrument: Chromatography tank. 8. Procedure: 8.1. A straight strip of Whatman no 1 filter paper (Merck) is cut to required breadth and a line is drawn an inch above the lower end of the paper. A dot is marked at the center of the line and encircled for sample application. 8.2. Two to three drops of the amino acid samples are placed on the circle marked with intermittent drying, using a capillary tube. 8.3. The paper is then stuck to the inner surface of the lid of the tank containing the mobile phase, such that the paper just touches the solvent. 8.4. After allowing the mobile phase to move for 12 hours, it is removed from the tank and hanged outside for drying. 8.5. After complete drying, the paper is tied in 90° to the previous run and again run in a different solvent. It was dried and then stained with ninhydrin. 8.6. After staining, the paper is again dried completely and then, is kept in the hot air oven at 90°C for a few seconds, to visualize the spot, and the Rf is calculated for the given amino acid sample. 9. Reference range: The spot obtained for the plasma/urine sample will be compared with the standard solutions. 10. Safety precautions: 10.1. Handle all samples as potentially infectious 10.2. Handle all reagents with care and avoid contact with eye, mouth and skin 10.3. Do not perform mouth pipette. 10.4. Discard used reagents and sample as per disposal procedure 11. Potential sources of variability: Lysed plasma specimens may give falsely elevated levels. BIBLIOGRAPHY Harold Varley. Practical Clinical Biochemistry 1980;5(1):494-96.

2D CHROMATOGRAPHY 1. Purpose: The technique of chromatography chemically closely similar substances like amino acids, sugar, etc. can be separated using the differences in their partitioning between two partially miscible liquids like butanol and water. They will have different Rf values. Using the Rf values they can be identified. Standards can also be used for identification. The work is useful in diagnosis of diseases like

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3. 4.

5.

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hyperphenylalaninemia, tyrosinosis, cystinuria, galactosemia, pentosuria, gyrate atrophy of the choroid and retina, etc. Principle: For high resolution, 2D chromatography is carried out. The amino acid sample to be examined is allowed to interact with two distinct entities—a mobile phase and a stationary phase. Both the phases are liquids. The mobile phase is prepared from two different mixtures. The first one is butanol, acetic acid and water in the ratio of 4:1:1, while the second one is pyridine, isoamyl alcohol and water in the ratio of 10:10:7. The stationary phase is water saturated with organic liquid. The mobile phase moves the solute through the region containing the stationary phase embedded in the interstices of the paper. The sample amino acid distributes itself between the mobile and stationary phases, depending on its partition coefficient between the two partially miscible solvents. Performance specifications: Sensitivity: Lower limit of detection is in microgram. Primary sample: 4.1. Urine specimen and ACD plasma. 4.2. Blood should be collected after overnight fasting or at least minimum of 4 hours fasting is mandatory. Collect 2.0 mL of venous blood sample using a sterile syringe fitted with 21 G needle and dispense the blood in a white plastic vial containing acid citrate dextrose as anticoagulant and mix gently. Transport the sample if necessary on ice. 4.2. Urine should be collected after overnight fasting or at least minimum of 4 hours fasting is mandatory. Collect 5–10 mL of fasting urine sample in the sterile container provided with azide. 4.3. Do not use lysed sample for testing as it may give very high results. 4.4. If the specimen is not analyzed within 3 hours, it should be stored at –20°C. 4.5. Transfer the sample to a plain dry test tube and centrifuge at 2500 rpm for 10 minutes to separate plasma. To an aliquot of the separated plasma add equal volume of 10% TCA, cyclomix and centrifuge at 2500 rpm for 10 minutes and store the remaining plasma sample and TCA supernatant at –20 °C for up to 15 days. 4.6. Do not use contaminated urine samples for testing. 4.7. Centrifuge urine sample at 2500 rpm for 10 minutes and store it at 2–8 °C for up to 3 days. Type of container and additive: Use white plastic vials containing acid citrate dextrose as anticoagulant for collecting plasma samples. Use sterile plastic containers with azide for collecting urine samples

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6. Reagents/Consumables: 6.1. 1st solvent: Butanol, acetic acid and water (4:1:1) 6.2. 2nd solvent: Pyridine, isoamyl alcohol and water (10:10:7) 6.3. Staining reagent: Ninhydrin - 100 mg; acetone - 100 mL 6.4. Standard amino acid solution (1 mg/mL) 6.5. Acid citrate dextrose (ACD): Trisodium citrate – 22 g; citric acid – 8 g; dextrose – 25 gm; water – 1000 mL; 300 μL of ACD for 2.0 mL blood. 7. Instrument: Chromatography tank 8. Procedure: 8.1. A square paper is cut from the Whatman no. 1 filter paper and a line is drawn horizontally, one inch from the lower end of the paper, and two equidistant spots are marked and encircled, one marked as standard and the other marked as test. 8.2. Place two-three drops of the test sample at the spot marked test using a capillary with intermittent drying. 8.3. Similarly, the standard solution containing 4 amino acids (mixture) applied at the spot marked standard. 8.4. Join the two ends of a paper by stitching, such that the sample applied was at the lower half. 8.5. Place the paper vertically in the tank containing the mobile phase—Butanol, acetic acid and water. 8.6. After allow running overnight, the paper is removed and dried. 8.7. After complete drying, the paper is opened by cutting the threads, then folded at the right angles to the previous fold and stitched again. 8.8. Now place the paper in the second tank containing the mobile phase—Pyridine, isoamyl alcohol and water. 8.9. The mobile phase runs perpendicularly to the first dimension (2nd dimension). 8.10. After allowing it to run overnight, the paper is taken out and dried. 8.11. After complete drying, the paper is stained with Ninhydrin. 9. Reference range: Compared with standard. 10. Safety precautions: 10.1. Handle all samples as potentially infectious 10.2. Handle all reagents with care and avoid contact with eye, mouth and skin 10.3. Do not perform mouth pipette. 10.4. Discard used reagents and sample as per disposal procedure 11. Potential sources of variability: 11.1. Urine with bacterial contamination should not be used. 11.2. Lysed blood sample should not be used.

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BIBLIOGRAPHY 1. Alan H Gowenlock, Janet R McMurray, Donall M McLaughlin. Varley’s Practical Clinical Biochemistry 1980;6:375-80.

HPLC ANALYSIS OF AMINO ACIDS 1. Purpose: Many of the inborn errors of metabolism (IEM) including urea cycle defects, organic acidemias and certain disorders in the amino acid metabolism are present in young infants. IEMs are mostly treatable and the successful outcome depends on the rapid diagnosis and early detection. Although screening test is there to detect the defect in amino acid metabolism, they are qualitative and not so sensitive. High Performance Liquid Chromatography (HPLC) analysis provides a quantitative and sensitive detection of amino acid in plasma and urine samples. The volume of sample required for analysis is also minimum thereby useful for young infants. 2. Principle: Primary amines react readily with OPA in the presence of mercaptopropionic acid to form one thio substituted 2-alkyl isoindoles. These isoindoles have been shown to be well-suited for HPLC separation. OPA derivatization procedures involve a rapid reaction and high sensitivity. 3. Performance specifications: 3.1. Linearity: This method is linear up to 1.0 nanomole 3.2. Measurement range: This method has a measurement range of 100 picomole to 1.0 nmol 3.3. Sensitivity: The minimum detection limit is 100 picomole 4. Primary sample: 4.1. Use only plasma and urine as specimen for the test 4.2. Plasma sample should be collected after overnight fasting or at least minimum of 4 hours fasting is mandatory. Collect 2.0 mL of whole blood in sterile vials containing ACD anticoagulant 4.3. Urine should be collected after overnight fasting or at least minimum of 4 hours fasting is mandatory. Collect 5–10 mL of fasting urine sample in a sterile container provided with sodium azide. 4.4. Do not use lysed sample for testing as it may give very high results 4.5. Do not use contaminated urine samples for testing. 4.6. Transfer the sample to a plain dry test tube and centrifuge at 2500 rpm for 10 minutes to separate plasma. To an aliquot of the separated plasma add equal volume of 10% TCA, cyclomix and centrifuge at 2500 rpm for 10 minutes and store the remaining plasma sample and TCA supernatant at –20 °C for up to 15 days.


96 4.7. 5.

6.

7. 8.

Centrifuge urine sample at 2500 rpm for 10 minutes and store it at –20° C for up to 15 days. Type of container and additive: 5.1. Use white plastic vials containing acid citrate dextrose as anticoagulant for collecting plasma samples. 5.2. Use sterile plastic containers with sodium azide for collecting urine samples. Reagents/Consumables: 6.1. Mobile phase A: Sodium acetate - 1.36 g; Milli Q water 500 mL; Adjust the pH to 7.20 ± 0.05 with 2% acetic acid; Triethanol amine - 90 mL; Tetra hydrofuran - 1.5 mL 6.2. Mobile phase B: Sodium acetate - 1.36 g; Milli Q water - 100 mL; Methanol - 200 mL (HPLC grade); Acetonitrile - 200 mL (HPLC grade) Adjust the pH to 7.20 ± 0.05 with 2% acetic acid 6.3. 10% Trichloroacetic acid 6.4. Borate Buffer: (Agilent technologies) – commercially 6.5. Orthophthalaldehyde Reagent: 15 mg/mL in 0.4 N borate buffer of pH 10.2 with 10 μL of beta mercaptopropionic acid/or commercially available (Agilent technologies). 6.6. Stock standard (Agilent technologies): The standard amino acids mixture of 1 nm and 250 pm concentrations provided by Agilent technologies is used as standard. 6.7. Acid citrate dextrose: Trisodium citrate – 22 g; Citric acid – 8 g; Dextrose – 25 gm; Water – 1000 mL; 300 μL of ACD for 2.0 mL blood. Instrument: Agilent 1100-HPLC Procedure: 8.1. Switch on the instrument and the computer for LAN connection to be established. 8.2. Click the HPLC instrument online and select the configuration of the instrument. 8.3. Select the method for amino acids. 8.4. Check the solvent reservoirs for adequate solvent. 8.5. Switch on the pump, column and the detector. 8.6. Equilibrate the column with solvent A in which the analysis is going to be done. 8.7. Check if the instrument is equilibrated by checking for zero line. 8.8. The instrument is calibrated using 1.0 nm and 250 pm standards individually. Mix 10 μL of the standard in 60 μL borate buffer and 10 μL of OPA reagent in dilution vial and cyclomix it. From

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8.10.

8.11.

8.12.

8.13.

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this mixture 50 μL is injected in the HPLC using Hamilton syringe. Each standard should be individually run in the gradient program mentioned below. The HPLC chromatogram for these will be obtained. The two consecutive runs that have the same retention time will be taken and the average of them is used for plotting the graphs in the calibration table. The procedure is termed as calibration and the curve obtained for the same is calibration curve. Use TCA supernatant for plasma amino acid analysis, for urine analysis dilute the supernatant 1 in 100 times with milli-Q water and use for the analysis. Mix 10 mL of the sample (TCA supernatant for plasma/1 in 100 diluted urine) in 60 μL borate buffer and 10 μL of OPA reagent in dilution vial and cyclomix it. From this mixture, 50 μL is injected in the HPLC using Hamilton syringes. After the sample (plasma/urine) is run, the chromatogram is obtained for the same. From the chromatogram the area of the peaks will be recorded and calibrated along with the standards. A standard is run at each time and is used for calculation. Results are expressed in mg/L. LC parameters: Reaction temperature: 40°C Flow rate: 0.5 mL per minute Detection wavelength: (VWD) 338 nm Injection volume: 50 μL Gradient program:

Time (min)

Solvent flow rate (mL/min)

B concentration (%)

0.00 25.00 30.00

0.5 0.5 0.1

0.0 100 0.0

9. Interferences: 9.1. Lysed plasma samples should not be used 9.2. Turbid urine should not be used. 10. Calculation of results: Molecular weight Test area Plasma = Std area × 320 × 2.5 × 10, 000 Molecular weight Test area Urine = Std area × 160 × 2.5 × 10, 000 11. Biological reference range: This is obtained by performing plasma and urine amino acid analysis in 10 control subjects and the mean is given as reference range aged below 10 years.

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For plasma amino acids in μmol/L

For urine amino acids in nmol/mg creatinine

Serine Histidine Glycine Threonine Alanine Arginine Tyrosine Valine Methionine Phenylalanine Isoleucine Leucine Lysine

Serine Histidine Glycine Threonine Alanine Arginine Tyrosine Valine Methionine Phenylalanine Isoleucine Leucine Lysine

24-52 226-268 47-109 19-43 43-165 17-31 35-63 58-110 19-35 27-47 18-48 42-78 32-72

821-3587 1089-3641 360-2722 361-1699 745-3471 59-1115 332-1282 35-419 0-1041 386-2020 0-420 128-720 13771-32493

12. Reportable intervals: 10 days 13. Critical/Alert level values: Not applicable. 14. Laboratory interpretation: Increase of all the amino acids, say in liver involvement or a particular amino acid like phenylalanine, tyrosine, cystine, methionine or ornithine is interpreted. 15. Safety precautions: 15.1. Handle all samples as potentially infectious 15.2. Handle all reagents with care and avoid contact with eye, mouth and skin 15.3. Do not perform mouth pipetting 15.4. Discard used reagents and sample as per disposal procedure 16. Potential sources of variability: 16.1. Lysed plasma samples and urine contaminated with bacteria may give falsely elevated values. 16.2. Samples should be stored properly at -20 °C 16.3. Do not use if the solvents are turbid and the pH is incorrect. 16.4. Do not use if the column is not equilibrated/deteriorated. BIBLIOGRAPHY 1. Bruckner H, Witner R, Godel H. Fully automated HPLC separation of DL amino acids derivatized with OPA together with N-isobutyryl-cysteine. Applications to food samples, chromatographic. 1991;383,32.

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Part V: Inborn Errors of Metabolism CARBOHYDRATES 1. Purpose: Qualitative urine analysis for abnormal constituents like glucose, galactose, proteoglycan fragments is useful in the diagnosis of some inborn errors of metabolism like renal glycosuria, galactosemia, deranged heteroglycan metabolism, etc. Such a diagnosis is necessary for therapeutic purpose to keep the disease process under control. 2. Principle: This is a general test for all carbohydrates, sugars and sugarprotein complexes. (muco and glycoproteins). Glucose is treated with alcoholic alpha naphthol and a few drops of concentrated sulfuric acid. The furfural, formed from glucose and H2SO4 gives purple color (in the form of ring ) with α naphthol. 3. Performance specifications Sensitivity: Qualitative method 4. Primary sample: 4.1. Urine specimen: Fasting urine – 10 mL 4.2. If the specimen is not analyzed within 3 hours it should be stored at 2-4°C 5. Type of container and additive: Use plain urine container with a pinch of sodium azide. 6. Instrument: Reagents consumables: Test tube, beaker, pipettes. Reagents: Concentrated sulfuric acid. Alpha naphthol: 1 gm of alpha naphthol in 100 mL of ethyl alcohol. 7. Procedure steps: Centrifuge the sample of urine at 2500 rpm in 5 minutes, take 2 mL of urine add 6 to 8 drops of Alpha naphthol reagent. Add 2 mL of concentrated sulfuric acid through the sides of the tube. The acid forms the lower layer. Observe a purple ring at the interface of layers. 8. Interferences: Protein-rich diet and amino-aciduria in heavy metal poisoning, de Toni Fanconi syndrome, Wilson's disease, galactosemia. 9. Interpretation: Positive reaction indicates fragments of Proteoglycans, deranged glycosaminoglycan metabolism. 10. Potential sources of variability: Urine with bacterial contamination should not be used. BIBLIOGRAPHY 1. Sulochana KN, RamaKrishnan S, Vasanthi SB, et at. First report of congenital or infantile cataract in deranged proteoglycan metabolism with released xylose. Br. J Ophtholmol 1997;81:519-23.

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GLUCOSE AND OTHER REDUCING SUGARS 1. Purpose: Qualitative urine analysis for abnormal constituents like glucose, galactose, proteoglycan fragments, is useful in the diagnosis of some inborn errors of metabolism like renal glycosuria, galactosemia, deranged proteoglycan metabolism, fructosuria, lactosuria and pentosuria. Such a diagnosis is necessary for therapeutic purpose to keep the disease process under control. 2. Principle: : Glucose and other reducing sugars like galactose, lactose, fructose and pentoses reduce alkaline copper reagent to a red precipitate of cuprous oxide from the cupric ions of the Benedict’s reagent. 3. Performance specifications Sensitivity: Qualitative method 4. Primary sample: 4.1. Urine specimen: Fasting urine – 10 mL 4.2. If the specimen is not analyzed within 3 hours, it should be stored at 2–4oC 5. Type of container and additive: Use plain urine container with a pinch of sodium azide. 6. Instrument/Reagents consumables: Not applicable test tube, beaker, pipettes Reagents: Benedicts’ qualitative reagent. Dissolve 173 g of sodium citrate and sodium carbonate 100 g in 600 mL of water. Separately dissolve 17.3 g of copper sulfate in about 100 mL of water and transfer this to the first solution slowly with stirring and make up to 1 liter with water. 7. Calibration: Not applicable. 8. Procedure steps: Add about 8 drops of urine to about 5 mL of boiled Benedict’s reagent;* keep in boiling water bath for 5 minutes. Reduction is obtained with glucose, galactose, fructose, lactose, maltose, and pentoses. No reduction is obtained with sucrose, as it is non-reducing sugar and heteroglycan fragments. If Benedict’s test is positive, we should test for sugars like glucose, galactose, fructose maltose, lactose and pentoses by paper chromatography. *Note: Boil 5 mL of Benedict’s reagent before urine is added. If there is a precipitate, it is a contaminated reagent so discard it. 9. Quality control procedure: The color obtained for the urine sample will be compared with the standard solution. 10. Interferences: Protein-rich diet and aminoaciduria in heavy metal poisoning, de Toni Fanconi syndrome, Wilson's disease, galactosemia. 11. Laboratory interpretation: Diabetes mellitus, lactosuria, galactosemia, disaccharidosis, pentosuria, fructosuria

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12. Potential sources of variability: Urine with bacterial contamination should not be used. BIBLIOGRAPHY 1. Harold varley. Practical Clinical Biochemistry, Fifth edition 1980; 421.

MAPLE SYRUP URINE DISEASE 1. Purpose: Qualitative urine analysis for abnormal amounts of keto acids of branched chain aminoacids is useful in the diagnosis of the inborn errors of the metabolism maple syrup urine disease. Such a diagnosis is necessary for therapeutic purpose to keep the disease process under control. 2. Principle: The keto acids of branched chain amino acids give brown color with dinitrophenyl hydrazine (DNPH). 3. Performance specifications: Sensitivity: Qualitative method 4. Primary sample: 4.1. Urine specimen: Fasting urine – 10 mL 4.2. If the specimen is not analyzed within 3 hours it should be stored at 2–4oC 5. Type of container and additive: Use plain urine container with a pinch of sodium azide. 6. Reagents/Instrument: Test tube, beaker, pipettes. 6.1. 2–4 DNPH: 0.1% in 2N hydrochloric acid 6.2. 0.4 N: sodium hydroxide. 7. Procedure steps: Test: Take 5 mL of centrifuged fresh urine in a test tube. Add 1 mL of DNPH and allow standing for 20 minutes. Then add 5 mL of 0.4 N sodium hydroxide. If the color turns brown, it is positive. 8. Interferences: Protein-rich diet and aminoaciduria in heavy metal poisoning, de Toni Fanconi syndrome, Wilson's disease, galactosemia. 9. Biological reference range: The color obtained for the urine sample will be compared with the Control Urine. 10. Laboratory interpretation: Maple syrup urine disease, a rare genetic disease in infants is due to the metabolic block by a nonfunctional oxidative decarboxylase which prevents the catabolism of all the three alpha keto acids of valine, leucine and isoleucine. The keto acids accumulate in the blood and urine imparting to urine the odor of maple syrup. The patient has functional impairment of the central nervous system. 11. Potential sources of variability: Urine with bacterial contamination should not be used.

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BIBLIOGRAPHY 1. John D Bauer. Clinical Laboratory Methods. Ninth edition; 696:1982.

CYSTINE 1. Cystinuria: Purpose: Qualitative urine analysis for abnormal amounts of cystine is useful in the diagnosis of the some inborn errors of metabolism like cystinuria (presently known as cystine-lysinuria). Such a diagnosis is necessary for therapeutic purpose to keep the disease process under control. 2. Principle: Cystine is reduced by alkaline cyanide, which gives a purplered compound when nitroprusside is added. There are three important hereditary diseases associated with the metabolism of the sulfurcontaining amino acid (a) cystinuria, (b) cystinosis (c) homocysteinemia 3. Performance specifications Sensitivity: Qualitative method 4. Primary sample: 4.1 Urine specimen: Fasting urine – 10 mL 4.2. If the specimen is not analyzed within 3 hours, it should be stored at 2–4o C 5. Type of container and additive: Use plain urine container with a pinch of sodium azide. 6. Instrument: Reagents: Test tube, beaker, pipettes. 1. 5% Sodium cyanide in water. 2. 5% Sodium Nitroprusside in water to stand for 10 minutes. 7. Test: To 5 mL of urine add 2 mL of 5% sodium cyanide solution. Mix and allow to stand for 10 minutes. Add 5% sodium nitroprusside drop by drop and shake. Red color shows the presence of cystine. 8. Interferences: Protein-rich diet and aminoacidurias in heavy metal poisoning, de Toni Franconi’s syndrome, Wilson's disease, galactosemia. 9. Biological reference: The color obtained for the urine sample will be compared with the Control urine. 10. Laboratory interpretation: Cystine-lysinuria. 11. Potential sources of variability: Urine with bacterial contamination should not be used. BIBLIOGRAPHY 1. John D Bauer. Clinical Laboratory Methods, Ninth edition, 1982:691.

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TYROSINE 1. Purpose: Qualitative urine analysis for abnormal amounts of tyrosine is useful in the diagnosis of the inborn errors of metabolism, tyrosinosis to keep the disease process under control. 2. Principle: Tyrosine gives red color complex when heated with mercuric sulfate followed by sodium nitrite. Tyrosinosis is a rare congenital defect in which Fumaryl aceto-acetyl hydrolase is deficient. Both acute and chronic forms of Tyrosinosis are known. There are three types. Type I: Acute tyrosinosis: Enzyme deficiently is Fumaryl acetoacetate hydrolase; Infants exhibit diarrhea and vomiting. Death will occur between 6–8 months. Type II: Is due to the paucity of the enzyme tyrosine transaminase. There may be raised levels of plasma tyrosine. Type III: Neonatal tyrosinosis is due to a hereditary deficiency of parahydroxy phenyl pyruvate oxidase. 3. Performance specifications sensitivity: Qualitative method 4. Primary sample: 4.1. Urine specimen: Fasting urine – 10 mL 4.2. If the specimen is not analyzed within 3 hours it should be stored at 2–4oC 5. Type of container and additive: Use plain urine container with a pinch of sodium azide. 6. Reagents consumables: Test tube, beaker and pipettes. Reagents: 10% Mercuric sulfate in 10% Sulfuric acid solution. 1% Sodium nitrite (100 mg in 10 mL of water). 7. Procedure: Take 1 mL of centrifuged fresh urine and add 1 mL of mercuric sulfate solution. Boil for 3 minutes. Then add 3 drops of 1% sodium nitrite solution. A red color is positive. 9. Interferences : Protein-rich diet, and aminoacidurias in heavy metal poisoning de Toni Fanconi syndrome, Wilson's disease, galactosemia. 10. Interpretation: Tyrosinemias. 11. Potential sources of variability: Urine with bacterial contamination should not be used. BIBLIOGRAPHY 1. John D Bauer, ninth edition. Clinical Laboratory Methods. 1982; 695.

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HOMOCYSTINURIA 1. Purpose: Qualitative urine analysis for abnormal amount of homocystine is useful in the diagnosis of some inborn errors of metabolism like homocysteinemia. Such a diagnosis is necessary for therapeutic purpose to keep the disease process under control. 2. Principle: A disorder in methionine metabolism is homocystinuria, in which there is a increased level of Homocysteine in the blood and increased excretion of Homocystine in urine. Only in type I, there is increased level of methionine also in blood. The disease is due to deficiency of cystathionine synthetase. The clinical findings especially in children are mental disorder, bilateral posterior dislocation of the lens, fair complexion with blue eyes and fine hair. The other types like type II, III, IV, etc. are due to deficiency of B12 or folate and do not have mental retardation. 3. Performance specifications Sensitivity: Qualitative method 4. Primary sample: 4.1. Urine specimen: Fasting urine – 10 mL 4.2. If the specimen is not analyzed within 3 hours it should be stored at 2–4oC 5. Type of container and additive Use plain urine container with a pinch of sodium azide. 6. Instrument/Reagents consumables: Test tube, beaker, pipettes. Reagents: Solid sodium chloride. Ammonia, 3 mL of ammonia diluted with 7 mL water. Silver Nitrate: 100 mg in 10 mL of reagent 2 (prepare freshly). Sodium nitroprusside solution: 100 mg in 10 mL distilled water (prepare freshly). Sodium cyanide: 70 mg in 10 mL of water (prepare freshly). 7. Procedure Steps: Saturate the urine with sodium chloride. Add 0.5 mL of silver nitrate solution to 5 mL of saturated specimen. For the control take 5 mL ammonia. Allow both to stand for exactly 1 min, and then add to each 0.5 mL of sodium cyanide. Terminal addition of cyanide is needed to bind the silver ion and allow the reaction of Homocysteine with the 0.5 mL of nitroprusside. At this point, excess cyanide begins to react with any cystine present to give slowly developing color. The test is positive for Homocysteine if a pink or purple pink color develops immediately in the test sample only. Note: If any test is positive, paper chromatography is done, as given in this manual, to confirm the findings. 8. Interferences: Protein-rich diet and aminoaciduria in heavy metal poisoning de Toni Fanconi’s syndrome, Wilson's disease, galactosemia

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9. Laboratory interpretation: Homocystinuria/Homocysteinemia 10. Potential sources of variability: Urine with bacterial contamination should not be used. BIBLIOGRAPHY 1. Harold Varley. Practical Clinical Biochemistry, Fifth edition 1990;520.

PHENYLKETONURIA 1. Purpose: Qualitative urine analysis for abnormal amount of phenylalanine is useful in the diagnosis of some inborn errors of metabolism like hyperphenylalaninemia (phenylketonuria), Such a diagnosis is necessary for therapeutic purpose to keep the disease process under control. 2. Principle: Phenylketonuria (PKU) is the result of transamination of phenyl alanine present in excessive amounts in hyperphenylalaninemia. Phenyl pyruvic acid (α keto acid) is formed in large amounts and excreted in urine. This gives the green color with ferric chloride. 3. Performance specifications: Sensitivity: Qualitative method 4. Primary sample: 4.1. Urine specimen: Fasting urine – 10 mL 4.2. If the specimen is not analyzed within 3 hours it should be stored at 2–4oC 5. Type of container and additive: Use plain urine container with a pinch of sodium azide. 6. Instrument/Reagents consumables: Test tube, beaker, pipettes. Reagents: Ferric chloride 10% –10 g in 100 mL; dissolved in distilled water. Store in refrigerator 7. Procedure: Test: Add 3–5 drops of reagent to 5 mL of fresh acid urine. Results: A positive test is indicated by a grayish green or blue green color appearing in 1–90 seconds and fading again in the same period of time. 8. Interferences: Protein-rich diet, aminoacidurias in heavy metal poisoning, de Toni Fanconi’s syndrome, Wilson's disease, galactosemia. 9. Laboratory interpretation: Hyperphenylalaninemia. 10. Quality control procedure: The color obtained for the urine sample will be compared with the standard solution. 11. Potential sources of variability: Urine with bacterial contamination should not be used.

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BIBLIOGRAPHY 1. John D Bauer. Clinical Laboratory Methods. Ninth edition 1982:693. 2. Ramakrishnan, Prassannan S, KG and Rajan R. Textbook of Medical Biochemistry, Orident Longman Limited Chennai third edition. 2001;3:530.

Part VI: Collection of Test Sample PROCEDURE FOR BLOOD COLLECTION Purpose: To organize the blood collection when a patient reports to the laboratory for investigations. Femoral tap: Generally for infants less than six months required amount of blood is collected from femoral vein by femoral tap. Only a physician does this procedure. Heel prick: Occasionally for infants blood is collected by heel prick. When only the blood glucose (F/R/PP) is asked a requisition form is sent from the concerned ward and a F-tube 3 mL syringe with needle is provided. The sample is then collected and sent to the laboratory. The technician wears a pair of gloves before doing the blood collection. The patient has to rest his either arm on the cushion kept on the table. A tourniquet is applied over the forearm. His fist should be closed so as to make the vein prominent. The anticubital area is cleaned with the cotton dipped in cetavlon solution. The technician using sterile syringe of proper volume and sterile disposable needle of proper gauge [generally 21’G and 1”] withdraws blood from the anti-cubital vein. After collecting the required amount, the technician removes the needle from the patient’s arm, making sure that the syringe is held horizontally, and keeps the syringe on the collection table. The technician then removes the tourniquet and immediately applies pressure with cotton dipped in cetavlon over the venipuncture site for the bleeding to stop and make sure that there is no oozing.

COLLECTION OF URINE SAMPLE Purpose: To collect urine specimen, for routine as well as for special investigations. Procedure: Urine collection is done in a toilet situated in the lab. For inborn error of metabolism (IEM) and 24 hours urine specimen, the patient collects urine at his residence.

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Specimen collection for urine: The patient is asked to collect the urine in this pre-labeled container provided by the technician and asked to deposit the container in a tray kept in the laboratory. Specimen collection for IEM: Generally this test is done for younger age group patients [children] hence the instructions are given to the accompanying adult. Three urine containers, each containing a pinch of sodium azide as preservative, are labeled with the patient’s name. One of them is marked as “Fasting”, and the other two is marked “Random” and all the three are handed over to the attender with the following instructions and lab requisition form. Collect from the patient early morning urine, at least 25 mL. Later on, the morning, to collect at least 25 mL of urine in each of the other two containers. This is random collection. To bring all the three urine specimens thus collected to the lab. 24 hours Urine Specimen: To diagnose certain diseases special investigations are done on 24 hours urine specimen. The patient is given a 2.5 liter bottle in which 10 mL of 6 M hydrochloric acid as a preservative is added. In addition, a small 500 mL bottle with 2 mL 6 M hydrochloric acid is also provided to the patient. Alternately, he may be given six 500 mL bottles each containing 2 mL of 6 molar hydrochloric acid. The technician gives the following instructions to the patient: Not to collect the first early morning urine in the container. This specimen is to be discarded. To collect the entire quantity of urine voided next in the container and to note the time of collection. To collect the entire quantity of urine subsequently voided in next 24 hours in the container/containers. This should include the first urine of the next early morning also. Then to bring the container/containers to the laboratory as early as possible for processing.

COLLECTION OF STOOL SAMPLE Purpose: To collect stool samples for investigation. Procedure: Occasionally stool examination is asked as an isolated test. The patient is discouraged from collecting the stool sample in the hospital premises. For routine stool exam: The lab provides a container labeled with patient name and the date of issue and asks him to collect stool sample at his

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residence. The patient is advised to bring the sample within two hours to the laboratory before the sample dries up.

FULLY AUTOMATED CHEMISTRY ANALYZERS The different biochemical parameters referred to above can also be analyzed using fully automated chemistry analysis with advantages due to refined calibration and speed.

The following are some of the important laboratory tests for which the patient needs some preparation before the tests. The patient will be instructed as follows: 1. GTT (Glucose tolerance test): Patient should come on 12 hours fasting in the morning. For an instance: He/She must come in the morning without any consumption of food (coffee, tea, breakfast, etc.) after the previous night food intake. 2. TGL (Triglycerides): Patient should come on 12 hours fasting in the morning. 3. IEM (Inborn Errors of Metabolism): Three containers will be provided for the patient, one of the three containers labeled ‘F’ (fasting) and the other two containers labeled ‘R’ (Random). Patient should collect the fasting urine in the ‘F’ container and the random urine in the ‘R’ containers.

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4. Vitamin A assay: Patient should not take carrot, leafy vegetables and Vitamin A tablets on the two days before blood collection. 5. Paper chromatography for Ornithine: Patient should come in fasting condition for blood collection. 6. Paper chromatography for galactose: Patient should come in fasting condition for blood and urine collection. 7. Homocystine plasma: Patient should come in fasting condition for blood collection. 8. PP (Post-prandial) sugar: Patient should take breakfast/lunch and the blood collection will be done 2 hours after the breakfast/lunch time reported by the patient. Fasting sugar: Patient should come for blood collection in fasting condition, i.e. without any food, coffee and tea. 9. 24 hours urine collection: The patient should void the first urine. Then the patient should start collecting the urine every time he passes for the whole day (24 hours). For e.g. If he starts collecting urine at 6:00 am on previous day, he should collect the urine till next day 6:00 am. 10. Aminoacid profile (plasma and urine): Patient should come in fasting condition without any breakfast, tea and coffee. 11. B12 and folate: Patient should come in overnight fasting condition (minimum 12 hrs). 12. Homocystinuria screening: Patient should come for urine collection in fasting condition i.e. without any food, coffee and tea. 13. Ornithine amino transferase: Patient should come for blood collection in fasting condition, i.e. without any food, coffee and tea. Specimen Acceptance Rejection Criteria Hematology, Clinical Pathology & Clinical Biochemistry S. No.

Type of sample

Criteria for Acceptance

Criteria for Rejection

Minimum Volume

1.

EDTA Blood

Properly mixed without any clot

Any clot, insufficient quantity, Hemolysis

1 ml

2.

Blood

Unlysed blood

Hemolysis, clotting

5 ml

3.

EDTA Plasma

Clear, no hemolysis no Particulate matter

Turbid, Foul smelling, Hemolysis, insufficient quantity

1 ml

4.

CSF

Sufficient quantity

Insufficient quantity, brought after 1 hour of collection

Adult: 2 ml Children: 1 ml

5.

Body fluids

Sufficient quantity

Insufficient quantity, brought after 1 hour of collection

2-5 ml

Contd...


110 Contd... 6.

24 hrs Urine Sample

Proper preservative Insufficient quantity and 25 ml total 24 hr volume contaminated must be mentioned

7.

Urine

Sufficient quantity in sterile container

Collected in routine container, insufficient quantity, brought after 1 hour

5 ml

8.

Stool

Sufficient quantity in sterile container

Brought after 1 hour

Not Applicable

9.

Serum/ • Clear Clotted • No hemolysis Sample • No particulate matter

• Turbid • Foul smelling • Hemolysis • Insufficient quantity • Sample brought After 2 hour of collection.

• Serum up to 5 test 1 ml • Above 5 tests 2 ml • Above 10 test 3 ml • Clotted sample Up to 5 test 2 ml above 5 test 4 ml Above 10 test 5 ml

DISPOSAL OF MATERIALS The syringes, which have been used for collecting the patient blood, are discarded in a container containing 1% sodium hypochlorite solution after the tips of the syringes are cut with the help of a cutting machine, rinsed in tap water, the excess water drained off and the syringes are discarded in color coded (red) bags. After collection of the blood specimen the bevel tip of the needles are charred with the help of an electric cutting machine. The eclipse needles are discarded in a container containing 1% hypochlorite solution autoclaved and discarded in polythene bag. The glass test tubes used are discarded in 1% sodium hypochlorite for 1 hour before washing. All the vacutainers tubes are autoclaved and discarded in red bags. The biomedical waste generated in the laboratory is segregated at the site of generation into color coded bags and disposed at the end of the day. Biomedical waste is disposed in accordance with the statutory regulations laid down by government of India chemistry of Environment and forest (Biomedical waste Management and Handling) Rules 1998. The following color bags are used for discarding lab materials. White bag

Red bag

Yellow bag

Blue bag

Paper waste, food waste, stationary waste

All types of tubings, catheters, blood bags, urine bags, Gloves, Disposable syringes

All infection wastes, Broken/unbroken cotton, dressings, bottles, ampules, soiled plaster casts, broken slides, disposable linen, cover slips tissue paper

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QUALITY CONTROL (QC) QC is done to detect and correct errors in the analytical procedures in the laboratory. It is done in the beginning of each shift; after an instrument is serviced; when reagent lots are changed; after calibration, and when patient results seem inappropriate. Quality control data is most easily visualized using a Levey-Jennings chart (Fig. 1.1). The dates of analyses are plotted along the X-axis and control values are plotted on the Y-axis. The mean and one, two, and three standard deviation limits are also marked on the Y-axis. Inspecting the pattern of plotted points helps to detect increased random error and shifts or trends in calibration. This is an internal QC program while external QC programe is also done to make comparison with the peer group globally who does it by same method and/or instrument. Inter lab comparison is another form of quality control.

Fig. 1.1: Levey Jennings Chart for SPC Period: 09/01/2011 – 10/04/2011 Lab: 285502, SNSC, Mrs Punitham, Clinical Biochemistry Phosphorus, Phosphomolybdate-UV, Siemens Dimension RxL, Factored, mg/dL, No Temperature Lot: 14190, Assayed chemistry, Scrum, Bio-Rad, 04/30/2012 Cum Mean/SD: [1] 3.23/0.05, [2] 7.24/0.09, Fixed Mean/SD: [1] 3.25/0.05, [2] 7.23/0.05 31. Test: Calibrate

BIBLIOGRAPHY 1. Carl A Burtis, Edwarrd R Ashwood. Tietz Text Book of Clinical Chemistry. 2nd Edn; (1994), WB Saunder Company, USA. 2. The Bio-medical waste segregation is done as per “The Bio-Medical Waste (Management and Handling) Rules 1998, as amended 2003” published by the Tamilnadu Pollution Control Board.

UNIT 2

Genetics

Kumaramanickavel G Sripriya S Soumittra N Vinita Kumari Madhavan Jagadeesan J Ramprasad VL

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INTRODUCTION Genetics deals with the concepts and techniques for identifying many mysteries of life starting from the appearance, behavior, inheritance of the phenotypes (both disease and normal), etc. from the parents/ancestors. Bateson et al first coined the term genetics in 1906 and later the basic principles underlying the field were revealed to the world by the works and principles of Gregor John Mendel. The factor responsible for the findings of Mendel was then identified as Deoxyribonucleic acid (DNA) that carries the sequence required for the cells to live, grow differentiate and multiply. DNA is also responsible for the variations observed in different organisms. Knowledge repository generated by the Human Genome Project made the genetic basis of the human diseases easily comprehensible to the world. DNA the molecule of heredity was first demonstrated as the basic genetic constituent of a cell. The whole genetic constitution of an organism referred as genome varies in its size ranging from 6,00,000 DNA base pairs in free-living organism (a bacterium) to 3 billion as seen in human and mouse genomes. The arrangement of the DNA ranges from simple (naked circular DNA of bacteria) to complex (compact packaging as in human). Each human has two sets of 23 chromosomes, one maternal and one paternal in origin. One of the 23 pairs of chromosomes are the sex chromosomes. A location on the chromosome is termed a genetic locus. At each locus, there may be distinct variants, called alleles. A pair of alleles (maternal and paternal) for an individual at a given locus is called the genotype. A genotype can be homozygous if the two alleles are the same allele and heterozygous if they are different alleles. The pattern of alleles on more than one locus for a single chromosome is called a haplotype. In the process of conception, each offspring receives at each locus only one of the two alleles from a given parent and alleles are transmitted randomly (that is, each with probability ½). BASIC CONCEPTS ON HUMAN DNA Human DNA is packed in an hierarchical fashion by associating with proteins (histones and transcription factors) inside the 23 chromosomes within the nucleus. Out of the total genomic DNA content of a human cell, only 2% are functional genes (20,000–25,000 in humans) and the remaining are noncoding regions that are proposed to provide structural integrity to the chromosomes, regulate gene expression, etc. The functional unit of a gene is called as the exon that codes for the amino acid sequence of a protein. The non-coding region of the gene called as the introns are spliced to give up the mature RNA. The other non-coding DNA produces non-coding RNAs, like transfer RNA, ribosomal RNA, small nuclear RNA, small nucleolar RNA, microRNAs, small interfering mRNAs, and small temporal RNAs. Non-coding DNA also contains numerous other functionally

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important sequences, including those necessary for maintenance of telomeres, proper segregation of chromosomes during cell division, and initiation and regulation of gene expression Some of the facts on our genome as deduced by the human genome project are as follows: Our genome has 3 Billions nucleotide bases and are concentrated in random areas along the genome, with intervening non-coding DNA. The repeat sequences that does not code for proteins accounts for 50% of the human genome are present in a greater proportion than that in fly (3%), worm (7%) and mustard weed (11%). Single nucleotide polymorphisms (SNPs) constitutes to about 90% of the human genetic variation and occurs at a frequency of one in 100 to 300 bases along the 3-billion-base human genome and can occur in both coding and noncoding regions of the genome. Two of every three SNPs involve the replacement of cytosine with thymine. Many SNPs have no effect on cell function, but could predispose people to disease or influence their response to a drug. Abnormality in the genome results in genetic disorders that are broadly classified into 4 different types namely (1) single-gene, (2) complex, (3) chromosomal, and (4) mitochondrial disorders. • Single-gene disorder (Mendelian or monogenic diseases): Diseases that are caused due to mutations in single gene are known as single-gene disorder. These diseases exist as autosomal dominant, autosomal recessive or sex-linked depending on the chromosomal location of the defective gene. There are more than 6,000 known single-gene disorders, which occur in about 1 out of every 200 births. Example: cystic fibrosis, sickle cell anemia, Marfan’s syndrome, Huntington’s disease, etc. • Multifactorial disorders (complex or polygenic diseases): This type is caused by the interaction of environmental factors with the risk predisposing genotypes inherited from the parents majority of the times. Examples include heart disease, high blood pressure, Alzheimer’s disease, arthritis, diabetes, cancer, and obesity. Multifactorial inheritance also is seen in heritable traits such as fingerprint patterns, height, eye color, skin color, etc. • Chromosomal: The abnormalities in chromosomes structure such as missing or extra copies, gross breaks and rejoining (translocations), can result in syndromic diseases. Some of the major chromosomal abnormalities can be detected by microscopic examination. Example: Down syndrome or trisomy 21 is a common disorder that occurs when a person has three copies of chromosome 21. • Mitochondrial: Mutations in the non-chromosomal DNA of mitochondria result in mitochondrial disorders, which are characteristically maternal in origin. Eye disorder, LHON is a classical example of this disease.

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It is thus shown that most of the diseases have a genetic component and can be either due to the direct effect of the mutant gene or the risk susceptibility conferred by the defective gene. The understanding of genetic and molecular mechanisms for the inherited diseases provides valuable guide for diagnosing/predicting disease susceptibility, disease intervention, development of treatment modalities, potential of gene therapy, etc. Significance of Understanding the Molecular Genetics of Diseases Analysis of the chromosomes or the DNA provides the information on the underlying genetic defect in an individual/family with a history of inherited diseases that provides information on not only on the cause of the disease but also on risk prediction for other family members, drug targeting, pharmacogenomics, gene therapy for specific diseases. Identification of the causative genes for different genetic diseases has resulted in the development of DNA tests categorized as carrier testing, prenatal testing, newborn screening, diagnostic testing and predictive testing. These tests are mainly done for carrier identification (in autosomal recessive diseases like cystic fibrosis, sickle cell anemia, etc.), prenatal diagnostics for predicting the risk in a fetus (diseases like Down syndrome), new born screening as a preventive measure, etc. In this chapter following techniques used for probing the genetic cause for a specific diseases are described: 1. Cytogenetic methods 2. Molecular cytogenetic methods 3. DNA based methods 4. RNA based analysis

CYTOGENETIC METHODS Cytogenetics involve the study of human chromosomes in health and disease. The chromosomes are visualized after staining in light microscopy as they display a characteristic dark and light banding pattern according to the DNA content that is unique for individual chromosome. It serves as an important laboratory diagnostic procedure in the field of prenatal diagnosis, in patients/families with history of mental retardation, multiple birth defects, abnormal sexual development, infertility or multiple miscarriages. The field also finds its application in malignancies and hematological disorders. The specimen for cytogenetics study varies according to the test involved; prenatal cytogenetics analysis requires chorionic villus sampling/amniotic fluid/cord blood, etc. Postnatal analysis requires blood or tissue material. Tumor cytogenetics demands the solid

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tumors or bone marrow specimen. The chromosomes are studied either in interphase /metaphase/ prometaphase stage. The interphase cells are used for molecular cytogenetic methods. The metaphase chromosomes are preferred for analyzing the numerical/structural chromosomal abnormalities. History of recurrent miscarriages with suspected balanced chromosomal translocation is evaluated using this method. Prometaphase studies are indicated whenever subtle chromosomal disarrangements like small deletions, duplications, and balanced or unbalanced translocations are suspected. PERIPHERAL BLOOD LYMPHOCYTE CULTURE1 Blood is the easily accessible tissue with abundant lymphocytes for analysis, usually preferred for most of the postnatal cytogenetic analysis. These quiescent are activated by stimuli that are transformed into a population of enlarged, de-differentiated lymphocytes that are ready for proliferation. The stimulation to these cells are given through mitogens that mimic the foreign antigens to transform the lymphocytes into mitotically active cells. Phytohemagglutinogen (PHA), poke weed mitogen, Epstein-Barr virus, etc. are some of the mitogens that could serve the purpose. The stimulation of mitogen occurs during the first 24h of exposure to the mitogen PHA during which morphological changes are not observed in the cell. The next 24h of the induction, mitotic activity is observed that is reflected by increased RNA synthesis, visibly enlarged nuclei and DNA synthesis that peaks at the 48h, 72h, or 96h corresponding to the 1st, 2nd, and 3rd cycles of cell division thus making maximum number of cells available for the analysis. I. Collection, Transport and Storage of Blood The quantity of whole blood required for a 10-mL culture is as follows: New born/Infant 0.1 mL Child < 5 years 0.5 mL Child > 5 years, adults 0.8 mL Sufficient blood sample is collected in a sterile glass or plastic tubes or vaccutainers according to the above specification in a suitable anticoagulant namely heparin or lithium heparin and mixed well. The samples are stored at 4°C until processed and could also be transported, as they are stable for several days with adequate care on avoiding extreme temperatures. II. Setting of Lymphocyte Culture A. Requirements Basal media: A basal media which is essentially a buffered salt solution containing amino acids, sugar, polysaccharides and nutrients. Media that

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are suitable for the purpose include, RPMI1640, Eagles MEM, Hams F10, DME, McCoy’s 5A, etc. However the RPMI1640 is the most preferred and widely used due to its low thymidine content making it suitable for thymidine synchronization procedures. The powdered commercially available media (widely preferred) are hydrated with deionized /distilled water and autoclaved for sterilization (appendix). They are stored in 4°C. Supplements: A complete culture media is prepared by the addition of certain supplements to the basal media, as the basal media alone cannot support the optimal lymphocyte growth. This complete media has a reduced shelf life of 2-4 weeks that are usually prepared in batches and stored at –20°C. 1. Fetal calf serum: It provides the needed growth factors for cell proliferation and makes 5–30% of the complete medium. It also helps in maintaining the pH at 7.2–7.4. The serum used should be sterilized by membrane filtration and screened for viral infection. The alternate for FCS is provided by pooled human serum. 2. Antibiotics: These are added to prevent microbial infection of culture. Penicillin is added to control gram-positive bacteria and streptomycin for gram-negative bacteria. Alternatively a broad-spectrum antibiotics like gentamycin/kanamycin are also preferred. 3. L Glutamine: An essential unstable amino acid that is converted to D Glutamine by the cells. 4. Buffering systems: Bi-carbonate/HEPES buffer are used. 5. Phytohemagglutinin: A mucoprotein that occurs naturally in red kidney bean, Phaseolus vulgaris to stimulate the T lymphocytes by binding to the T cell receptor complex. The optimum concentration of PHA has to be maintained in the culture since little concentration results in poor mitotic response and increased concentration generates toxic effects on the cell. The activity of the PHA reduces by 30% over a month period of reconstitution in sterile distilled water. PREPARATION OF RPMI 1640 MEDIA RPMI 1640 medium is a basic cell culture medium used for a wide range of applications. To optimize the cell culture of certain cell types it can be supplemented with serum, growth factors, cytokines, vitamins and amino acids. The preparation is done in Class II laminar airflow hood to prevent airborne contamination and reduced exposure of the operator to particles dispersed within the cabinet. Sartorius Filter with Millipore membrane (pore size 0.22 μ) is used for the sterilization process. Reagents Penicillin Sodium bicarbonate

200 mg 2.2 g

Genetics L-Glutamine RPMI-1640 (Base) Autoclaved Milli Q Water

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600 mg 10.3 g 1000 mL

Equipment Filtration apparatus with suction flask filter holder and funnel (Sartorius). (Filtration apparatus is wrapped with aluminum foil and autoclaved the previous day and kept in a sterile environment). Vacuum pump Laminar flow hood (Clean Air). Ultra violet (UV) light in the Laminar flow hood is switched on for 30–60 min prior to the preparation of the medium. Millipore membrane - pore size 0.22μ. pH indicator strips (5 to 7). Glasswares Conical flask - 2 nos. Milk dilution bottles - 12 nos Description of the filtration apparatus: Filtration apparatus has a sterile suction flask with an opening on the sides, which is connected to a vacuum pump and sartorius filter provided with a holder and a funnel. Filtration process is carried out by creating vacuum with the help of a suction pump. Filtration is a physical method of removing microorganisms from the liquid medium. The filter holds back microbes by serving as microscopic sieves; however, the effectiveness depends on the pore size (0.22μ). Procedure First day: The filtration apparatus is assembled and wrapped with aluminum foil and Milli Q water are autoclaved. Both are kept inside the Laminar airflow hood in the culture room. Second day: An hour before the preparation of the media, UV light is switched on. All the components are weighed and dissolved in 500 mL of autoclaved Milli Q water. Check for pH (7.0) with the pH strip. If the pH is not correct, adjust it with 1N hydrochloric acid and then made up to 1000 mL with autoclaved MilliQ water. Then medium is passed through the filter, which is carried out in the Laminar Air Flow Hood. This method of sterilization is applicable to media containing heat labile components. Aliquot 80 mL of the medium into the milk dilution bottles (up to the mark). The bottles are also labeled as “RPMI-media” (Roswell Park Memorial Institute) with the date of preparation. Store in refrigerator. This stored RPMI media should be used within one month of preparation. At the time of setting up of culture, add 20 mL of Fetal Calf Serum (FCS) to

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80ml of the medium. From this reconstituted medium, 10 mL is taken for each culture vial while setting up the culture (Proc. No. 3). Procedure for Discarding the Used Glasswares and Other Items The used glass pipette is discarded in the 2% Sodium hypochlorite solution kept in the discarding mug. The aluminum foil wrapped in the sterile glasswares and cotton are thrown into the dustbin. Check for the sterility: Take 10 mL of media in a culture vial and incubate it in the 5% Carbondioxide (CO2) incubator at 37°C. Every day it should be checked for any growth or turbidity in the media. This has to be repeated for a week. If the media remains clear then it is sterile. Aliquot 100 mL of the medium into the milk dilution bottles (up to the mark). The bottles are also labeled as “RPMI-media” (Roswell Park Memorial Institute) with the date of preparation. Store in refrigerator. This stored RPMI media should be used within one month of preparation. At the time of setting up of culture, add 20 mL of Fetal Calf Serum (FCS) to 80 mL of the medium. From this reconstituted medium, 10 mL is taken for each culture vial while setting up the culture. Discarding the used glasswares and other items: The used glass pipette is discarded in the 2% Sodium hypochlorite solution kept in the discarding mug. The aluminum foil wrapped in the sterile glass wares and cotton are thrown into the dustbin. Safety Precautions As a safety measure Facemask is worn while preparing the culture media. Appropriate gloves are worn during media preparation. RPMI media is prepared in Class II laminar airflow hood. REQUIRED GLASSWARES Culture vial with screw cap 3 1 10 mL pipette Mantoux syringe (disposable) 1 Vial stand 1 All the above should be sterile and dry and also ensure sterilization by Ultraviolet (UV). The basal media and FCS are kept in water bath for warming and Phytohemagglutinin (PHA) is allowed to thaw at room temperature before use. i. The culture vials are labeled by using stickers with the name of the patients, MRD Number and date of setting culture. ii. The mouth of the vials are flamed and left in the vial stand for cooling.

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iii. The mouth of the media and the FCS bottles are flame sterilized. The pipette is also flame sterilized and used after cooling. iv. 20 mL of FCS is added to 100 mL of RPMI medium. From this reconstituted medium 10 mL is transferred to the culture vial using a graduated 10 mL glass pipette, after flaming and cooling them. The left out media is used for future purposes. v. With a Mantoux syringe add 0.2 mL of PHA. vi. Then add the blood sample drop by drop (25–30 drops = 0.5 mL). vii. Shake and mix well. viii. Flame the screw cap and close the culture vial. The cap is loose so as to allow CO2 when left inside the CO2 incubator. ix. Mix the contents of each culture tube gently. x. Incubate the cultures at 37°C with 5% carbondioxide, in a slanting position on a cotton bed kept inside a breadbox. This position creates more surface area between the liquid and gaseous phases and allows the cells to settle over a larger area of the culture tube, which provides optimal culture conditions for cell growth and proliferation. After this, clean the hood with rectified spirit and leave the UV on for 30 minutes The culture, which has been set, is checked every morning if there is any contamination till the harvesting time is reached. In the evening around 4 o’clock the cultures are shaken well every day till the harvesting. HARVESTING OF CHROMOSOMES2 Cells are subjected to hypotonic treatment, which increases their volume, and disrupts the cell membrane of the red blood cells allowing their removal. A fixative solution is added to the cell suspension to preserve the cells in their “swollen” state and to remove the water, thus “hardening” the biologic material. The common fixative (3:1 methanol:acetic acid) removes lipids and alters/denatures proteins thus making the cell membrane remnant very fragile, which is important for subsequent chromosome spreading. Metaphase Chromosomes The mitotic spindle formation is blocked, usually by adding colcemid to the culture, and the cell division is stopped at the metaphase level. Prometaphase Chromosomes Methotrexate is used to block the cells in the late S phase to get elongated chromosomes with a resolution of 850 bands when compared to the metaphase chromosomes. The S phase block is released by thymidine synchronization.

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Requirements 1. Methotrexate (MTX) solution 3 MTX stock solution (1 × 10– M) MTX [(+) Amethopterin] 1 mg Distilled water (Sterile) 2.2 mL Store in frozen condition in dark vials MTX working solution (1 × 10–5 M) MTX stock solution (1 x 10–3 M) 0.1 mL Distilled water (Sterile) 9.9 mL MTX is relatively unstable and can be used for one month. Store frozen in dark vials. Sterilization is usually not required. 2. Thymidine (1 × 10-3 M) Thymidine 2.5 mg Distilled water (Sterile) 10 mL Filter sterilize and store frozen in small aliquots. Solution is stable and can be used for one month. Both the solutions have to be left in dark for thawing before use. 3. 0.02% colchicines - 1 mg in 5 mL of sterile water (available commercially). Store frozen in amber colored bottles (4°C ) 4. 0.56% Potassium chloride solution (0.075 M) - Prepared freshly and prewarmed (37°C) before use. 5. Carnoy’s fixative - 1:3, acetic acid: methanol. For one tube approximately 20 mL of fixative is required for one tube. Based on the number of tubes fixative is prepared freshly before use and left in the freezer till use. 6. Centrifuge tubes 3 Nos 7. Pastuer pipettes 3 Nos 8. Micropipettes (5–50 μL) 1 No 9. Timer 1 No 10. Cold slides 4 slides per tube (approx.) 11. Forceps 1 No. 12. Diamond marker 1 No. 13. Hot plate 14. Centrifuge 15. Water bath set at 37°C 16. CO2 incubator. Procedure 1. After 72 hours of incubation, add 0.1 mL of MTX working solution (1x 10-5 M) to the culture. (Final concentration of MTX is 1x10 - 7 M.) 2. Incubate the cultures at 37° C for 17 hours (overnight) 3. Centrifuge the cultures at 1,800 rpm for 8 minutes and discard the medium containing MTX.

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4. Wash the cells twice by suspending them in fresh unsupplemented medium (medium without serum), by centrifuging (1,800 rpm for 10 minutes) and then discarding the supernatant each time. After the final wash, suspend the cells in complete culture medium containing the serum and antibiotics at usual concentrations. 5. Transfer the contents to a culture vial and add 0.1 mL of thymidine solution to each culture containing 10 mL of medium. 6. Sterilize the mouth of the culture vials and incubate the cultures at 37°C for 5 hours in the carbondioxide incubator. 7. After the completion of 5th hour, take the culture vial and leave it in vial stand in the laminar airflow hood. 8. Sterilize the mouth of the vials and allow them to cool. 9. Add 100 μL of colchicine to culture containing 10 mL of medium, gently mix and incubate at 37oC in the carbon dioxide (CO2 ) incubator for 10 minutes. 10. Take the tubes outside the incubator and leave them in the laminar airflow hood. 11. Flame the mouth of the vials and allow them to cool. 12. Take the sterile centrifuge tubes and label them with the name of the patient. 13. Flame the mouth of the centrifuge tubes and allow it to cool. 14. Then transfer the contents from the vial to the centrifuge tubes. 15. Centrifuge the tubes at 1,800 rpm for 10 minutes. 16. Discard the supernatant by pipetting leaving as little medium as possible over the cell button. 17. Resuspend the cell button in 5 mL of prewarmed hypotonic solution. Close the mouth of the centrifuge tubes with aluminium foil and incubate for 30 minutes in a water bath at 37°C. 18. Add 4–5 mL freshly made cold fixative to each tube and mix gently. Centrifuge at 1,800 rpm for 10 minutes. [Cells should be handled very gently following hypotonic treatment. Any harsh treatment may rupture the cells, leading to many incomplete metaphases in final preparations. To prevent any such undue damage to the cells, avoid passing the suspension through narrow-tipped droppers or pipettes at this stage]. 19. Discard the supernatant. Disturb the pellet thoroughly by tapping at the bottom of the tube. Resuspend the pellet in 5 mL of fresh cold fixative. 20. Again centrifuge the tubes, discard the supernatant, and suspend the cells in fresh cold fixative. Repeat this twice. 21. After the final centrifugation, suspend the cells in a small volume of fixative (0.5–1.0 mL) to make a suspension.

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22. Place 3–4 drops evenly on a cold wet slide and allow it to dry. Place the slide on a slide warmer immediately after the cell suspension is dropped on the slide (50–60°C). After the slide is completely dry, examine under low magnification (10 X) to check the cell density and spread of metaphase chromosomes. If the cell density is too high, add a few more drops of fixative to the cell suspension. If the cell density is low, centrifuge the suspension and re-suspend the pellet in a smaller amount of fixative. The slides for use are described in Process No. 1) 23. Then the slides are marked at one end (usually at right hand corner) with the following details: G-No (Genetic reference No.), Date, method of culture with the slide numbers (96 hr represented by “HR” meaning “High Resolution”; slide Nos given according to the order of the slides prepared (HR-1, HR-2, etc.). 24. After all the slides are checked, dried and kept inside the slide box. 25. Allow the slides to age for at least one week or incubate the slides at 60oC for sixteen to seventeen hours to do the staining and banding. GIEMSA BANDING3 Giemsa banding (G-Banding) is the most commonly used technique for the routine staining of mammalian chromosomes. The structural and functional composition of chromosomes leads to the differential banding patterns. The slides are treated with a protease (Trypsin) and stained with giemsa. Dark bands correlate with pachytene chromomeres, generally replicate their DNA late in S-phase, contain A+T rich DNA, appear to contain relatively few active genes and may differ from light bands in terms of protein composition. Differential extraction of protein during fixation and banding pre-treatments from different regions of the chromosome may be important in the mechanism by which G-bands are obtained. Older slides will require higher trypsin concentration or more aggressive treatments to obtain good banding. Requirements Giemsa buffer (pH 6.8) 0.2 M Sodium dihydrogen phosphate (Na H2 PO4) (M.W-156) 0.2 M Disodium hydrogen phosphate (Na2 H PO4) (M.W-142) Na H2 PO4 - 14 mL Na2 H PO4 - 36 mL Mix well and make it up to 100 mL with distilled water. Store at room temperature.

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Giemsa Stain Giemsa stock solution Giemsa powder 3.8 g Glycerol (98%) 250 mL Absolute methanol (HPLC) 250 mL Weigh 3.8 g of Giemsa powder and transfer to a mortar. Measure 10 mL of glycerol and add to the powder. Grind the stain powder and transfer to a dry brown bottle with 50 dry glass beads. Remaining 240 mL of glycerol is used as above. Keep the bottle in water bath maintained at 60°C for 2 hours. Allow it to cool to room temperature. Measure 250 mL of absolute methanol and add slowly by stirring. Keep the stain in water bath at 60°C for 1 hour. Allow it to cool. Store at room temperature for one week. After a week decant and remove sediment. Store in a dark bottle at room temperature for 2-3 weeks and then use. Filter before use. Giemsa working solution (50 mL) Giemsa buffer 48 mL Giemsa stain 2 mL (to be filtered before use) Mix well and remove the froth using blotting paper Sorensen’s buffer (pH - 7.0) Disodium hydrogen phosphate 5.112 gm/600 mL Potassium dihydrogen phosphate 3.264 gm/400 mL Mix both the solutions and store at 4 °C Trypsin-EDTA (Ethylene Diamine Tetra Acetic Acid) solution Trypsin 20 mg EDTA 10 mg Sorensen’s buffer 50 mL Cyclomix well for 20–30 min at room temperature. Prepared freshly when required. Distilled water (to fill four coplin jars) Coplin jars - 5 Measuring cylinder (100 mL)-3 Prepared slides (according to the Process No. 3) Timer Microscope with oil immersion


126 Method 1. 2. 3. 4. 5. 6. 7.

All the solutions are kept in coplin jars. Preheat the slides to 50°C in the slide warmer. Incubate the slides for 10–15 seconds in the trypsin-EDTA solution. Rinse the slides thrice in distilled water (in three different coplin jars). Stain the slides in Geimsa solution for 2–3 minutes. Rinse the slides in distilled water and air-dry. The slides can be mounted and used for scanning.

Note: If the chromosomes appear fuzzy, insufficient aging of slides is the most likely explanation. Over-trypsinized chromosomes appear fat and pale while under trypsinized chromosomes are dark with poor distinction between light and dark bands. If the chromosomes appear pale and blue in colour, check the pH of buffers. Chromosomes which appear to be stained too darkly can often be improved simply by rinsing the slide in distilled water. Slide Mounting Mounting of slides is done to store the harvested chromosomes as a permanent record. 1. Requirements Slides prepared after harvesting DPX mountant Cover slips All the slides stained by the above methods (except Quinacrine banding) should be mounted for permanent preparations. DPX Mountant is required for mounting. 2 Process A layer of DPX mountant is poured on the slides. Then place a cover slip and press so as to release all the air locked between the slide and the cover slip. Wipe the excess mountant with a tissue and allow them to dry in the room temperature for 2-3 days. The mounted slides, which are the permanent preparations, are stored in the slide boxes till they are reported. Then they are transferred to the slide cupboard. Those slides, which were not stained/banded, are kept along with the stained/banded in the slide boxes. They are also transferred to the slide cupboard later along with the stained/banded slides. These slides are taken for scanning and identifying the chromosomes for karyotyping. 25 plates are scanned per patient, if abnormality is noticed, 50 more plates are scanned.

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KARYOTYPING4

To identify the chromosomes according to the International standard for cytogenetics nomenclature (ISCN) and study the cytogenetic abnormalities. Table 2.1 shows the symbols used for representation of karyotypes. Table 2.1: Symbols and abbreviations used in karyotyping ,

Separates chromosome modal number, sex chromosomes, and chromosome abnormalities

-

Loss of a chromosome

()

Surround structurally altered chromosomes and breakpoints

+

Gain of a chromosome

;

Separates rearranged chromosomes and breakpoints involving more than one chromosome

/

Separates cell lines or clones

//

Separates recipient and donor cell lines in bone marrow transplants

del

Deletion

der

Derivative chromosome (used when only one chromosome from a translocation is present, or when one chromosome has two or more structural abnormalities)

trp

Triplication of a portion of a chromosome

dic

Dicentric chromosome

dn

Chromosomal abnormality not inherited from parents (de novo)

dup

Duplication of a portion of a chromosome

fra

Fragile site (usually used with Fragile-X syndrome)

h

Heterochromatic region of chromosome

i

Isochromosome (both arms of the chromosome are the same)

ins

Insertion of a portion of a chromosome.

inv

Inversion

ish

Precedes karyotype results from fluorescence in situ hybridization (FISH) analysis.

mar

Marker chromosome (unidentifiable piece of chromosome)

mat

Maternally derived chromosome rearrangement

p

Short arm of a chromosome

pat

Paternally derived chromosome rearrangement

psudic

Only one centromere is active (pseudo dicentric)

q

Long arm of a chromosome

r

Ring chromosome

t

Translocation

ter

Terminal end of arm (i.e. 2qter - end of the long arm of chromosome 2)

tri

Trisomy

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Classification of chromosomes: Chromosomes are grouped based on their size and position of the centromere. Based on the descending order of length: Group A Chromosome pairs 1 to 3 Group B Chromosome pairs 4 and 5 Group C Chromosome pairs 6 to 12 and the X chromosome Group D Chromosome pairs 13 to 15 Group E Chromosome pairs 16 to 18 Group F Chromosome pairs 19 and 20 Group G Chromosome pairs 21, 22 and Y chromosome Based on the position of the centromere: Metacentric: Centromere at the center of the chromosome so that both the arms are of equal size, e.g. 1, 3, 16, 19, 20 Sub-metacentric: Centromere positioned towards one arm so that one is short and the other arm is longer, e.g. 2, 4, 5, 6-12, X, 17, 18 Acrocentric: Centromere positioned towards the end of the chromosome, e.g. 13-15, 21, 22, Y Identification of chromosomes under the microscope: Chromosomes have to be identified under the microscope based on the size, the position of the centromere and the banding patterns. The following is the key to identify each chromosomes. Chromosome 1 G-banding: Largest chromosome, metacentric. Short arm (p): lighter at distal end. Long arm (q): dark heterochromatin may be visible just below the centromere; two distinguishable dark bands below, separated by a goodsized light band. Chromosome 2 G-banding: Largest sub-metacentric chromosome. Short arm (p) one dark band just above the centromere and three more above that, almost equidistant from each other. Long arm(q): two-double dark bands appear, one pair in the middle region of the long arm and one sub terminally, separated by a lighter, often double band. Chromosome 3 G-banding: Metacentric; similar in length to the B group (chromosomes 4 and 5); larger than the C group. Short arm (p): proximal area dark; dark band just before a terminal light band on the distal end. Long arm (q):

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almost identical to the short arm pattern except that the distal dark band is twice as broad as its counterpart on the short arm but light bands better defined. Chromosome 4 G-banding: Very submetacentric; larger than C group (Chromosomes 6– 12); indistinguishable from chromosome 5. Short arm (p): dark band in the center, which can often be seen as double, distinguishing it from chromosome 5. Long arm (q): distinguishing band just below the centromere, which either is not present or is smaller and lighter on chromosome 5. Telomeres are much better defined. Chromosome 5 G-banding: Submetacentric; largest of C group chromosomes indistinguishable from chromosome 4. Short arm (p): very dark band in center, which may have blurred edges. Long arm (q): characterized by three bands that fuse in a dark area in the middle of this arm; area followed by a light band, a subterminal dark band (which may be split in two), and a light telomeric band; very clear dark band on the short and light telomeric bands. Chromosome 6 G-banding: Submetacentric, largest of C group chromosomes short arm (p): broad light band in the middle. Long arm (q): two dark band in the center, relatively indistinct. Negative telomere on the short arm is more distinct and more distinct bands appear on the long arm. Chromosome 7 G-banding: Submetacentric, slightly smaller than chromosome 6. Short arm(p): distinct, dark nearly terminal band, which often has a flattened appearance. Light telomeric material distal to this band is usually invisible. Long arm (q): two very distinct dark bands, followed by a third less intense dark band and a light telomere. Usually with clearly distinguished light telomeric bands. Chromosome 8 G-banding: Very submetacentric like chromosomes 10 and 12. Short arm (p): two narrow dark bands with an almost equal-sized light band between them. Long arm (q) : two dark bands: the first in the center of the arm, and the second, usually darker, band more distal, but with more distinct bands seen.

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Chromosome 9 G-banding:Varies from slightly submetacentric to very submetacentric, depending on the amount of heterochromatin located in the region just proximal to the centromere on the long arm. This area may also stain lightly and may appear stretched. Short arm (p): quite dark overall, with a central darker band. Good banding on longer chromosomes will show two distinct bands. Long arm (q): a variable, proximal, thin light area may be present— it can be as long as the short arms or almost non-existent without having any noticeable phenotypic effect, this region is called the secondary constriction. Two heavy dark bands are distal to the secondary constriction; sometimes the more distal band is divided into two dark bands. Bands are better defined with more contrast and the distal dark band on the q arm is usually double. Chromosome 10 G-banding: Submetacentric similar to chromosome 8 Short arm(p): dark overall with a small dark band at the center. Long arm (q): Prominent distinguishing dark band proximally, followed by two dark equidistant bands, the second of which may appear terminal but is actually followed by a light band, often unclear. The centromere stains darkly and the light telomeres are much better defined. Chromosome 11 G-banding: Submetacentric, but closer to metacentric than the other C group chromosomes. The area under the centromere may appear as a secondary constriction. Short arm (p): strong band just below the center, which fades out distally into a thin telomere. Long arm (q) : a proximal dark band distinguishes this chromosome from chromosome 9. This is followed by a broad, light band; a central, broad, dark band that is sometimes resolved into two dark bands; and a light telomere. The central short arm dark band and the long arm broad dark band are each seen as two close sub bands. Chromosome 12 G-banding: Very submetacentric; usually the shortest short arms in the C-group Short arm (p): dark central band. Long arm (q): proximal dark band followed by a light band, then a broad dark band, and a light telomeric region. The broad dark band on this arm is frequently divided into several sub-bands; the light telomeres are usually better defined. The long arm proximal light band is narrower than that on chromosome 11.

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Chromosome 13 G-banding: Large acrocentric, indistinguishable from chromosomes 14 and 15. Short arm (p): may or may not show short arms and/or satellites of variable size. Long arm (q): proximal half of arm light but divided in two by a narrow dark band. Distal half has two prominent dark bands separated by a narrow light band, followed by a light telomeric region with a small dark band in it. Chromosome 14 G-banding: Large acrocentric, indistinguishable from chromosome 13 and 15. Short arm (p): may or may not show short arm and /or satellites. Long arm(q): two distinguishing dark bands, one proximal and the other distal but subterminal. In particular, the light telomere of the long am is much more distinct. Chromosome 15 G-banding: Large acrocentric, indistinguishable from chromosomes 13 and 14. Short arm (p) : may or may not show short arm and/or satellites. Long arm (q) : may show two bands like chromosome 14. The first one is almost central, and the second, if present, is terminal. Chromosome 16 G-banding: Varies from almost metacentric to submetacentric, owing to a block of heterochromatin on the long arm adjacent to the centromere; usually similar in size to chromosomes 17 and 18. Short arm (p): one central dark band with flanking light bands. Long arm (q) : Short arm(p) : uniformly dark. Long arm (q) : centromeric heterochromatin stains very darkly; two more dark bands below heterochromatin block; distal dark band appears terminal. Chromosome 17 G-banding: Same size as chromosome 18, but less submetacentric. Short arm (p): prominent central dark band Long arm (q) : light with two sub terminal dark bands; hard-to-see light telomeric band. Chromosome 18 G-banding: Similar in size to chromosome 17, but more submetacentric; often the same shape as chromosome Y Short arm (p) : uniformly dark. Long arm (q) : two characteristic dark bands, one proximal, one distal. There is a narrow light telomere that is hard to distinguish except for the light short arm, the dark centromere, the two dark bands on the long arm

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that may each separate into two dark sub bands, and the more distinctly showing light telemore. Chromosome 19 G-banding: Smallest metacentric. Indistinguishable from chromosome 20. Short arm (p) : lightly stained chromatin. Long arm (q) : same as short arm, but with a proximal dark band, which may appear as part of the centromere. Narrow dark band in the center of the short arm and another one on the proximal one-third of the long arm. Chromosome 20 G-banding: Smallest metacentric, indistinguishable from chromosome 19. Short arm (p) : dark terminal band. Long arm (q) : two narrow dark bands. Light telomeres observed on both the arms. Chromosome 21 G-banding: Acrocentric, indistinguishable from chromosome 22. Short arm (p): possible short arms and/or satellites. Long arm (q): proximal region, which dominates, fading out to almost invisible light telomere with an additional narrow dark band often appearing in the light telomeric region. Chromosome 22 G-banding: Acrocentric; indistinguishable from chromosome 21. Possible short arms and satellites. Long arm (q): Dark centromeric region; light arm with dark central band. General Guidelines for Representation of a Karyotype The karyotype should represent the following details: modal number, sex chrom, abn abbrev (first chrom; second chrom) (arm band number; arm band number) Modal number: Total count of number of chromosomes Band number: Numerical description of the location on a chromosome arm, in order from the centromere out to the end of the chromosome. These numbers are according to the standard determined by International System for human Cytogenetic Nomenclature (ISCN). Figure 2.1 shows the karyotype with numerical (2.1A) and structural abnormality (2.1B). MOLECULAR CYTOGENETICS The routine cytogenetic method has certain disadvantages pertaining to specific applications like prenatal diagnosis, detection of microdeletions, etc.

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Fig. 2.1A: Ideogram/Karyotype of a Down syndrome male patient showing trisomy 21 47, XY + 21

Fig. 2.1B: Ideogram/Karyotype of retinoblastoma patient showing deletion in q 14 region of 13th chromosome: 46, XY, del 13q14

The advent of molecular cytogenetic methods that is more rapid and sensitive, which unites molecular biology and cytogenetics has revolutionized the conventional cytogenetic methods. The molecular cytogenetic methods are based on the fluorescent in in situ hybridization (FISH) that exploits the complementarity of the DNA sequences by using a DNA probe of specific length that binds to the specific DNA sequence (a part of the gene) in the chromsomes. The tagging of a fluorescent label to the probe facilitates the visualization of the signal that is produced due to the binding of the probe to its complementary region. The technique is widely applied in the field of prenatal and preimplantation genetic diagnosis to detect numerical and structural chromosomal abberations. The major advantage of FISH over conventional cytogenetic method is that can be performed in cells at any stage of cell

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cycle including the interphase stage. Thus FISH is preferable for prenatal and preimplantation genetics in which the availability of the sample is also less in quantum. The technique also facilitates the detection of chromosomal abberations at an increased resolution compared to the conventional technique. While with the conventional technique the smallest detectable chromosomal abnormality ranges ~2000–3000 kb, FISH facilitates the detection of abnormality ranging as low as 0.5 kb on metaphase chromosome. This increased resolution of FISH makes the technique more suitable for microdeletion syndromes like Prader-Willi syndrome. Thus the gap in resolution between the conventional cytogenetic (in Mb) and molecular genetic methods (in Kb) has been much shortened by the molecular cytogenetic techniques. The other approaches involving FISH technique include comparative genome hybridization (CGH) and Spectral Karyotyping (SKY), which are used for characterizing chromosomal imbalance in solid tumors, and complex chromosomal rearrangements and thus widely applied in the field of cancer genetics. The method involves denaturation as a first step to separate the complementary strands within the chromosome followed by a hybridization step to facilitate the site specific ending of the probe to its complementary specific region on the chromosome. FISH probes are of 4 different types namely gene specific probes, repetitive sequence probes, whole genomic DNA probes, and chromosome painting probes. The FISH signals are visualized by fluorescent microscope that uses a light source (mercury vapor and xenon lamps) to excite the fluorochromes with which the probes are labelled. A variety of filter sets are provided with the FISH microscope that are specific for the different fluorochromes. In the routine cytogenetic methods to prepare an ideogram, the images are captured in high magnification and printed.The specific chromosomes are cut neatly from the print and stuck in a specific format. In contrast, the images are captured by a digital imaging system such as a CCD camera provided with the microscope and then analyzed using specific systems and are also stored for future purposes. Thus automation is the key note that adds on to the advantage of FISH over conventional method. Method Reagents required 20X SSC 2X SSC Methanol 100% Ethanol 12N HCL

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1N NaOH Formamide, ultra pure grade Autoclaved filtered milli Q water Reagent preparation 1. 20X SSC (vysis) 20X SSC : 132 g Filtered Autoclaved MilliQ Water : 400 mL Mix thoroughly 20X SSC in 400 mL of autoclaved filtered MilliQ water. Measure pH and adjust to pH 5.3 with HCl. Add autoclaved filtered MilliQ water to bring final volume to 500 mL. Store at 2-8º C. Discard stock solution after 6 months, or sooner if solution appears cloudy or contaminated. 2. 2X SSC (For 500 mL) 20X SSC : 50 mL Filtered Autoclaved MilliQ Water : 350 mL Adjust the pH to 7.0 ± 0.2 with 1N NaOH. Make up to 500 mL with autoclaved MilliQ Water. Store at room temperature. Discard stock solution after 6 months, or sooner if solution appears cloudy or contaminated. 3. Denaturation solution (70% Formamide/2X SSC) Formamide : 49.0 mL 20X SSC (pH5.3) : 7.0 mL Purified water : 14.0 mL Measure pH using pH indicator strips to verify pH is 7.0–8.0. Between uses, store covered at 2–8ºC. Discard after 7 days. 4. Ethanol solution (75%, 85%, 100%) Prepare v/v dilutions of 100% ethanol with purified water. Between uses, store covered at 2–8ºC. Discard after 7 days. 5. Formamide wash solution (50% Formamide/2X SSC) Formamide : 105 mL 20X SSC (pH 5.3) : 21 mL Purified water : 84 mL pH is measured using pH indicator strips to verify ( 7.0–8.0). Pour equal volume of solution into each of the three glass coplin jars with lids. Label the jars “1”, “2”and “3”. Between uses, store covered at 2–8°C. Discard after 7 days. 6. 2X SSC/0.1%NP-40 wash solution 20X SSC (pH 5.3) Autoclaved filtered MilliQ water NP-40

: 10 mL : 90 mL : 0.1 mL


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Store at 2–8°C temperature. Discard stock solution after 6 months, or sooner if appears cloudy or contaminated. Procedure 1. A slide with good metaphase spread was taken. 2. Humidified box prepared and kept in 37°C incubator an hour prior to beginning of the procedure. 3. Place the jars containing the denaturation solution in a 73±1°C waterbath approximately 30 minutes prior to start of experiment. 4. Mark hybridization areas with a diamond tipped scribe under the specimen slide. 5. Ensure that the denaturation solution is 73±1°C. 6. To age the slides, immerse the slides in 2X SSC for 3 minutes. 7. Dehydrate slides for 1 minute in 70% ethanol, followed by 1 minute in 85% ethanol and 1 minute in 100% ethanol. 8. Immerse the slides in the denaturation solution for 5 minutes. Preparation and denaturation of the probe mixture Add to the following for each target area, to a micro centrifuge tube at room temperature • 7μl LS /WCP Hybridization Buffer • 1μl probe • 2 μL purified water 1. Centrifuge tube for 1–3 seconds. 2. Vortex and then centrifuge again. 3. Hold tube in a 73±1°C waterbath for 5 minutes to denature the probe. 4. Remove tube from water bath. Hybridizing the probe to the specimen target 1. Remove the slide from 100% ethanol. 2. Dry slides by touching the bottom edge of the slides to a blotting sheet and wiping the underside with a tissue paper. 3. Apply 10 μL of probe mixture to one target area and immediately apply coverslip without any air bobble. Repeat for additional target area. Seal the edges of the coverslip with clay. Place slides in the prewarmed humidified box, cover with aluminium foil and replace in the 37°C incubator and incubate for 12–16 hours. Washing procedure: 1. Prepare wash solutions and Keep it in 46°C the water bath 45 minutes prior to end of incubation. 2. At the end of the incubation, remove the humidified chamber from the 37°C incubator, carefully take the slide from the chamber.

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3. Remove the clay and the cover slip is lifted up carefully using the surgical blade. 4. Immerse the slide in wash solution “1” of 50% formamide/2X SSC Agitate slides for 1–3 seconds and incubate for 10 minutes. 5. The slide is blotted onto a tissue paper and transferred to the wash solution 2. Agitate slides for 1–3 seconds and incubate for 10 minutes. 6. Immerse slides in jar “3” of wash solution. Agitate slides for 1–3 seconds. Remove slides after 10 minutes. 7. Immerse slides in 2X SSC. Agitate slides for 1–3 seconds. Remove slides after 10 minutes. 8. Immerse slides in 2X SSC/0.1NP-40. Agitate slides for 1–3 seconds. Remove slides after 5 minutes. Visualizing the hybridization 1. Air-dry slides completely in darkness. 2. Apply 10 μL of DAPI II to the center of the hybridized area and apply coverslip without any air bubble. 3. Seal the corner of the coverslip with nail polish. 4. Store at room temperature for 45–60 minutes in darkness before viewing. 5. First view the cells under the 10X using filter 2 (DAPI), then to view the signal use filter 5 under oil immersion. Results of Hybridization In a normal metaphase spread, signals are observed on both chromatids on the two homologous chromosomes. The abnormal chromosomes show either single signal when there is a deletion or more than two signal on gain of a chromosome. Chromosomal translocations are also visible by this method. Figure 2.2 represents the FISH results for a metaphase spread (2.2Aand 2.2B) and interphase nuclei (2.2C and 2.2D).

Contd....


138 Contd....

Figs 2.2A to D: FISH for a metaphase spread (A and B) and interphase nuclei (C and D) from normal and retinoblastoma patient respectively

BIBLIOGRAPHY 1. McNeil N, Ried T (2000). Novel molecular cytogenetic techniques for identifying complex chromosomal rearrangements: technology and applications in molecular medicine. Exp. Rev. Mol Med. http://www.expertreviews.org/ 00001940h.htm

REFERENCES 1. Lymphocyte culture in human cytogenetics a practical approach – volume I Constitutional Analysis, second edition. Ed by DE Rooney, 1992;1:32–7. 2. Lymphocyte culture in human cytogenetics a practical approach – volume I Constitutional Analysis, second edition. Ed by DE Rooney, 1992;1:37–45. 3. Lymphocyte culture in human cytogenetics a practical approach – volume I Constitutional Analysis, second edition. Ed by DE Rooney, 1992;1:31–6. 4. An introduction to human chromosomes and their analysis in human cytogenetics a practical approach – volume I Constitutional Analysis, second edition. Ed by DE Rooney, 1992;1:14–30.

DNA BASED METHODS GENOMIC DNA EXTRACTION FROM BLOOD Principle: DNA extraction is a procedure to extract DNA for subsequent molecular analysis. Because of the large size and fragile nature of chromosomal DNA it is very difficult to isolate DNA in an intact, undamaged form. These DNA preparations are stable, of high molecular weight and relatively free of RNA and protein. DNA can be extracted from various samples like blood, saliva, hair, nail, tumors, etc. Blood samples are preferred for the ease with which they can be obtained as well as the quantity. The anticoagulants used are either heparin or acid citrate dextrose.

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The basic steps in DNA extraction are • Break open cells and remove membrane lipids using a detergent such as triton X. • Dissociation of protein DNA complexes and denaturation of the cellular proteins: Proteinase K is used for deproteinization. EDTA also helps in this step. The EDTA serves two purposes. First, it binds divalent metal ions (Cd++, Mg++ and Mn++) that could form salts with anionic phosphate groups of DNA. Second, it inhibits deoxyribonucleases that have a requirement for Mg++ or Mn++. The mildly alkaline medium (pH 8) acts to reduce the electrostatic interaction between the DNA and the basic histones and polycationic amines. The relatively high pH also tends to diminish nuclease activity and reduces the positive character of the histones. Detergents like triton X are used at the stage to disrupt the ionic interactions between positively charged histones and the negatively charged backbone of DNA. Phenol helps in the denaturation of proteins as well as their removal from the interphase in a soluble form. The phenol used is equilibrated, as DNA partitioning is pH dependent. Depending on the pH of the phenol, DNA will partition into either the organic phase or the aqueous phase. If the pH is greater than 7.0, both DNA and RNA will partition into the aqueous phase. If it is below 7.0 DNA gets denatured and will partition into the organic phase and interphase while the RNA will partition into the aqueous phase. To facilitate the process of DNA extraction, phenol is equilibrated to increase its pH from acidic to alkaline. Chloroform aids the DNA extraction by improving separation of nucleic acid into the aqueous phase and also dissolve lipids • Precipitation of DNA: Ice cold absolute ethanol or isopropanol is used to precipitate the DNA where it is insoluble and clings together. A final wash with 70% ethanol removes the salt precipitated with ethanol. Reagents used 1. Digestion Buffer: Tris (1M), Triton X, Proteinase K with MilliQ water digests most of cell components. 2. Equilibrated Phenol 3. Tris EDTA (TE Buffer): Isolated DNA should be stable than in the cells. The best way for storage is in TE for stability. 4. Equilibrated Phenol: Chloroform: Isoamyl alcohol (25:24:1) 5. Chloroform: Isoamyl alcohol (24:1) 6. Saturated NaCl 7. 100% Ethanol 8. 70% Ethanol

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Preparation of Reagents Tris(1M) pH 8 Tris - 60.55 g Final volume made up to 500 ml with water Digestion buffer: Tris (1M) - 250 mL Proteinase K - 10 μL Triton X - 50 mL MilliQ water - 700 mL EDTA (0.5M) pH 8 EDTA - 1.8612 g MilliQ water - 100 mL Equilibrated Phenol • The phenol crystals stored at -20°C are brought to room temperature and liquefied at 60°C and equilibrated with equal volume of 0.5M Tris HCl (pH 8) for 15 minutes. For equilibration, 250 mg of 8-hydroxy quinolone 125 mL of MilliQ water and 125 mL of 1 M tris are added to liquefied phenol and stirred with a magnetic stirrer for 15 minutes at room temperature. 0.1% 8 hydroxy quinolone was added to prevent the oxidization of phenol. • The upper aqueous phase is aspirated and the above two steps are repeated with the organic layer till the pH reaches 7.8. • The aqueous phase was removed and the above step was repeated until the pH of the phenol reaches 7.8. This pH facilitates the partitioning of DNA to the aqueous phase. To the final organic phase 0.1 volume of 0.1M Tris chloride (pH 8.0) containing 0.2% β-Mercaptoethanol was added. • The phenol equilibrated by the above procedure is stored at 4°C. Protocol for DNA Extraction • 3 mL of heparinised whole blood from a vacutainar container was taken in a 15-mL centrifuge tube. • To the blood 1 mL of 1M Tris as buffer solution, 250 μL of 0.5 M EDTA and 1 mL of Digestion Buffer was added. • This solution is then incubated at 65°C for 1 hour. • Equal volume of equilibrated phenol was added and mixed and centrifuged at 2000 rpm for 10 min. • The solution was deproteinized by adding 3 mL of equilibrated phenol: chloroform: iso amyl alcohol (25: 24: 1) followed by centrifugation at 2000 rpm for 10 min. This step ensures complete dissociation of the DNA protein complex and removes bound cationic amines.

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• Upon centrifugation, three layers were formed: an upper aqueous layer, lower organic layer, and a compact layer of denatured proteins at the interface between aqueous phase and the organic phase. The upper aqueous phase containing nucleic acids was then separated. This step was repeated till the interface was minimal. • This solution was mixed and centrifuged with chloroform: iso amyl alcohol (24: 1) mixture. Chloroform causes surface denaturation of the proteins and removal of the oil. Iso amyl alcohol reduces foaming and stabilizes the interfaces between the aqueous phase and the organic phase where the proteins collect. • The supernatant layer containing the DNA was collected and two volumes of ice chill absolute ethanol was used to precipitate the DNA. Because of the ionic nature of DNA, it becomes insoluble if the aqueous medium is made less polar by adding an organic solvent. • The DNA formed a thread like precipitate that could be collected by spooling onto a glass rod. Proteins can be removed by dissolving the spooled DNA in saline medium (1/10 volume of saturated NaCl). This spooled DNA solution was incubated at - 20°C for 16 hrs. • RNA does not normally precipitate like DNA, but it still could be a minor contaminant. RNA may be degraded during the procedure by treatment with ribonuclease or it separates itself because of its instability. • Following incubation and centrifugation at 2000 rpm for 8 min, the supernatant was discarded. The settled pellet was washed once with 100% ethanol and centrifuged at 2000 rpm for 5 min. • The DNA pellet obtained was washed twice with 70% ethanol. • The tube with DNA pellet alone was sealed with paraffin film to reduce contamination and incubate at 37°C for 4–5 hrs for drying the ethanol. • After complete removal of alcohol, DNA was redissolved in 550 μL of TE buffer, which protects from DNAses. This was then incubated at 37°C overnight and transferred to vials. • DNA solution can be stored in frozen state, but repeated freezing and thawing tense to damage long molecules by shearing. • 10 μL of the stock DNA was added to the vial containing 990 μL of MilliQ water. Quantification was performed using Beckman Spectrophotometer at 260 nm and at 280 nm. After quantification the stock DNA was stored at 4°C for further use. Quantification of DNA The different methods of DNA quantification includes 1. Spectrophotometer methods based on UV absorbance referred to as the OD method

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2. Fluorescent based methods namely PicoGreen dsDNA Quantitation Reagent which selectively binds to dsDNA Detection of fluorescent signal from a 5' exonuclease assay The wide availability of the spectrophotometer and minimal use of reagents accounts for the advantage of the method while the major disadvantage being the utilization of large volume of the extracted DNA for estimation in addition to its inability to distinguish ssDNA and dsDNA. The fluorometric methods demands less volume of extracted DNA thus limiting the consumption of DNA yet giving the accurate and precise value on the amount of extracted DNA. Quantification of nucleic acid is done in spectrophotometer at a wavelength of 260 nm and 280 nm according to the principle of BeerLambert’s law. The ratio between the optical density (OD) reading at 260 nm and 280 nm provides an estimate of the purity of DNA and should have a value of 1.8. If there is contamination with protein or phenol the OD 260/OD 280 will be significantly lesser than the value mentioned above and accurate quantification of nucleic acid will not be possible. For the calculation of the DNA, concentration, OD value was taken for 10 μL of the stock DNA in 1 mL of sterile water. The dilution factor was then calculated for DNA concentration of 50 ng/μL. POLYMERASE CHAIN REACTION (PCR) PCR is a technique used to amplify the number of copies of a specific region of DNA in order to produce enough DNA to be adequately tested. PCR has become one of the most widely used techniques in molecular biology. It is a rapid and simple means of producing relatively large numbers of copies of DNA molecules from minute quantities of source DNA material. PCR is a rapid and versatile in vitro method for amplifying defined target DNA sequences present within a source of DNA. PCR was invented in the mid-1983 by Kary Mullis and the technique was made possible by the discovery of the thermostable enzyme, Taq polymerase, the DNA polymerase that is used by the bacterium Thermus aquaticus that was discovered in hot springs. As the reaction mixture contains primers complementary to both strands of DNA, the products of the DNA synthesis can themselves be copied with the opposite primer. PCR helps in the detection of low level bacterial infections or rapid changes in transcription at the single cell level, as well as the detection of a specific individual’s DNA in forensic science. The purpose of PCR is to make a huge number of copies of a gene. This is necessary to have enough starting template for sequencing which is used for screening genetic disorders, site specific mutation of DNA, or cloning or subcloning of cDNAs.

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Requirements for PCR Template : DNA sequences (e.g. total genomic DNA or a complex cDNA population). Primers: Short stretches of nucleotides, which are often about 18–25 nucleotides long act as oligonucleotide primers (amplimers) and will be specific for the target sequence. The primer sequence information are got from the target sequences. The specificity of amplification depends on the extent to which the primers can recognize and bind to sequences other than the intended target DNA sequences. For complex DNA sources, such as total genomic DNA from a mammalian cell, it is often sufficient to design two primers about 20 nucleotides long. Taq polymerase and deoxynucleoside triphosphates (dNTPs) : After the primers are added to denatured template DNA, they bind specifically to complementary DNA sequences at the target site. In the presence of a suitably heat-stable DNA polymerase and DNA precursors (dNTPs: dATP, dCTP, dGTP, and dTTP), they initiate the synthesis of new DNA strands which are complementary to the individual DNA strands of the target DNA segment, and which will overlap each other. Buffer and MgCl2: Magnesium chloride acts as the cofactor for the Taq polymerase enzyme while the buffer provides the required salt concentration and pH for the PCR. The PCR is a chain reaction because newly synthesized DNA strands will act as templates for further DNA synthesis in subsequent cycles. After about 25 cycles of DNA synthesis, the products of the PCR will include, in addition to the starting DNA, about 105 copies of the specific target sequence, an amount which is easily visualized as a discrete band of a specific size when submitted to agarose gel electrophoresis. A heat-stable DNA polymerase is used because the reaction involves sequential cycles composed of three steps: Denaturation (at 94 ºC). During this step, the double strand melts open to single stranded DNA, and all enzymatic reactions stop. Hybridization or Annealing (ideal at 50–55 ºC). At this step, the singlestranded sample DNA is “mixed” with a probe, and under the right conditions, hydrogen bonds form between this probe and its complementary sequence in the sample DNA. The double-helix structure is re-created. DNA synthesis or extension (at 72 ºC). This step presents the ideal temperature for the polymerase. Here, the primer (a piece of DNA that is base paired to the template strand in such a way that the 3’ end of it is available to serve as the starting point for the new DNA) starts the process of “multiplying” the amount of DNA by attracting bases to attach to it. Primers that are on positions with no exact match get loose again and don’t give an extension of the fragment.


144 Types of PCR

Different types of PCR are adopted for improving the specificity of the reaction. Accidental matching at the 3’ end of the primer is critically important: spurious products may derive from substantially mismatched primer-target duplexes unless the 3’ end of the primer shows perfect matching. Nested Primers The products of an initial amplification reaction are diluted and used as the target DNA source for a second reaction in which a different set of primers is used, corresponding to sequences located close, but internal, to those used in the first reaction. Hot-start PCR Mixing of all PCR reagents prior to an initial heat denaturation step allows more opportunity for nonspecific binding of primer sequences. To reduce this possibility, one or more components of the PCR are physically separated until the first denaturation step. Touch-down PCR The annealing temperature is lowered incrementally during the PCR cycling from an initial value above the expected Tm to a value below the Tm. Requirements • DNA: This is diluted from stock DNA with TE buffer. • 10X PCR buffer (100 mM tris pH 9.0, 500 mM KCl, 15 mM MgCl2, 0.1%gelatin) • Taq polymerase • dNTPs: Each dNTP- 10 μM/100 μL. (40 nM of dNTP per reaction) • Primers: Specific primers both forward and reverse primers • Autoclaved milli Q water • 0.2 mL vials, microtips, micropipettes, PCR machine. The procedure for preparing PCR cocktail is given in Table 2.2. Table 2.2: Cocktail preparation for setting PCR Reagents

Concentration

Volume (μL)

Genomic DNA Forward primer (FP) Reverse primer (RP) dNTPs PCR buffer Taq DNA polymerase Autoclaved milli Q water Total

100 ng 20 pmoles/μL 20 pmoles/μL 40 nmoles 10 x 3 U/μL

2.0 0.2 0.2 2.0 2.0 0.2 13.4 20.0

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Formula for Calculation of Annealing Temperature (AT) 2 (A + T) + 4 (G + C) = AT [AT of FP + AT of RP] ÷ 2 – 5 = working Formula for Calculating Working Primer Dilution Working primer concentration Required concentration Stock concentration n

= = = =

0.325 × n × 2 = 20 pmoles/μL 0.325 × n × 2 (X μg ) Y μg/100 μL number of bases (mer length of the primer) Required concentration × stock volume Working primer dilution = stock concentration

X g × 100 Y g = Z mL Z μL made up to 100 μL gives the working primer with the required concentration.

=

PCR Protocol

Electrophoretic Techniques Electrophoresis is a technique used to separate proteins and nucleic acids — that differ in size, charge or conformation. When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge. In contrast to proteins, which can have either a net positive or net negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone, and migrate toward the anode. Proteins and nucleic acids are electrophoresed within a matrix or “gel”. The gel is cast in a slab, with wells for loading the sample. The gel is immersed within a buffer that provides ions to carry current and maintain the pH at a relatively constant value. The gel is composed of either agarose or polyacrylamide according to the requirements. Agarose is a polysaccharide extracted from seaweed that is used at concentrations of 0.5 to 2%. Agarose gels have a large range of separation, but relatively low resolving power and the concentration can be varied according to the size of the molecules to be separated.

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Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains is based on the concentration of acrylamide usually used between 3.5 and 20%. Polyacrylamide gels are prepared between glass plates or cylinders to prevent their contact with oxygen, which inhibits the polymerization process by acting as a free-radical scavenger. Acrylamide is a potent neurotoxin and thus handled with care by using disposable gloves and mask when handling the powder/solutions of acrylamide. Polyacrylamide gels have a rather small range of separation, but with very high resolving power. A polyacrylamide gel forms when a dissolved mixture of acrylamide and cross-linker monomers polymerize into long chains that are covalently linked. The most common cross-linker is N, N’-methylenebisacrylamide (bis). The polymerization of acrylamide gel can be initiated either by chemical peroxide or by a photochemical method. The most common method uses ammonium persulfate as the initiator peroxide and the quaternary amine, N,N,N’,N’-tetramethylethylenediamine (TEMED) as the catalyst. For photochemical polymerization, riboflavin and long-wave ultraviolet (UV) light are the initiator and TEMED is the catalyst. The photochemical reaction is started by shining long-wavelength ultraviolet light on the gel mixture, usually from a fluorescent light. Since the polymerization process generates heat that can result in breaking of the glass plates, the concentration of initiator-catalyst reagents are adjusted for high concentration gels so that complete polymerization requires 20 to 60 minutes. Agarose Gel Electrophoresis Requirements Agarose (2%) 10X TBE buffer Tris – 54 gm Boric acid – 27.5 gm EDTA – 3.72gm Distilled water – 500 mL Ethidium bromide (2 mg/mL concentration) Tracking dye- Bromophenol blue (BPB) Bromophenol blue – 0.01 gm 1X TBE buffer – 10 mL Sucrose – 4 gm in 10 mL water Mix equal volumes of 0.1% BPB and sucrose. Preparation of agarose gel • The gel trough was cleaned with ethanol and the ends were sealed with cellophane tape.

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• Combs were placed in the respective positions to form wells. • 0.5 gm of agarose was weighed and dissolved in 25 mL of 1X TBE Buffer (2% agarose gel). • The agarose was melted in microwave oven and 8 μL of ethidium bromide was added to the molten agarose. • This was mixed and poured on to the sealed trogh and allowed to set in dark. • After 20 min when the gel gets solidified, the cellophane tapes and combs were removed and the trough placed in electrophoresis tank containing 250 mL of 1X TBE Buffer. • 10 μL of amplified DNA product was mixed with 5 μL of 0.1% bromo phenol blue and loaded on to the wells. • 1 μL of Molecular weight marker diluted with 4 μL of 1X TBE Buffer and mixed with 5 μL of bromo phenol blue and loaded onto the wells. • The electrophoresis was run at 100 V for 30 to 45 min. The flow of current is confirmed by observing bubbles coming off the electrodes. The distance DNA has migrated in the gel can be judged by visually monitoring migration of the tracking dyes. Bromophenol blue migrate through agarose gels at roughly the same rate as double-stranded DNA fragments. When adequate migration has occurred, DNA fragments are visualized under gel documentation system. The gel was captured in the Liscap software and analyzed using Imagemaster Totallab gel documentation system. UV sample tray and orange filter was used to visualize ethidium bromide staining. Note: The fluorescent dye (Ethidium bromide) intercalates between bases of DNA and RNA. It is often incorporated into the gel so that staining occurs during electrophoresis, but the gel can also be stained after electrophoresis by soaking in a dilute solution of ethidium bromide. To visualize DNA or RNA, the gel is placed on a ultraviolet transilluminator. Be aware that DNA will diffuse within the gel over time, and examination or photography should take place shortly after cessation of electrophoresis. Factors Affecting Mobility in Agarose Gels Agarose Concentration: Higher concentrations of agarose facilitate separation of small DNA fragments, while low agarose concentrations allow resolution of larger DNAs. The image below shows migration of a set of DNA fragments in three concentrations of agarose, all of which were in the same gel tray and electrophoresed at the same voltage and for identical times. It could be noticed from the gel that larger fragments are better resolved in the 0.7% gel, while the small fragments separated best in 1.5% agarose. The 1000 bp fragment is indicated in each lane.

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Voltage: As the voltage applied to a gel is increased, larger fragments migrate proportionally faster that small fragments. Thus the best resolution of fragments larger than about 2 kb is attained by applying no more than 5 volts per cm to the gel (the cm value is the distance between the two electrodes, not the length of the gel). Electrophoresis Buffer: Several different buffers have been recommended for electrophoresis of DNA. The most commonly used for duplex DNA are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength. Buffers not only establish a pH, but provide ions to support conductivity. If you mistakenly use water instead of buffer, there will be essentially no migration of DNA in the gel! Similarly, if you use concentrated buffer (e.g. a 10X stock solution), enough heat may be generated in the gel to melt it. Effects of Ethidium Bromide: Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels, as shown by all the images on this page. As described above, it can be incorporated into agarose gels, or added to samples of DNA before loading to enable visualization of the fragments within the gel. As might be expected, binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility (Figure 2.3). Polyacrylamide Gel Electrophoresis (PAGE) Requirements Reagents for gel preparation • Stock Acrylamide (30%)

Fig. 2.3: 2% Agarose gel with PCR amplified products for myocilin gene exons. Lanes 2–5, 7–10, 15–17 and 19–21: amplified products; Lanes 6 and 18: Phi X 174 DNA/Hinf I digest (molecular weight marker)

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• Acrylamide – 29.2 g • Bis acrylamide – 0.8 g • Dissolved completely in 50 mL water and then made up to 100 mL and then filtered. Tris pH 8. 8 = dissolve 18 g of TRIS in 100 mL of water. Tris pH 6.8 = 6 g of TRIS in 100 mL water. Note: The solution is made up to 100 mL after adjusting the pH with 1N HCl. 10% Ammonium per sulfate (APS)= 10 g in 100ml water. TEMED: commercially available.

Equipment Vertical PAGE tank, Glass plates, Spacers, combs, Power pack. Protocol for preparing 12% gel: Note: Percentage of the PAGE can be varied according to the size of the products. Clean the glass plates thoroughly with soap water, dry them and wipe them with ethanol. • Fix the plates with spacers to the support stand and clamp tightly. • Pour a small amount of molten Agarose to seal the bottom of the plates. When solidified, pour the separating gel over it to three-fourth and allow it to polymerize at room temperature for 20-30 minutes. • Pour the stacking gel over the separating gel, and place the comb over it and allow it to polymerize for 10–15 minutes. • Mix equal amount of the amplified product with bromophenol blue and load into the wells and subject it to electrophoresis at 100 V for 30–40 min. • Fix the gel in 10% ethanol with 0.5% acetic acid for 3 min. Repeat the step twice. • Place the in 0.1% silver nitrate solution (prepared in AMQW) for 15 min and rinse with two changes of autoclaved milliQ water.

Fig. 2.4: PAGE showing products (97 – 131bp) of microsatellite marker (GT repeat in the promoter of bTNF β gene Lanes 1 – 5; 7 – 9: PCR amplified products; Lane 6 molecular weight marker (100bp ladder)


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Table 2.3: Preparation of stacking and separating gel for PAGE Reagents

Separating Gel

Dis. Water (mL) Tris HCl (mL) 30% Acrylamide (mL) 10% Ammonium per sulfate TEMED

3.5 2.5 (pH 8.8) 4.0 50 μL 5 μL

• Transfer the gel to 1.5% sodium hydroxide solution containing 0.1% formalin solution for 20 min. • Fix the gel in 0.75% Na2 CO3 for 10 min. • Place the gel in a white tray and captured using blue filter and analyzed in VDS Image master Gel documentation system (Pharmacia biotech.) Figure 2.4 shows silver stained PAGE gel with PCR products of a (GA)n repeat marker. BIBLIOGRAPHY 1. Sambrook J, Fritsch EF, Manniatis T. Gel electrophoresis of DNA and pulse field Agarose gel electrophoresis. In: Molecular cloning: A Laboratory Manual, Vol 2; Cold Spring Harbour Press, USA, 1997;6;6.3:6.5

TECHNIQUES FOR SCREENING THE NUCLEOTIDE CHANGES IN CANDIDATE GENES Mutation screening of the candidate genes in patients is the most sensitive and well-adopted method to confirm the linkage data for a disease. PCR based approaches are the most common methods employed for such purposes in which the genomic DNA or the cDNA of interest are amplified and screened by different techniques. The difference in the annealing pattern of the primers designed specifically for the wild type/mutant sequence followed by gel analysis based on which the Allele specific PCR amplification system (ARMS) works is used to detect the known sequence changes. The differences in the electrophoretic mobility between the single strand of wild type and mutant upon denaturation that is in turn governed by their conformation (Single-strand conformation polymorphism) or the heteroduplex formation on reannealing (Heteroduplex analysis) is used for detecting unknown mutations as a rapid method of mutation detection. An advanced use of chromatographic column based on similar principle mentioned above called as the denaturing high performance liquid chromatography (DHPLC) is used to analyze samples for detecting the known/unknown nucleotide variations. The other rapid techniques for the detection of known mutations include restriction enzyme digestion in which

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the alteration in the recognition site for restriction enzyme due to mutation is exploited for detecting the nucleotide change. The direct sequencing of the genomic DNA or the cDNAs the gold standard for screening unknown mutations, despite being an expensive technique. Sanger Dideoxy Sequencing DNA polymerases copy single-stranded DNA templates by adding nucleotides to a growing chain. Chain elongation occurs at 3’ end of the primer,an oligonucleotide that anneals to the template. The extension product grows by the formation of phosphodiester bond between the 3’ hydroxyl end at the growing end of the primer and the 5’ phosphate group of the incoming deoxynucleotide. The growth is in the 5’→ 3’ direction. DNA polymerases can also incorporate analogues of nucleotide bases. The dideoxy method of DNA sequencing devoloped by Sanger et al (1981) takes advantage of this ablity by using 2’,3’-dideoxynucleotides as substrates. When a dideoxy nucleotide is incorporated at the 3’ end of the growing chain, chain elongation is terminated selectively at A,T,Gor C because the chain lacks 3’ hydroxyl groups. In automated fluorescent sequencing, fluorescent dye labels are incorporated into DNA extension products using 5’-dye labeled primers or 3’-dye labeled dideoxynucleotide triphosphate (Table 2.4; dye terminators). The most appropriate labelling method to use depends on sequencing objectives, the performance characteristic of each method,and on personal preference. PE Applied Biosystem DNA sequencer detects fluorescence from four different dyes that are used to identify the A,G,T and C extension reactions. Each dye emits light at a different wavelength when extited by an argon ion laser. All four colours and therefore all four bases can be detected and distinguished in a single gel lane or capillary injection. Table 2.4: The four nucleotide bases with the respective acceptor dyes and color emission Terminator

A C G T

Acceptor dye

Color of raw data on ABI PRISM310 electrophoretogram

dR6G dROX dR110 dTAMRA

Green Red Blue Black

ABI PRISM 310 Genetic Analyzer (Figurs 2.5A and B) which is an automated instrument was used for analyzing fluorescently labeled DNA

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Figs 2.5A and B: (A) Region of laser emission that detects the fluorescent signals, (B) processed sequence of a gene segment

fragments by capillary eletrophoresis. The sequencing reaction sample tubes are placed in an autosample tray that holds 48 samples. The autosampler successively brings each sample into contact with the cathode electrode and one end of a glass capillary filled with a separation polymer. An anode eletrode at the other end of the capillary is immersed in buffer. The sample enters the capillary as current flows from the cathode to the anode. The short period of electrophoresis conducted while the capillary and the cathode are immersed in the sample is called electrokinetic injection. The end of the capillary near the cathode is then placed in buffer. Current is applied again to continue electrophoresis. When the DNA fragments reach the detector window in the capillary, a laser excites the fluorescent dye labels. Emitted fluorescence from the dye is collected once per second by cooled, charge-coupled device (CCD) camera at particular wavelength bands (visual filters) and stored as digital signals on a Power Macintosh computer for processing. The Sequencing Analysis software interprets the results, calling the bases from the fluorescence intensity at each datapoint. The protocol and reaction conditons for cycle sequencing reaction is given in Tables 2.5 and 2.6. Purification of Extension Products The extension products were purified to remove the unincorporated dye terminators before subjecting the samples to capillary electrophoresis. Excess dye terminators in sequencing reactions obscure data in the early part of the sequence and can interfere with base calling. Procedure 2 μL of 125 mM EDTA and 2 μL of 3M sodium acetate (pH 4.6) were mixed to the cycle sequenced products followed by the addition of 50 μL of absolute ethanol and incubated at room temperture for 15 minutes followed by

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Table 2.5: Protocol for cycle sequencing reaction Components

Volume (μL)

Amplified products Sequence buffer Primer (2pmoles/μL) RRMIX Water

2.0 2.0 2.0 1.0 3.0

The amount of buffer and RRMIX are varied according to the product size. Table 2.6: PCR conditions for cycle sequencing for 25 cycles PCR step Initial denaturation Denaturation Annealing Extension

Temperature (°C)

Time

96 96 50 60

1 min 10 sec 5 sec 4.00 min

centrifugation at 8000 rpm for 20 minutes to precipitate the amplified product and remove the unutilized ddNTPs, primer (short length molecules), etc. The pellet was washed twice with 75% ethanol followed by air drying. The purified samples are suspended in formamide and subjected for capillary electrophoresis in ABI PRISM 310/3100/Z genetic analyzer. The sequences were then analyzed in Sequence Navigator software (version 1.0.1; ABI Prism 310) or Seq scape manager (version 2.1; ABI Prism 3100, AVANT Figure 2.6)

Fig. 2.6: Electrophoretogram of OPTN sequence with IVS7+24G>A polymorphism run in ABI prism 3100 Avant Genetic analyser. (A) WT (G/G) (FP), (B) Heterozygous (G/A) (FP), (C) Homozygous (A/A) FP, (D) Homozygous (T/T) RP

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Genetic Linkage Linkage analysis is a technique aimed at finding the estimated chromosomal location of any type of disease gene. Two loci that are on the same chromosome, are said to be in syntenic. Alleles at the two loci which are close enough either maternal or paternal in origin tend to pass to the same gamete (sperm or egg) and hence are transmitted together to an offspring; resulting in a cosegregation at the two loci. However, when the chromosomes pair up together at the time of gamete formation (a process known as meiosis), portions of the paternal and maternal chromosomes (in each of the parent) interchange by a process known as crossing over. Then the alleles received by the offspring at the two loci from one parent are no longer identical to those that occur in one of the parental chromosomes. In fact, they have recombined. The closer the loci are together, the lesser the probability of a recombination, and thus the larger the probability of cosegregation, a phenomenon called genetic linkage. The fraction of gametes in which recombination is likely to occur between two loci is the recombination fraction, usually denoted as θ. If the two loci are far apart, segregation at one locus is independent of that at the other, and θ = 1/2; all four different types of gametes are produced in equal frequencies. When linkage occurs, 0< θ <1/2, and when gametogenesis the chance for maternal and paternal-type gametes are more frequent than the recombined gametes. In linkage analysis, two parameters are tested to see if they are linked, locus for genetic marker is the measured one for which the genotype is known with a high degree of certainty, and the other is a disease locus with unknown genotype, for which the genotype is inferred only through the disease or trait phenotype. When genotyping of marker loci at known locations is done we can test each marker for linkage to a disease or trait and approximate the location of the disease or trait to the chromosomal region harboring the linked marker. The further apart two loci are, the higher the probability of recombination between them and this is the measure of genetic distance. Genetic distance is correlated with physical distance, but there is no simple function that relates them because of the varying recombination frequencies across the chromosomes. The unit of recombination is the Morgan, which is defined as the distance in which exactly one crossover is expected to occur. A Morgan is divided into centiMorgans (cM), where 1 Morgan = 100 cM. As recombination is the measure of genetic distance, identifying recombinants and estimating recombination fractions is the only obvious way to construct the genetic map. For this there should be markers to identify double heterozygotes for identifying recombinants. In principle any Mendelian trait can be a marker. But disease-disease, trait-trait mapping is not possible in humans.

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Researchers have used different markers for the identification of disease genes starting from blood groups to serum proteins, which are not DNA markers. Initially gene-mapping studies used restriction fragment length polymorphisms (RFLP), which were the first generation DNA markers, but the advent of PCR and the enormous information given by the human genome project facilitated the concept of microsatellite regions to be used extensively as DNA markers. Microsatellite DNA sequences consist of short repeats of one to five base pairs together with satellites and minisatellites they belong to a large family known as tandemly repetitive sequences. Microsatellites or STRs (short tandem repeats) comprise of simple mononucleotide to pentanucleotide repeats, varying from a few tens of bases up to typically one hundred.1 They are spread almost evenly across the entire human genome. Microsatellites are thought to arise by a process that has been multiply referred to as “DNA slippage”, “polymerase slippage”, or “slipped strand mispairing”. In essence, this slippage is thought to occur within the complex of proteins that mediates DNA replication, as a consequence of mispairing (by one repeat unit or occasionally more) between the original template strand and the newly synthesized DNA strand. Microsatellites are the most widely used and ideal genetic markers for genetic studies in human diseases. They are abundant, highly polymorphic and, more importantly, they are spread across the entire euchromatic part of the genome. As a result they have largely replaced minisatellites and represent the markers of choice for many genetic applications. As they are widely used in many different applications that a universally accepted system for naming and cataloguing have evolved. The naming of human microsatellite markers are in standard formats—for example, D20S324, where 20 is the chromosome on which the marker is located and 324 comprises a unique identifier.2 Important information pertaining to each marker, like cytogenetic location, heterozygosity, allele frequencies, and assay conditions, primer sequence, product size can be obtained using online public databases. Some of the major applications which use microsatellite genotyping are linkage analysis mainly used for gene mapping, triplet repeat expansion studies for anticipatory diagnostic tests, loss of heterozygosity, which is a technique used to identify the possible locations of tumor suppressor genes and in gene mapping diagnostics. The basic concept of genetic linkage analysis is relatively simple, relying fundamentally on two assumptions: 1. At a given locus each parental allele has an equal probability of being transmitted to a child as a result of the random meiotic recombinations. 2. The lesser the genetic distance between two loci the higher the chance that they will be co-inherited.

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So particular microsatellite marker alleles, which are inherited preferentially by those family members with the disease, are said to be linked, and hence co-localize with the disease gene. In contrary, the alleles of unlinked markers located far away from the disease gene locus should be inherited equally between affected and unaffected family members. So one of the most important steps in gene mapping by positional cloning approach is genotyping of marker alleles (at present the description would restrict to microsatellites). A decade ago microsatellite genotyping was done using traditional techniques only. DNA fragments separated in polyacrylamide have been visualized by techniques such as autoradiography or silver staining. However, for several reasons, such gels had drawbacks and limitations, which have now been resolved by the use of fluorescence technology and robust software fragment calling. In this technology reference size standards and PCR primers used to amplify the microsatellite sequences are covalently labeled with different fluorophores. These fluorophores emit fluorescence after absorbing the light energy supplied by a laser and the fluorescent signal is captured by CCD camera and interpreted by a computer and displayed as different colors in real time. After automated fragment sizing, allele calling is a task of relabelling fragments of the same size as the same allele. This is called as binning process. The computer software-sizing algorithm generates non-integer values, which have to be rounded to an integer value uniformly.3 The technology described above is now standard in many laboratories. But it is predicted that in due course atleast in certain applications it may change, and microsatellites may themselves be outdated as the markers of choice. There is debate that for linkage analysis single nucleotide polymorphisms may possess certain advantages over microsatellites, and could ultimately replace them when haplotyped. Significance of LOD Scores In 1955, Morton developed a statistical method to calculate the overall likelihood of the pedigree, on the alternative assumptions that the loci are linked (recombination fraction, θ=0) or not linked (recombination fraction, θ=0.5). The ratio of these two likelihood gives the Odds of linkage, and the logarithm of Odds is the LOD score. Morton demonstrated that LOD scores represent the most efficient statistic for evaluating linkage in pedigrees and derived formulae to give the LOD score. LOD scores, thus provides a measure of the likelihood that any preferential inheritance seen for a given marker is the result of genuine linkage as opposed to simple chance. In linkage analysis, the LOD score (Z) is used as a measure of strength of linkage between loci. The LOD score is the log10 of the odds for linkage, and is calculated in association with a value of the recombination fraction (θ) between loci. It compares the likelihood of the observed data if the loci

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are linked and separated by a distance of θ, with the likelihood of the data given that the loci are unlinked (θ=0.5). LOD = Z = log10 [likelihood of data linked at θ/likelihood of data if loci unlinked (θ=0.5)].Likelihood L = θR(1- θ)NR, R= Number of recombinants, NR=Number of non-recombinants. So Z = log10 [θR(1 - θ)NR/ 0.5R(0.5)NR] Standard LOD score analysis is usually unsuitable for complex/nonMendelian diseases as they do not have a specific inheritance pattern. Moreover, standard LOD score analysis called parametric/model based analysis requires a precise genetic model, detailing the mode of inheritance, gene frequencies and penetrance of each genotype. As long as a suitable model is available, parametric linkage provides a wonderfully powerful method for scanning the genome. For Mendelian diseases, specifying an adequate model should be no big problem, whereas nonmendelian conditions/complex diseases, however, are not the same. There are many algorithms like LIPED, LINKAGE, MLINK (LCP), MERLIN, GENEHUNTER, and FASTLINK, etc. for calculating the LOD score. Genotyping of Microsatellite Markers in ABI Prism 310/3100 AVANT Genetic Analyzer The primers were designed to amplify the polymorphic microsatellites and labelled with dyes mentioned NED(yellow)/ FAM (blue)/ VIC (green) Figure 2.7. The length of the amplified product determines the separation that are differentiated by the fluorescent labels and product size. The reaction protocol: 16 ng/μL of DNA (1 in 3 dilution of the 50 ng/μL DNA), 40 nM of dNTP and 15 mM MgCl2, 10x assay buffer and respective primers are used to set gene scan PCR of total reaction volume of 5.0 μL. The Gene scan PCR protocol and the reaction conditions are given in Tables 2.7 and 2.8. The amplified gene scan products are then pooled with the molecular sizing marker namely the LIZ500 (orange dye) or ROX400 (red dye) size standards. The initial PCR products were diluted with water (made up to 20 μL). These products are further diluted with 12.0 μL of injection mix (addition of 12.0 μL of Hi Di formamide and 0.5 μL of size standard) and denatured at 100°C and then subjected to capillary electrophoresis. After the electrophoresis the size of the products were analyzed by Gene scan (version 3.1; ABIPrism 310) /Gene mapper (version 3.5; ABI Prism 3100) software (Figure 2.7) and then binned for the linkage analysis. The LINKAGE programs require two input files namely “pedfile” that contains the pedigree data, and a “datafile” which describes information on the loci, locus order, affection status, etc. Generation of pre file: The first step in the linkage analysis is the creation of pedigree file using a text editor (word processor) capable of producing


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Table 2.7: PCR protocol for amplifying microsatellite marker for Genescan analysis Reagents

Volume (μL)

DNTPs Tris buffer with 15 mM MgCl-2 25 mM MgCl2 Primer Taq (5U/μL) DNA Water

0.5 0.5 0.2 0.2 0.1 1.0 2.5

Table 2.8: Genescan PCR conditions Temperature

Time

95°C 94°C 55°C 72°C 89°C 55°C 72°C 94°C 55°C 72°C 89°C 55°C 72°C 72°C 55°C 72°C 89°C 55°C 72°C 72°C

12 min 15 sec 15 sec 30 sec 15 sec 15 sec 30 sec 15 sec 15 sec 30 sec 15 sec 15 sec 30 sec 10 min 15 sec 30 sec 15 sec 15 sec 30 sec 10 min

Cycles 10

20 10 20 10 20

ASCII files. One row of information for each individual that describes the following information per column is entered: • Pedigree name (or number) • ID name (or number) of given individual • ID name (or number) of that individual’s father (0 if father is not in pedigree) • ID name (or number) of that individual’s mother (0 if mother is not in pedigree; either both or no parents must be given) • Sex of individual: 1 = male, 2 = female • Phenotype at locus 1 • Phenotype at locus 2, etc.

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Dye/Sample Peak

Minutes

Size

Peak Height

Peak Area

Data Point

B, 1

13.38

89.41

26

80

3649

B, 2

20.50

307.91

B, 3

20.56

G, 1

13.67

97.69

70

322

3728

G, 2

13.74

99.69

202

1130

3747

G, 3

13.78

100.66

55

325

3757

G, 4

13.81

101.41

458

2626

3765

G, 5

13.88

103.22

284

1556

3784

G, 6

15.30

142.15

425

2536

4173

G, 7

15.24

142.98

134

881

4155

G, 8

15.63

155.62

44

258

4261

G, 9

15.69

157.70

125

714

4279

49

317

5590

362

2749

5606

G, 10

15.76

159.77

226

1417

4297

G, 11

17.26

204.82

143

940

4705

G, 12

17.32

206.73

538

3832

4722

G, 13

17.50

212.48

56

325

4773

G, 14

17.54

213.50

38

104

4782

G, 15

17.56

214.29

144

915

4789

Fig. 2.7: Electrophoretogram of PCR amplified microsatellite markers with VIC (green) fluorescent tagged primers run in DNA sequencer and analyzed by gene scan software; homozygous genotype for D13S795 of fragmentation size (101.41/ 101.41bp) and heterozygous genotype of fragmentation size (142.15/159.77) and (204.82/206.73) for D11S902 and D13S1280 respectively

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Thus generated file will have the extension *.pre like the example given in Table 2.9. Table 2.9: Pre file (*.pre) generated in the text document 141001212 142002100 143122223 144122223 145122223 146122112 147121223 148121222 149121223 1410121222

Conversion of pre file to pedigree file with MAKEPED program The information of *. pre-file was then converted into *.ped-file, compatible with the MLINK program which is used to calculate the two-point LOD scores for each marker using the MAKEPED command. This step also emphasis on consanguinity if existing in the pedigree (Table 2.10). Table 2.10: Pedigree file *.PED generatedd through the “MAKEPED” command 1410030011212 Ped: 14 Per: 1 1420030020100 Ped: 14 Per: 2 1431204420223 Ped: 14 Per: 3 1441205520223 Ped: 14 Per: 4 1451206620223 Ped: 14 Per: 5 1461207720112 Ped: 14 Per: 6 1471208810223 Ped: 14 Per: 7 1481209910222 Ped: 14 Per: 8 149120101010223 Ped: 14 Per: 9 14101200010222 Ped: 14 Per: 10 Table 2.11: Data file through the “PREPLINK” command 1 2 1 2 < < AFFECTION, No. of ALLELES 0.999999 0.000001 < < GENE FREQUENCIES 1 < < No. of LIABILITY CLASSES 01.0000 1.0000 < < PENETRANCES 3 3 < < ALLELE NUMBERS, No. of ALLELES 0.333333 0.333333 0.333333 < < GENE FREQUENCIES 0 0 < < SEX DIFFERENCE, INTERFERENCE (IF 1 OR 2) 0.1000 < < RECOMBINATION VALUES 10.10000 0.45000 < < REC VARIED, INCREMENT, FINISHING VALUE

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DATA file and PREPLINK program: The datafile (entered as *.dat-file) is generated to give the information on the loci for each individual using the PREPLINK program. The information on total number of alleles, gene frequency depicting the type of inheritance, allele frequencies and recombination fractions needed for MLINK analysis are provided by this command. An example of a pedigree and data file is given in Tables 2.10 and 2.11. The other details that are entered in the preplink program vary according to the pedigree details and are given along with the program. The linkage program is then run by the command LCP that integrates the data from *.ped and *.dat file to generate the LOD score at different recombination fractions. Positive lods give evidence in favor of linkage and negative lods give evidence against linkage. Only recombination fractions between 0 and 0.5 are meaningful, and that all LOD scores are zero at θ = 0.5 (because then we will be measuring the ratio of two identical probabilities, and log10 (1) = 0). The most likely recombination fraction is the one at which the LOD score is highest. If there are no recombinants, the LOD score will be maximum at θ = 0. If there are recombinants, LOD scores will peak at the most likely recombination fraction. Regarding the threshold of significance. Z = 3.0 is the threshold for accepting linkage, with a 5% chance of error. Linkage can be rejected if Z < -2.0. Values of Z between -2 and +3 are inconclusive. For most statistics p < 0.05 is used as the threshold of significance, but Z = 3.0 corresponds to 1000: 1 odds (log10(1000) = 3.0). For a whole-genome scan, the appropriate significance threshold is a value where the probability of finding a false positive anywhere in the genome is 0.05. Over the years many modifications have been proposed for the threshold significance of LOD scores especially for genome wide scans in complex diseases. REFERENCES 1. Bennett P. Demystified microsatellites. Molecular pathology. 2000;53:177-83. 2. Ellergen H. Microsatellites: Simple sequences with Complex Evolution Nature Reviews Genetics 2004;5:435-45. 3. Ziegle JS, SuY, Corcoran KP, Nie L, Marlyand PE, Hoff LB, MC Bride LJ, Kronick MN, Diehl SR. Application of automated sizing technology for genotyping microsatellite loci. Genomics. 1992;14:1016-31.

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QUANTIFICATION OF GENE EXPRESSION GENE EXPRESSION In genetics and molecular biology investigation/analysis, quantifying the expression of the gene also becomes essential for better understanding of the disease and its pathophysiology. Generally gene expression studies involve quantifying the amount of mRNA (messenger RNA) present in particular sample/disease condition. The first step in this process is extracting the total RNA or mRNA from the sample. One method of mRNA quantification is reverse transcription PCR (RTPCR), wherein the mRNA is used as template for cDNA (complementary DNA)synthesis using the reverse transcriptase enzyme followed by amplification of the cDNA for gene of interest. This is a semi-quantitative method as it is an end-point analysis. The input mRNA is usually made equal across the samples and the result is observed in an agarose gel as amplified product. The intensity of the amplification can be compared and quantitated by densitometric analysis. The second method, the quantitative real-time PCR (qPCR) also depends on reverse transcription of the mRNA to quantify gene expression, but the data is obtained at the exponential phase of the amplification and analysed either by standard curve or comparative threshold (Ct) method as instructed by respective manufacturer. Specific instrumentation with various chemistries to perform Real-Time PCR are available. RNA EXTRACTION • The tissue is processed for RNA extraction using a monophase solution containing a mixture of guanidine thiocyanate and phenol 1 (TRI Reagent®, Sigma Aldrich, Bengaluru, India). • Sample is homogenized in TRI REAGENT (1 mL per 50–100 mg of tissue). • 0.2 mL of chloroform per mL of TRI REAGENT is added and shaked vigorously for 15 seconds and allowed to stand for 8–10 minutes at room temperature. • The resulting mixture is centrifuged at 12,000 rpm for 15 minutes at 4ºC. • The aqueous phase is transferred to a fresh tube containing 0.5 mL of isopropanol per 1 mL of TRI REAGENT and mixed thoroughly. The samples were allowed to stand at room temperature for 10 minutes and centrifuged at 12,000 rpm for 10 minutes at 4ºC. The RNA precipitate will form a pellet on the side and bottom of the tube. • The supernatant is removed and the pellet washed in 1 mL of 75% ethanol and centrifuged at 8,000 rpm for 5 minutes at 4ºC.

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• The pellet is air dried and dissolved in 50–75 μL of DEPC treated water by repeated pipetting. • The RNA extracted is quantified using Beckman DU 640 spectrophotometer and stored at -80ºC. RNA Purification • Isolated RNA is treated by TURBO DNase (Ambion, Genetix Biotech Asia Pvt. Ltd, Chennai, India) to remove the DNA in the sample. • 50 μL reaction containing 7.5–10.0 μgm of RNA is mixed with 1 μL of TURBO DNase (2U) and 0.1 volume 10X TURBO DNase buffer is incubated at 37 ºC for 30 minutes. • To the reaction mixture 0.1 volume of resuspended DNase inactivation reagent is added and mixed thoroughly during the incubation period of 2 minutes. • The mixture is centrifuged for 1.5 minutes at 10,000 rpm and supernatant transferred to a fresh tube. • The TURBO DNase treated RNA is quantified before reverse transcriptase PCR. Reverse Transcription • For all samples 1μg of total RNA is used to synthesize first-strand cDNA using SuperScript II® reverse transcriptase2,3 (Invitrogen, Joyvel, Chennai, India) and random primers. • Each component is mixed and centrifuged before use. • Following components are added accordingly (Table 2.12). Table 2.12: Protocol for setting RT reaction Component

Sample

- RT control

+ RT control

1 μg of total RNA Control RNA (50 ng/μL) Random hexamers (50 ng/μL) 10 Mm dNTP DECP treated water

n μL

n μL

-

-

-

1 μL

2 μL 1 μL 12 μL

2 μL 1 μL 12 μL

2 μL 1 μL 12 μL

• The sample is incubated at 65ºC for 5 minutes and then in ice for 1 minute. • The following reaction mixture is prepared and 7 mL is added to each reaction: Component Each reaction 5X first-strand buffer 4 μL 0.1M DTT 2 μL Ribonuclease 1 μL Inhibitor (40 units/ μL)


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• Incubate at 25ºC for 2 minutes. • Add 1 μL of Superscript II reverse transcriptase (200 units/mL) and mixed gently. • Incubate at 25ºC for 10 minutes. • Incubate at 42ºC for 50 minutes. • Inactivate the reaction by heating at 70ºC for 15 minutes. • The cDNA is quantified and diluted to contain 50 ng/μL. Real-time RT-PCR Analysis • 100 ng of cDNA is used to quantify the gene expression using ABI 7300 Real time PCR system. • Assays — TaqMan MGB probes (FAM dye-labeled) were used to quantify the on–Demand mRNA expression of different genes and cell markers. • Reaction protocol: 20 μL reaction containing TaqMan Universal PCR MasterMix,NoAmpErase UNG (2x) 10 μL, 20X Assay- on-Demand Gene Expression Assay Mix 1 μL and cDNA diluted in RNase – free water 9 μL. • Cycling conditions were as follows: 2 min at 50°C, 10 min at 95°C and 40 cycles of 15 seconds at 95°C plus 1 min at 60°C. REFERENCES 1. Yih-Horng Shiao. A new reverse transcription-polymerase chain reaction method for accurate quantification. BMC Biotechnology 2003;3:22. 2. Subbu Dharmaraj RT-PCR: The Basics, Technical Resources, Ambion Inc http:/ /www.ambion.com/techlib/basics/rtpcr/index.html. 3. Chomczynski P. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue sampels. Biotechniques 1993;15(3):5224,536-7. 4. Kotewicz ML, D’Alessio JM, Driftmier KM, Blodgett KP, Gerard GF. Cloning and overexpression of Moloney murine leukemia virus reverse transcriptase in Escherichia coli. Gene 1985;35(3):249-58. 5. Gerard GF, D’Alessio JM, Kotewicz ML, Noon MC. Influence on stability in Escherichia coli of the carboxy-terminal structure of cloned Mononey murine leukemia virus reverse transcriptase. DNA 1986;5(4):271-9.

STERILIZATION PROCEDURES PURPOSE The process for complete elimination or killing of all micro organisms is called sterilization. Sterilization means the use of a physical or chemical procedure to destroy all microbial life, including highly resistant bacterial

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endospores. The agents, which destroy microbes, can be broadly sub divided into physical and chemical. PRINCIPLE The major sterilizing agents used in hospitals are (a) moist heat by steam autoclaving, (b) ethylene oxide gas, and (c) dry heat. However, there are a variety of chemical germicides (sterilants) that have been used for purposes of reprocessing reusable heat-sensitive medical devices and appear to be effective when used appropriately, i.e. according to manufacturer’s instructions. These chemicals are rarely used for sterilization, but appear to be effective for high-level disinfection of medical devices. PERFORMANCE SPECIFICATION The effect of the chemical agents is influenced by factors like Dilution, pH and volume whereas the physical agents are influenced with temperature, duration, etc. STEP-BY-STEP PROCEDURE Cleaning, drying and sterilizing the glasswares are done in the wash room i. There are different types of glasswares used in the laboratory like the beakers, conical flasks, measuring cylinders, culture vials, test tubes, centrifuge tubes, reagent bottles, Pasteur pipettes, graduated pipettes, microscopic slides, etc. According to the purpose, the glasswares are grouped together and the method of washing will also be same for them. ii. New glasswares are also washed and sterilized before put to use. USED GLASSWARES 1. The discarded glasswares are thoroughly rinsed in tap water. 2. Then the glasswares are soaked in a large bucket containing soap solution over night. 3. The next day the glasswares are washed in running tap water 4. Soaked in the distilled water over night. 5. Then left in the hot air oven for dry heat sterilization for a minimum of 3 hours at 160°C. The machine is later switched off and the glasswares are taken out after cooling them so as to prevent water condensation. PREPARATION OF MATERIAL FOR AUTOCLAVING Materials must be properly wrapped before putting in autoclave for sterilization. Aluminium foil is used to cover all the glasswares and labelled

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with a signalloc as an indicator for perfect sterilization. Table 2.13 lists out the sterilization methods for different objects. Pasteur pipettes: To sterilize, plug mouthpiece with non-absorbent cotton wool. Place them in a pipette container and keep them for autoclaving after sticking the signalloc stickers. Note: Do not fill the liquid to the top, leave one-quarter space for the liquid to expand or boil without touching the lid. If it is necessary to use rubber stoppers, screw caps or plastic caps, they should be set in place loosely in order to allow air to escape, to prevent the containers from bursting or blowing off the caps as steam is generated, and to allow steam to penetrate easily. STERILIZATION PROCESS WITH VERTICAL AUTOCLAVE i. Fill the bottom of the boiler with water (up to the basket support). Do not let the water touch the basket; if necessary, remove excess water. ii. Put the basket with materials to be sterilized in the chamber and close the lid. Caution: make sure that the rubber washer is in its groove. iii. Screw down the lid clamps evenly and firmly but not too tightly. iv. Open the air outlet valve and turn on the burner to heat the water inside the boiler. v. Continue heating until a jet of steam appears through the air outlet valve. Wait for 5 minutes to drive out the air and for the boiler to contain saturated steam. vi. Close the air outlet valve. vii. Tighten the lid clamps and reduce the heat slightly. viii. Watch the pressure gauge until 15 pounds. is reached. See that the needle on the dial remains constant. Start the timer when the pressure is 15 pounds. ix. Continue sterilizing the material for 45 minutes. x. Turn off the heat as soon as the required time is up. xi. Open the air outlet valve. This will equalize the pressure inside and outside the chamber. xii. Unscrew the clamps when the hissing sound stops, indicating the drop of steam pressure to zero. xiii. Take off the lid and transfer the materials in hot air oven. xiv. Remove the basket when it is cool. The condensed water on the surface of the sterilized materials can be dried in an incubator. Do not open the package until they are ready to be used. xv. The sterility check for the autoclave is done monthly and records will be maintained.

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PRECAUTIONS FOR USING THE AUTOCLAVE i. Do not touch the valves (drainage valve or safety valve) when the autoclave is under pressure. ii. Do not open the autoclave until the pressure has gone down to “0”.Stay away from the steam when the door (or lid) is opened. It may cause serious burns. iii. Always watch the pressure, which may cause accidents if the safety valve is not properly functioning. iv. Do not leave the autoclave unattended when the steam pressure is on the rise. v. It is not good practice to leave the autoclave to cool for a long period (overnight) following sterilization. If it is left for several hours without the outflow valve being opened, a vacuum forms and the sterilized material may break. MICROSLIDES Sets of new microslides are soaked in dichromate cleaning solution. The Biochemistry department prepares the dichromate solution and supplies the Genetics department. Before put to use, it has to be washed in running tap water and then in distilled water. SCREW-CAPPED BOTTLES Wash the liners separately. Dry quickly to avoid corrosion. Sterilize metal caps and their bottles in hot air oven. Rubber liners are autoclaved. NEW GLASSWARES All the new glasswares are left in soap solution for few hours (minimum 2 hours). Then they are washed thoroughly in running tap water soaked in distilled water over night. Sterilization of new glassware is as same as for the used glassware. PLASTIC PASTEUR PIPETTES The new pipettes are packed in plastic bags and sent for gas sterilization, which is done, in the sterilizing unit for the operation theaters. The used ones are soaked in soap solution over night and washed in running tap water. Left in the distilled water and air-dried. If required for any purpose where sterilization is not necessary they are used after gas sterilization.

168

Manual of Medical Laboratory Techniques Table 2.13: Sterilization methods for different objects

Items

Autoclave

Hot air oven

Glass wares like Centrifuge tubes, beakers, conical flasks, measuring cylinder, cultural vials, test tubes, reagent bottles, Pasteur pipettes, graduated pipettes

+

+

Sartorius filter apparatus

+

–

Milli Q Water

+

–

Screw caps

+

–

Tips

+

–

Cotton

+

–

BIBLIOGRAPHY 1. Introduction to sterilization, disinfection and infection control. Sterilization by steam at increased pressure. 2nd edition. Authors: Joan F Gardner, Margaret M Peel. pg 80-4.

UNIT 3

Hematology and Clinical Pathology

SB Vasanthi S Krishna Kumar Doreen Gracias

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Part I: Hematology COMPLETE BLOOD COUNT PURPOSE Quantitative enumeration of different formed elements of blood and hemoglobin in whole blood by automated cell counter by the principle of Impedance Variation. This test includes total WBC count, total RBC count, hemoglobin, hematocrit, MCV, MCH, MCHC, platelet count. It is the enumeration of cellular elements of blood, evaluation of red cells indices and determination of cell morphology by means of stained smears. PRINCIPLE Coulter Method The Coulter method accurately counts and sizes cells by detecting and measuring changes in electrical resistance when a particle (such as a cell) in a conductive liquid passes through a small aperture. As each cell goes through the aperture, it impedes the current and causes a measurable pulse. The number of pulses signals the number of particles. The height of each pulse is proportional to the volume of that particle. While number of pulses indicates particle count, the amplitude to the electrical pulse produced depends on the cell’s volume. Theoretical analysis of the behavior of particles within an aperture shows that the height of the electrical pulse produced by the cell is the characteristic that most nearly shows proportionality to the cell volume. WBCs are counted after lysing the RBCs by a detergent (ionic or nonionic) which does not lyse the WBCs themselves. After dilution (1: 250 for WBC count, 1:6250 for RBC count Indices and platelet count) the diluted sample is directed to two sides of the instrument and particles on the red cells side that are larger than 36 fl are counted as RBCs. Cells from 2to 20 fl are counted as platelets. For WBC counting lysing reagent is added to the sample in the system to lyse RBCs and causes differential shrinking of WBCs, thereby allowing them to be separated into a partial differential (3 part differential) and white cells are shown in 3 groups lymphocytes (3590 fl) mononuclear cells (90 -160 fl ) and granulocytes ( 160 -450 fl). Hct is calculated based on RBC pulse height value. It is measured by height of impulse generated by a passage of cells through a micro aperture, which is directly proportional to the volume of analyzed cells. RBCs are lysed and the freed hemoglobin combines with potassium cyanide to form cyanmethemoglobin, which is measured by Spectrophotometry at 525 nm.


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MCH and MCHC are calculated from RBC count, Hb and Hct Hct : Calculated from RBC and MCV MCV: Derived from RBC histogram MCH: Calculated from HGB and RBC MCHC: Calculated from Hgb and Hct MPV AND PLT: Derived from PLT histogram Performance specification Linearity WBC ( × 103 Cells/μL) 99.9 RBC ( × 106 Cells/μL) 7.0 Platelets ( × 103 Cells/μL) 999 HGB (g/dL) 25

Measurement range

Minimum detection range

0 –99.9 0–7.0 0–999 0–25

0 0 0 0

PRIMARY SAMPLE • • • • •

Use whole blood as sample collected in K2 EDTA. Mix well by rotation using hemomixer before processing for 5 minutes Process the samples within 2 hours of collection Do not use decomposed or contaminated sample Reject clotted samples.

CONSUMABLES/REAGENTS • Cell diluent • Lytic reagent • Shutdown diluent INSTRUMENT (FIG. 3.1)

Fig. 3.1: Coulter ACT diff analyzer

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PROCEDURE • Check the background values and QC values to see whether they are within the range specified. • Run any one of the three levels (low, normal and high) of controls before analyzing the samples to verify whether the system is within the acceptable limits. • Press the ID key to enter the lab reference number. • Press the enter key to save the lab reference number. • Mix the blood samples thoroughly using hemomixer before processing for 5 minutes • Keep the sample tube under the sample probe and press the key located behind the sample probe. • Result will be displayed on the LCD screen. POTENTIAL SOURCES OF VARIABILITY The following factors will affect counting: • Fibrin micro clots or cryoprecipitate in the specimen • Inadequate lysis of RBC • Inadequate dilution of specimen and inadequate calibration of the analyzer • Deterioration of diluting solution. • Presence of dust particles in the diluent—Ensure the diluents and buffer solutions used are free of dust particles especially for platelet counting • Partial or total obstruction of the aperture • Wrong threshold setting of the instrument • Carry over from one to next measurement • Fluctuation of electric current. REFERENCE RANGE Parameter

Range

WBC count

Adults 4000–11000 cells/cu.mm At birth 10,000–25,000 cells/cu.mm Infants (one year) 6000–18000 cells/cu.mm 4–7 years 6,000–15,000 cells/cu.mm 8–12 years 4,500–13,500 cells/cu.mm

RBC count

Male : 4.5 to 6.5 × 106cells/cu.mm Female: 3.9 to 5.6 × 10 6 cells/cu.m

Hemoglobin

At Birth At 1 year 10–12 years Women Men

: 13.6–19.6 g % : 11.3–13.0 g% : 11.5–14.8 g% : 11.5–16.5 g% : 13.5–18.0 g%

Hematology and Clinical Pathology Platelet count

150,000 – 450,000/cu.mm

Hematocrit

At birth At 1 year At 10 years Women Men

MCV

76–96 μm3

MCH

27–32 pg

MCHC

30–35 %

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: 44–63% : 35% : 37.5% : 30–40% : 40–54%

TOTAL ERYTHROCYTE (RBC) COUNT PURPOSE Quantitative estimation of total RBC in human by manual method using Hemocytometer. Total RBC count is increased in conditions such as a) hemoconcentration due to Burns, Cholera, etc. b) In central cyanotic states as seen in chronic heart disease, conditions of increased lung functions such as emphysema c) In Polycythemia. Decreased erythrocyte count is seen in Old age, Anemia due to any cause and in pregnancy. PRINCIPLE The blood specimen is diluted 1:200 with the RBC diluting fluid and cells are counted under high power (x 40) by using a counting chamber. The number of cells in undiluted blood are calculated and reported as the number of cells per cumm of whole blood. PRIMARY SAMPLE • Use whole blood as specimen for the test • Collect 1 mL of venous blood in tubes containing 2 mg of K2 EDTA for every 1 mL of blood (prepared by taking 20 ul of 10 % K2 EDTA for 2 mL of blood) • Do not use clotted specimens or contaminated serum • Process the specimen on the same day within 2 hours of collection • Mix the blood sample gently by rotation for 2 minutes in a hemomixer and between the palms of hand prior to testing. REAGENTS/CONSUMABLES • RBC diluting fluid prepared by adding — Sodium citrate – 3.8 g — Formalin – 1.0 mL — Distilled water to – 100 mL

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EQUIPMENT • Light microscope • Improved Neubauer Counting chamber • Pipette

Fig. 3.2: 01—RBC pipette, 02—WBC pipette

PROCEDURE • Take 4 mL of Formal citrate solution and add 0.02 mL of EDTA anticoagulated blood to get a dilution of 1:200. • Charge Neubaur’s chamber and allow the cells to settle for five minutes. • Count cells in the RBC chamber - four corner squares and the centre square. • Multiply the total number by 1000 to get the number of cells present/ mm3 CALCULATION The following formula is used to calculate the number of red cells: Total red blood cells/cu mm =

No. of red cells counted × Dilution Area counted × Depth of fluid

Where Dilution = 1:200 Area counted = 80/400 (1/5) sq.mm Depth of fluid = 1/10 mm INTERPRETATION OF RESULTS The results are calculated as per the above formula and the number of RBC is reported as per cu.mm of whole blood.


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PRECAUTIONS • Ensure the counting chamber is clean and dry. • Take care to ensure air bubbles do not enter while drawing blood or the diluting fluid into the RBC pipette • Discard the initial few drops before loading the counting chamber • Load the counting chamber in one application and ensure fluid does not overrun the surrounding moat • Ensure there are no air bubbles under the cover slip and that the diluting fluid does not over run the cover slip • Use a cover slip that is thick and flat • Count after allowing the cells to settle for 2–3 minutes and include all cells lying within the square and also those cells lying on the lines or touching the lines. Safety Precautions • • • •

Handle all samples as potentially infectious Handle all reagents with care and avoid contact with eye mouth and skin Do not perform mouth pipetteing Discard used reagents and sample as per disposal procedure.

POTENTIAL SOURCES OF VARIABILITY • Errors may occur in counting can be caused by dirt, dust particles and yeast cells in RBC diluting fluid as it may lead to falsely elevated RBC counts. Hence take extra care during preparation and storage of RBC diluting fluid • Errors may also occur due to clumping of RBC or WBC or cellular debris • Falsely elevated counts may occur due to collection of blood from an area where there is hemoconcentration, inadequate wiping of the pipette, improper mixing, uneven distribution of the specimen in the counting chamber • Falsely low counts may occur if the blood sample gets diluted with tissue fluid due to squeezing, due to improper pipetteing and dilution (too much blood sample or too little diluting fluid) contamination by saliva, non standardized counting chamber. REFERENCE RANGE Male: 4.5–6.5 million/cu.mm Female: 3.9–5.6 million/cu.mm. BIBLIOGRAPHY 1. A Laboratory Manual for Rural Tropcial Hospitals - Monica Cheesbrough & John McArthur.Pg: 37-38. 2. www.hemcindia.com/sundries-blood-chemistry.html (pictures)


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TOTAL WBC COUNT PURPOSE Quantitative estimation of total WBC in human by manual method using Hemocytometer. Total WBC count is increased (Leukocytosis) in transiently in bacterial, viral protozoal infections parasitic infections such as Filaria, and also in severe hemorrahage. The degree of leukocytosis depends on the severity and type of infection. Leukopenia (decrease in leukocyte count) occurs in certain viral infections such as Hepatitis, Influenza and Measles, Protozoal infections such as Malaria and certain bacterial infections such as Typhoid fever and in Leukemias, bone marrow depression due to any cause and in iron deficiency and megaloblastic anemias. PRINCIPLE The glacial acetic acid lyses the red cells while the gentian violet slightly stains the nuclei of the leukocytes. The blood specimen is diluted 1:20 in a WBC pipette with the diluting fluid and the cells are counted under low power of the microscope using a Neubauer counting chamber. PERFORMANCE SPECIFICATIONS The variation in Total WBC count by this method is 10% and the error is 5 % if 400 cells are counted. PRIMARY SAMPLE • • • • •

Use whole blood as specimen for the test Collect 2 mL of venous blood in 3.6 mg K2EDTA vacutainer Do not use clotted specimens or contaminated serum Process the specimen on the same day within 2 hours of collection Mix the blood sample gently by rotation for 2 minutes in between the palms of hand prior to test.

REAGENTS/CONSUMABLES WBC diluting fluid prepared by: • Adding Glacial acetic acid 2.0 mL • 2 drops of 1% Gentian violet • Made up to 100 mL with distilled water. • Store the WBC diluting fluid at RT 25°-35 °C Equipment • Light Microscope • Improved Neubauer Counting chamber • Pipette


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PROCEDURE • Make 1: 20 dilution of blood by mixing 20 μL of K2 EDTA blood and 0.38 mL of WBC diluting fluid in a glass tube cork the tubes and mix for 1 minute fill the chamber by means of a capillary tube/ Pasteur pipette. • Mix the contents in the pipette well and after 5 minutes, discard the first few drops, fill the Neubauer counting chamber and allow the cells to settle for 2–3 minutes. • Focus on one of the ‘W’ marked areas by turning objective to low power (10X). (Each having 16 small squares) • Count cells in all four ‘W’ marked corners. CALCULATION No. of WBC/cu.mm of whole blood =

No. of white cells actually counted × Dilution Area counted × Depth of fluid

Dilution = 20 Depth = 0.1 Area = 4 x1= 4 sq.mm =

No. of white cells actually counted × 20 4 × 0.1

= N × 20 = N × 50 cells/cumm 4 × 0.1 INTERPRETATION OF RESULTS The results are calculated as per the above formula and the number of WBC is reported as per cu.mm of whole blood. SAFETY PRECAUTIONS • Handle all samples as potentially infectious • Handle all reagents with care and avoid contact with eye, mouth, and skin • Do not perform mouth pipetteing • Discard used reagents and sample as per disposal procedure Precautions • Ensure the counting chamber is clean and dry. • Load the counting chamber in one application and ensure fluid does not overrun the surrounding moat. • Ensure there are no air bubbles under the cover slip and that the diluting fluid does not over run the cover slip. • Use a cover slip that is thick and flat.


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• Count after allowing the cells to settle for 2–3 minutes and include all cells lying within the square and also those cells lying on the lines or touching the lines. POTENTIAL SOURCES OF VARIABILITY • Errors may occur in counting can be caused by dirt, dust particles and yeast cells in WBC diluting fluid as it may lead to falsely elevated WBC counts. Hence take extra care during preparation and storage of WBC diluting fluid • Errors may also occur due to clumping of RBC or WBC or cellular debris • Falsely elevated counts may occur due to collection of blood from an area where there is hemoconcentration, inadequate wiping of the pipette, improper mixing, uneven distribution of the specimen in the counting chamber • Falsely low counts may occur due to improper pipetting and dilution (too much blood sample or too little diluting fluid) REFERENCE RANGE Adults 4000 – 11,000 cells/cu.mm At birth 10,000 – 25,000 cells/cu.mm Infants (one year) 6000–18000 cells/cu.mm 4–7 years 6000 – 15,000 cells/cu.mm 8–12 years 4,500 – 13,500 cells/cu.mm BIBLIOGRAPHY 1. Practical Haematology by JV Dacie and SM Lewis, 10th Edition. Pg. 680-1. 2. District Laboratory Practice in Tropical Countries-Monica Cheesebrough- part 2-Pg. 314-6.

DIFFERENTIAL COUNT PURPOSE Differential count is used to identify changes in the distribution of white cells, which may be related to specific types of disorders like infection (bacterial , viral or parasitic) or leukemias. The increase or decrease in specific white cells are also detected. PRINCIPLE The blood smear is prepared on a microscopic slide which is dried, fixed and stained. The fixative does not allow any further change in the cells and


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make them adhere to the glass slide. The polychromic staining solutions containing acidic and basic dyes induce multiple colors when applied to cells. Basic components of the cells are stained by acidic dyes, acidic components by basic dyes and the neutral components by both the dyes. Stained cellular components are then viewed under the microscope. PERFORMANCE SPECIFICATIONS The film must be smooth at the end. There should be no lines extending across or down through the film and it should not contain holes. PRIMARY SAMPLE • Use whole blood as specimen • Collect 3 mL blood in a tube containing 0.2 mL of 4% K2 EDTA. Mix well gently. • Do not use clotted sample for testing. CONSUMABLES/REAGENTS • • • • • •

Leishman’s stain Leishman buffer Immersion oil Glass slides Cotton Slide rack

INSTRUMENT Light microscope. PROCEDURE Preparation of Smear • Take a clean, dry slide. • Transfer a small drop of blood near the edge of the slide. • Place a smaller piece of glass with a width slightly less than that of the slide called as spreader at an angle of about 45º to the surface of the slide. • Pull back the spreader until it touches the drop of blood. • Push the spreader forward to the end of the slide with a smooth movement. • Dry the blood smear at room temperature. • Write the patient’s lab reference number and the date with a pencil and place it on a staining rack, making sure that the smeared surface faces upward for staining.

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Staining of the Blood Film • Place the slide with the smear side up on a staining rack horizontally (without sloping). • Add Leishman stain to cover the entire slide. Then dilute it with working buffer. • Mix the reaction mixture adequately by blowing on it through a pipette to produce fine ripples, which will mix the buffer and stain well. • Wait for 15 minutes. • Fifteen minutes later drain off the stain and wash the slide under running tap water. • Place it on a rack to dry. Examination of Film • As the distribution of granulocytes are more in the edge and tail of the smear and lymphocytes more towards center, counting is done by what is known as a Battlement method. • First examine the stained smear under low power. In an ideal smear three zones appear: a) Thick area b) Body c) Thin tail end of the smear • Choose the portion slightly before the tail end where the red cells are beginning to overlap. • Place a drop of immersion oil (cedar wood oil) on the smear. • Count 100 cells by systematic way. Counts are made with a ‘Digital Counter’ and expressed as percentage of each cell. Examine the film by moving from one field to the next systematically.

Interpretation of Results A total of 100 leukocytes are counted and the different types of white cells are reported as the percentage of cells. PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure


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Fig. 3.3: WBC

POTENTIAL SOURCES OF VARIABILITY • Dirty slides do not give an even smear. • An appropriate size of blood drop should be used. • Smear should be made immediately after putting the drop of blood on the slide; a delay will cause uneven distribution of white cells on the film. • Jerky movement of the spreader slide and the loss of contact between the spreader and the smear slide will yield poor smears. REFERENCE RANGE Cells

At birth

4 weeks

4 years

6 years

Adult

Neutrophils Band Eosinophils Basophils Monocytes Lymphocyte

37–57% 0–1% 1–3% 0–1% 4–8% 25–35%

25–35% 0–1% 1–3% 0–1% 5–9% 45–65%

25–45% 0–1% 1–3% 0–1% 4–8% 40–60%

45–60% 0–1% 1–3% 0–1% 4–8% 40–45%

40–80% 0–1% 1–6% 0–1% 2–10% 20–40%

BIBLIOGRAPHY 1. Practical Hematology Dacie and Lewis. 10th Edition, Pg. 33–36. 2. www.Unomaha.edu/hpa/blood.

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PERIPHERAL SMEAR STUDY PURPOSE Peripheral smear study is used in reporting any abnormal morphology of RBC and WBC and in the diagnosis of various anemias, leukemias and presence of blood parasites, e.g. malaria. PRINCIPLE The blood smear is prepared on a microscopic slide which is dried, fixed and stained. The fixative does not allow any further change in the cells and make them adhere to the glass slide. The polychromic staining solutions containing acidic and basic dyes induce multiple colors when applied to cells. Basic components of the cells are stained by acidic dyes, acidic components by basic dyes and the neutral components by both the dyes. Stained cellular components are then viewed under the microscope. PERFORMANCE SPECIFICATIONS • The film must be smooth at the end. There should be no lines extending across or down through the film and it should not contain holes.

Fig. 3.4: Wedge-type blood film preparation


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PRIMARY SAMPLE • Use whole blood as specimen • Collect 1 mL blood in a tube containing 2 mg of 10% K2 EDTA. Mix well gently. • Do not use clotted sample for testing and samples without an ID number. CONSUMABLES/REAGENTS • • • • • •

Leishman’s stain Leishman buffer Immersion oil Glass slides Cotton Slide rack

INSTRUMENT • Light Microscope. PROCEDURE • Preparation of smear: i. Take a clean, dry slide. ii. Transfer a small drop of blood near the edge of the slide. iii. Place a smaller piece of glass with a width slightly less than that of the slide called as spreader at an angle of about 45º to the surface of the slide. iv. Pull back the spreader until it touches the drop of blood. v. Push the spreader forward to the end of the slide with a smooth movement. vi. Dry the blood smear at room temperature. Staining of the Blood Film: • Place the slide with the smear side up on a staining rack horizontally (without sloping). • Add Leishman stain to cover the entire slide. Then dilute it with working buffer. • Mix the reaction mixture adequately by blowing on it through a pipette to produce fine ripples, which will mix the buffer and stain well. • Wait for 15 minutes. • Fifteen minutes later drain off the stain and wash the slide under running tap water. Place it on a rack to dry. • Examination of stained smears: The air-dried stained smear is viewed 1st under dry 10X and later under 100X oil immersion objective of a light microscope

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INTERPRETATION OF RESULTS Look under oil immersion for the following features: a. Size of RBCs: Normal red blood cell is about 7.2 microns in diameter. Normal cells are called normocyte. When the cells are smaller than normal that is less than 6 micron in diameter, it is called a microcyte and when large than normal – more than 9 micron in diameter, it is called a macrocyte. When the variation in size is greater than normal it is called anisocytosis. b. Shapes of RBCs: Normal red cells are biconcave in shape and appear circular on smear. Abnormal cells may assume different shapes. Variation in shapes is known as poikilocytosis. These shapes are described as follows: i. Ovalocytes/elliptocytes- oval in shape. A small percentage may be found normally. Almost all the cells are oval in size in condition called on hereditary ovalocytosis and may be associated with haemolytic ovalocytosis and may be associated with hemolytic anemia ii. Pencil shaped cells — Elongated thin cells found in iron deficiency anemia. iii. Target cells — These are cells with a central round area stained pink around which there is a colorless zone, rimmed by a border of normal pink. Found in Thalassemia, sickle cell anemia, hypochromic anemia. iv. Sickle cells — These cells are sickle shaped and are rarely found in regular blood films. These cells assume such shape due to crystallization of the abnormal haemoglobin when the oxygen tension is reduced. v. Schistocytes — These are regular red cells fragment found in hemolytic anemias. vi. Crenated cells — Crenation is a term applied to the shrinking of red cell. vii. Acanthocytes — These are cells with irregular margins and have pointed projections. viii. Burr cells — Very small irregularly shrunken cells with rounded, pointed projections of varying size. ix. Crescent bodies — These are pale, curved bodies seen in the thin part of the films and are commonly seen in aneamia. c. Color-Look for chromacity. Normal cells have a central area of pallor. When this normal pallor is increased, it is called hypochromia. Some cells appear purplish or pinkish and are the polychromatic erythrocytes. Some cells appear to be hyperchromic –without central area of pallor.


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Fig. 3.5: Abnormal RBC morphology a. d. g. j. m. p. s.

Hypochromasia Microcytosis Spherocytosis Target cell Schistocytosis Tear drops Howell Jolly bodies

b. e. h. k. n. q. t.

Polychromasia Macrocytosis Stomatocytosis Elliptocytosis Echinocytes Rouleaux formation Basophilic stippling

c. f. i. l. o. r.

Anisocytosis Dimorphic blood picture Acanthocytosis Cigar cells Sickle cells Red cell agglutination

d. Inclusions: i. Basophilic stippling: These are small blue or black granules seen in red cells. Found in cases of hemolytic anemia, mercury or lead poisoning, pernicious anemia, megaloblastic anemia. ii. Howell-Jolly bodies: These appear small, round, densely staining, dark blue particles. Usually found near the periphery of the cells. iii. Cabot rings: These are pale staining rings or figures of 8 seen in conditions like hemolytic, megaloblastic anemia and leukemia. iv. WBCs: Look for number, morphology, distribution, abnormal cells or immature forms. v. Platelets: Look for number, platelet distribution, morphology and aggregation. vi. Parasites: Look for intra-cellular (malaria) and extra-cellular parasites (MF).


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PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure POTENTIAL SOURCES OF VARIABILITY • Dirty slides do not give an even smear. • An appropriate size of blood drop should be used. • Smear should be made immediately after putting the drop of blood on the slide; a delay will cause uneven distribution of white cells on the film. • Jerky movement of the spreader slide and the loss of contact between the spreader and the smear slide will yield poor smears. BIBLIOGRAPHY 1. District Laboratory Manual in Tropical Countries; Monica Cheesbrough-Part 2, Pg 327,329 2. Hematology Atlas- Department of Hematological Pathology. University of Stellenbosch and Tygerberg Academic Hospital (pictures) 3. www.ruf.rice.edu/.../sds-page/bloodcytology.html, Experimental Biosciences, Introductory Laboratory, BIOS -211 (pictures) 4. Practical Hematology. Dacie and Lewis. 10(5)80-111.

ERYTHROCYTE SEDIMENTATION RATE PURPOSE To determine the rate at which the red cells sediment by the Westergren’s Method. ESR is a non-specific test. ESR is increased in all conditions where there is tissue breakdown or when foreign proteins enter the blood. It reflects changes in plasma protein, which accompany most of the acute and chronic infections. Conversely, normalization of the ESR indicates possible recovery from the diseased state. PRINCIPLE When the anticoagulated blood is taken in a tube and left undisturbed in a vertical position, the erythrocytes tend to settle down at the bottom. The level of the column of red cells is noted in the beginning (0 hr) and after 1 hr. The first is the stage of aggregation where the red cells form rouleaux. This is followed by the stage of sedimentation in which the falling of the


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red cells takes place. The larger the aggregation in the first stage, the faster the rate of fall. Two layers are formed, the upper plasma layer and the lower one of red cells. The distance (mm) the column has moved is the erythrocyte sedimentation rate. PRIMARY SAMPLE • Use whole blood as specimen. • Collect 2 mL of blood in 3.6 mg of K2EDTA Vacutainer. REAGENTS/CONSUMABLES • • • • •

Plain tubes 12 mm × 70 mm Westergren tube Stop watch Stand for holding the tube 3.2% Trisodium citrate

PROCEDURE • Make certain that the Westergren ESR rack is exactly leveled. • Make sure that the blood collected in E tube is well-mixed, by rolling the tube in between the palms. • Pipette 0.4 mL of tri-sodium citrate anticoagulant in a small tube. • To this add 1.6 mL of well-mixed blood from E tube and mix well. • Pipette out this mixture in the Westergren’s pipette exactly to its “O” mark, making sure that there are no air bubbles. • Place the pipette in the ESR rack making sure that the pipette fits snugly and evenly into the grooves provided for it. • Allow the pipette to stand for exactly 60 minutes and note the number of millimeters that the red cells have fallen. This result is the ESR. PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure POTENTIAL SOURCES OF VARIABILITY • ESR is influenced by various laboratory factors like temperature, time, anticoagulant and length of ESR tube. • Slanting of the tube affect the ESR.


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REFERENCE RANGE Male: Up to10 mm/1 hour Women: Up to 15 mm/1 hour BIBLIOGRAPHY 1. District Laboratory Practice in Tropical Countires, Volume-2, Monica Cheesebrough. Edn. 2000, Pg 330-1. 2. Practical Hematology. Dacie and Lewis, 10th Edition, Pg-595-604.

PLATELET COUNT PURPOSE Quantitative estimation of platelet count in human by manual method using Hemocytometer. Decreased platelet count is associated with prolonged bleeding and poor clot retraction. It also occurs in aplastic anemia, megaloblastic anemia, hypersplenism, acute leukemia and in immune thrombocytopenia. Increased platelet count is observed in polycythemia Vera, following splenectomy and in chronic myelogenous leukemia. PRINCIPLE The blood specimen is diluted with the platelet diluting fluid (ammonium oxalate reagent) and cells are counted under high power (x 40) by using a counting chamber. The diluent prevents coagulation, fixes the platelets and prevents them from clumping. The number of cells in undiluted blood are calculated and reported as the number of cells per cumm of whole blood. PRIMARY SAMPLE • • • • •

Use whole blood as specimen for the test Collect 2 mL of venous blood in 3.6 mg K2 EDTA. Do not use clotted specimens or contaminated serum Process the specimen on the same day within 2 hours of collection Mix the blood sample gently by rotation for 2 minutes in between the palms of hand prior to testing.

REAGENTS/CONSUMABLES • Reagent: (i) Ammonium oxalate (ii) Distilled water

: :

1 gm 100 mL


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EQUIPMENT • Light microscope • Improved Neubauer Counting chamber • Pipette PROCEDURE • • • • •

• • • • •

Mix the blood specimen with the anticoagulant. By using a pipette take 0.05 mL of blood. Add the blood into 0.95 mL of platelet diluting fluid and mix well. Prepare a moist chamber by placing a wet piece of filter paper in a covered plastic box. [A used visiting card box.] Charge one side of the hemocytometer and leave it in a moist chamber, to avoid the evaporation of the fluid in the counting chamber, and to allow the platelets to settle. After 20 minutes or so remove the hemocytometer from the moist chamber and wipe its underside with a clean dry tissue paper. Place the counting chamber carefully on the stage of the microscope. Under low power focus red cell counting area. Move to view the corner of the red cell area and change to high power objective. The platelets will appear like highly refractile particles. Count platelets in all 25 small squares. The area covered by 25 squares is equivalent to 1 sq.mm.

CALCULATION No. of platelets/cu.mm of whole blood =

No. of platelets actually counted × Dilution Volume of the fluid

Dilution = 20 Volume of fluid = 1 × 0.1=0.1 cu.mm Number of platelets × 20 0.1 = Number of platelets × 200

Platelets per cumm (uL) =

INTERPRETATION OF RESULTS The results are calculated as per the above formula and the number of platelets is reported as per cu.mm of whole blood. PRECAUTIONS • Ensure the counting chamber is clean and dry • Take care to ensure air bubbles do not enter while pipetting.

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• Load the counting chamber in one application and ensure fluid does not overrun the surrounding moat • Ensure there are no air bubbles under the cover slip and that the diluting fluid does not over run the cover slip • Use a cover slip that is thick and flat • Count after allowing the cells to settle for 2–3 minutes and include all cells lying within the square and also those cells lying on the lines or touching the lines SAFETY PRECAUTIONS • Handle all samples as potentially infectious • Handle all reagents with care and avoid contact with eye, mouth and skin • Do not perform mouth pipetteing • Discard used reagents and sample as per disposal procedure POTENTIAL SOURCES OF VARIABILITY • Particles of dust, bacteria and fragments of RBC can be mistaken for platelets. • Platelet count should be carried out within 2 hours of blood collection. Delay causes clumping and disintegration of platelets. • As the platelets are very small in size, they agglutinate and break up easily. They also tend to adhere to glassware and articles in the diluting fluid. Therefore, in order to obtain an accurate platelet count, clean and dry syringes and blood collection bottles should be used. REFERENCE RANGE 1, 50,000–400,000 platelets /cu.mm. BIBLIOGRAPHY 1. District Laboratory Manual in Tropical Countries- Monica Cheesbrough 31718. 2. Practical Hematology. Dacie and Lewis, 10th Edition. pg 681-2.

ABSOLUTE EOSINOPHIL COUNT PURPOSE Quantitative estimation of Absolute Eosinophil Count in human by manual method using Hemocytometer. Increased Eosinophil count is observed in wide variety of conditions like allergy, drug, reaction, parasitism, collagen


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vascular disease and in certain leukemias. Eosinophils are decreased in hyper-adrenalism and Cushing’s syndrome. PRINCIPLE On diluting the blood with eosinophil diluting fluid, the RBCs and other types of leukocytes except eosinophils gets lysed. Eosinophils take the eosin stain and appear red. These cells are then counted by using a Neubauer chamber under low power (10 x) of Microscope. PERFORMANCE SPECIFICATIONS Definition of terms: EDTA - Ethylene Diamine Tetra Acetic acid; RBC Red Blood Corpuscles; WBCs - White Blood Corpuscles; mm3 - Cubic millimeter. PRIMARY SAMPLE • • • • •

Use whole blood as specimen for the test Collect 2 mL of blood in K2EDTA vacutainer Do not use clotted specimens or contaminated serum Process the specimen on the same day within 2 hours of collection Mix the blood sample gently by rotation for 2 minutes in a hemomixer and between the palms of hand prior to testing.

REAGENTS/CONSUMABLES • Diluting fluid (Hingleman’s solution) prepared by adding – Yellow eosin – 0.5 g – 95% phenol – 0.5 ml – Formalin – 0.5 ml – Distilled water – 99 mm • Micro pipettes and glass test tubes and pipettes EQUIPMENT • Light microscope • Neubauer chamber PROCEDURE • Pipette 0.36 mL of eosinophil diluting fluid in a test tube and add 0.04 mL of K2 EDTA blood. • Mix well for not more than 30 seconds and keep for 10 mts and charge the Neubauer counting chamber. • Keep it in a moist chamber for 2-3 min. • Count the cells under low power objective with reduced light in all 9 squares of Neubaurer chamber.


192 Calculations

No. of cells counted × 10 0.9 Dilution = 10 Volume of fluid = area counted × depth = 9 sq. mm × 0.1 = 0.9

Total no. of eosinophils, cu mm (μl) =

INTERPRETATION OF RESULTS The results are calculated as per the above formula and the number of eosinophils are reported as per cu.mm of whole blood. PRECAUTIONS • Ensure the counting chamber is clean and dry • Take care to ensure air bubbles do not enter while diluting blood • Load the counting chamber in one application and ensure fluid does not overrun the surrounding moat • Ensure there are no air bubbles under the cover slip and that the diluting fluid does not over run the cover slip • Use a cover slip that is thick and flat • Count after allowing the cells to settle for 2–3 minutes and include all cells lying within the square and also those cells lying on the lines or touching the lines Safety Precautions • • • •

Handle all samples as potentially infectious Handle all reagents with care and avoid contact with eye, mouth and skin Do not perform mouth pipetting Discard used reagents and sample as per disposal procedure

POTENTIAL SOURCES OF VARIABILITY • Eosinophils disintegrate in the diluting fluid hence counting should be done with in 30 minutes of the diluting the sample with the diluting fluid REFERENCE RANGE 40-440 cells/cumm. BIBLIOGRAPHY 1. Practical Haematology - Sir John V Dacie & SM Lewis, 6th Edition. Pg 42-3.


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RETICULOCYTE COUNT PURPOSE Quantitative estimation of reticulocyte in human by manual supravital staining method. The number of reticulocytes in peripheral blood is a reflection of red cells forming activity (erythropoietic activity) of bone marrow. Increased reticulocyte count is observed in hemolytic anemia and decreased count is observed in aplastic anemia. PRINCIPLE The RNA in the polychromatic erythrocyte is precipitated by staining them. This appears as dark blue network or reticulum. PERFORMANCE SPECIFICATION The error in counting can be minimized if more RBCs are counted PRIMARY SAMPLE • • • • •

Use whole blood as specimen for the test Collect 2 mL of venous in 3.6 mg K2 EDTA Vacutainer. Do not use clotted specimens or contaminated serum Process the specimen on the same day within 2 hours of collection Mix the blood sample gently by rotation for 2 minutes in between the palms of hand prior to testing • If capillary blood is used as specimen, collect capillary blood from the pulp of the middle finger after cleaning the site well with rectified spirit CONSUMABLES/REAGENTS • • • • • • • •

Pasteur pipettes Glass slides Test tube Reticulocyte diluting fluid is prepared by adding Brilliant cresyl blue - 1 gm Sodium citrate – 0.6 gm Sodium chloride – 0.7 gm Distilled water to 100 mL

EQUIPMENT Light microscope.

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PROCEDURE • Take 2–3 drops of reticulocyte diluting fluid in a test tube and add 2–3 drops of well mixed K2 EDTA sample. • Mix well and keep the test tube at 37oC for 15–20 min. • Then take the tube out, mix well and make a thin smear of the stained blood. • Allow the smear to dry and count under oil immersion. A dark blue reticulum or network will be present in the reticulocytes. • Count 1000 red cells and note the number of reticulocytes among those. CALCULATION Reticulocyte percentage =

No. of reticulocytes counted × 100 Number of red cells counted

INTERPRETATION OF RESULTS The results are calculated as per the above formula and the number of reticulocytes is given as percentage of the total RBC counted PRECAUTIONS • Handle all samples as potentially infectious • Handle all reagents with care and avoid contact with eye, mouth and skin • Do not perform mouth pipetting • Discard used reagents and sample as per disposal procedure POTENTIAL SOURCES OF VARIABILITY • The sample should not be stained less than 30 minutes. REFERENCE RANGE Adults Infants Children up to 5 yrs

0.2 – 2% 2 – 6% 0.2 – 5.0 %

BIBLIOGRAPHY 1. District Laboratory Practices in Tropical Countries-Monica Cheesebrough. pg 332-3. 2. Medical Laboratory Technology -fourth edition –Ramnik Sood. pg 212. 3. Practical Hematology. Dacie and Lewis, 10th Edition. pg 36-40.


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RED BLOOD CELL INDICES PURPOSE To calculate red blood cell indices. DEFINITION OF TERMS MCV: Mean corpuscular volume, MCH: Mean corpuscular Hemoglobin, MCHC: Mean corpuscular Hemoglobin concentration, PCV: Packed cell volume, Hb: Hemoglobin, pg=picograms,μm 3 =micrometer cube, %= percentage. PRINCIPLE Red blood cell indices are useful in the diagnosis of anemia. Mean corpuscular volume (MCV) is defined as the volume of the average red cell expressed in μm3 or femtoliters(fl). Mean corpuscular Hemoglobin (MCH) is defined as the weight of hemoglobin in average red cells and expressed in picograms (pg). Mean cell hemoglobin concentration (MCHC) is defined as the ratio of hemoglobin to the volume of packed red cells. METHOD The following formula is used: Mean Corpuscular Volume (MCV): Mean Corpuscular Hemoglobin (MCH):

PCV × 10 = μm3 RBC count Hb × 10 = pg RBC count

Mean Corpuscular Hemoglobin Concentration:

Hb × 100 = percentage (%) PCV NORMAL RANGES MCV : 76–96 μm3 MCH : 27–32 pg MCHC : 30–35 % BIBLIOGRAPHY 1. Arthur Simmons Hematology: A combined Theoretical and Practical Approach. Second Edition. pg 27-31. 2. Practical Hematology. Dacie and Lewis, 10th Edition. Pg. 45-6.

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PACKED CELL VOLUME PURPOSE Quantitative determination of the amount of packed red blood cells in whole blood sample. Increased hematocrit values are observed in polycythemia, dehydration, emphysema and congenital heart disease. Decreased hematocrit values are observed in anemias and hydremia. PRINCIPLE When anti-coagulated blood is centrifuged in a sealed capillary tube, the erythrocytes sediment at the bottom. The red cell column is called as Packed Cell Volume (PCV) or hematocrit. The volume of packed cell is expressed as a percentage of the original volume of blood. PRIMARY SAMPLE • Use 2 mL of blood collected in 3.6 mg K2EDTA vacutainer. CONSUMABLES/REAGENTS • Micro hematocrit capillary tubes • Plasticine clay EQUIPMENT/INSTRUMENT • Micro haematocrit centrifuge PROCEDURE • Fill the Micro hematocrit capillary tubes with anti-coagulated blood collected in E tube • Seal one end with clay and centrifuge for 5 minutes at 10,000 rpm. INTERPRETATION OF RESULTS The percentage of packed cells is read by placing the spun capillary on the specially provided scale. PCV is recorded in “%” unit. PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure POTENTIAL SOURCES OF VARIABILITY • Hemolyzed specimen will yield false low values.


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• Inadequate mixing of blood and incompleteness of packing may lead to erroneous results. REFERENCE RANGE Normal values: At birth At 1 year At 10 years Women Men

: 44–63% : 35% : 37.5% : 30–40% : 40–54%

BIBLIOGRAPHY 1. Monica Cheesbrough. District Laboratory Manual in Tropical countries-part 2. Pg 310-2. 2. Practical Hematology. Dacie and Lewis, 10th Edition. Pg 16-7, 44-5.

HEMOGLOBIN ESTIMATION (CYANMETHEMOGLOBIN METHOD) PURPOSE To determine the Hb concentration in blood by Cyanmethemoglobin method. A decrease in Hb below normal range is an indication of anemia. An increase in Hb concentration occurs in hemoconcentration due to loss of body fluid in severe diarrhea and vomiting. High values are also observed in congenital heart disease (due to reduced oxygen supply) in emphysema and also in polycythemia. PRINCIPLE When blood is mixed with Drabkin’s reagent (potassium cyanide and potassium ferricyanide) Hb reacts with ferricyanide to form methemoglobin, which is converted to stable Cyanmethemoglobin by the cyanide. The intensity of the color is proportional to hemoglobin concentration and is measured at 540 nm. PERFORMANCE SPECIFICATIONS This method is linear up to Hb concentration of 19g/dl. For values beyond 19 g/dl, dilute the sample with 0.9% Isotonic Saline/= NaCl (1 :—) and repeat the test. Multiply the result obtained by dilution factor (x —) to get the actual value.

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PRIMARY SAMPLE • Collect 2 mL of blood in 3.6 mg K2EDTA vacutainer • Process the specimen on the same day within 2 hours of collection. REAGENTS/CONSUMABLES • Drabkin’s reagent • Cyanmethemoglobin Standard (HiCN) corresponding to Hb concentration of 15 g/dl EQUIPMENT Hemoglobinometer. PROCEDURE • From EDTA tube, containing blood for hematological investigations, using a 20 μL fixed volume micropipette withdraw blood and mix with 5 mL of Drabkin’s solution in a test tube. • Allow to stand the mixture for a minimum of 4-5 minutes to develop the color. • Within next 8–10 minutes measure the intensity of the color in a hemoglobinometer. PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure • Cyanide is poisonous and hence has to be used very carefully POTENTIAL SOURCES OF VARIABILITY • Presence of Hb C/Hb S, lipemia, abnormal plasma proteins and a high leukocyte count will produce turbidity and interfere with the test. • Deterioration of Drabkin’s reagent will produce erroneous results. REFERENCE RANGE Normal values At birth At 1 year 10–12 years Women Men

: 13.6–19.6 g % : 11.3–13.0 g% : 11.5–14.8 g% : 11.5–16.5 g% : 13.5–18.0 g%


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BIBLIOGRAPHY 1. Monica Cheesbrough. District Laboratory in Tropical Countries – Part 2 Pg 300-01. 2. Dacie and SM Lewis. Practical Haematology JV. 4th edition. pg. 12 3. The procedure has been modified according to the Hb meter manual. 4. Dacie and SM Lewis. Practical Hematology. 10th edition. pg. 26-30.

HEMOGLOBIN ELECTROPHORESIS PURPOSE Identification of abnormal hemoglobins in human blood sample by electrophoresis technique. The congenital disorder of hemoglobins (Hemoglobinopathies) that originate from a defect in the globin chain, are identified by this technique. PRINCIPLE Electrophoresis is the movement of the charged molecules in an applied electric field. Molecular migration in an electric field is influenced by the size, shape, charge and chemical composition of the molecule. Hemoglobin, obtained from hemolyzed red cells, is placed on a supporting medium (buffer-saturated gel type matrix) and allowed to migrate towards the anode at different rates in an electrophoretic apparatus, which enables their identification. PERFORMANCE SPECIFICATION The hemoglobin content in the hemolysate should be 10.0 g %. If it is more than 10.0 g %, add a few drops of distilled water to get the hemoglobin content of 10 g %. PRIMARY SAMPLE • Use venous blood as specimen. • Collect 2 mL of venous blood in a tube containing 3.6 mg of K2 EDTA. vacutainer. CONSUMABLES/REAGENTS • • • • • •

EDTA vacutainer Cathode buffer (pH 8.6) Anode buffer (pH 9.1) Carbon tetrachloride [Commercially available] Normal saline (NS) 1% Agarose

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• 3% acetic acid • Amidoblack EQUIPMENT/INSTRUMENT • Electrophoretic apparatus • Centrifuge PROCEDURE Preparation of the Hemolysate i. Collect 2 mL venous blood in K2 EDTA vacutainer ii. Transfer 1 mL of blood into the test tube - 12 × 100 mm and add 4 mL of NS mix gently and centrifuge the sample. iii. Discard the supernatant and repeat the washing of RBCs with saline thrice. iv. Then remove all the saline and add 2 to 3 drops of distilled water to the deposit. v. Close the tube with a rubber stopper and shake it vigorously for 10 minutes. This will lyse the RBCs. vi. Then add 1 mL of carbon tetrachloride and shake the tube again for another 10 minutes. vii. After this centrifuge the tube for 30 minutes. On centrifugation 3 layers are separated. viii. The top layer, the hemolysate, the middle layer that is the stroma and the bottom layer is of carbon tetrachloride. ix. Pipette out the hemolysate using a pasteur pipette and check its hemoglobin content by cyanmethemoglobin method. x. If the hemoglobin content is less than 10.0 g % the entire process is repeated using another sample. Electrophoresis i. Prepare hemolysate like the above mentioned procedure ii. Prepare 1% agarose. Take four clean microscopic slides and pour 2 mL of that onto the slide, making sure to avoid any air bubbles. Allow it to solidify without drying. iii. Write the sample identification mark on the back of slide with a diamond marker pencil. iv. The adjusted hemolysate is applied at the cathode end of the slide using a cover slip. v. Place the slide inside the tank, cut Whatman No: 1 filter paper into 25 × 25 mm and used it to bridge the slide and buffer.


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vi. The current (2 mA/sample and a voltage of 250 V) is passed for all four slides from cathode to anode vii. Run the electrophoresis for 4 hours viii. Take the slides out, fix them in Methanol for 30 minutes ix. Then dry the slides by incubating at 37°C for 12 – 18 hours x. Stain the dried slides in 1% amido black for 8 min and then destain in the mixture of 90% methanol containing 10% acetic acid until the background is clear xi. Destain again with pure methanol for 5 min air dry the slides and label them. INTERPRETATION OF RESULTS The abnormal hemoglobins are identified by comparison of their migration with that of Hb A which is found in normal adults. PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure POTENTIAL SOURCES OF VARIABILITY BIBLIOGRAPHY 1. Practical Haematology - Sir John V Dacie and SM Lewis, 10th Edition. Pg. 282 and 180 2. Practical Clinical Biochemistry - Harold Varley.

BLOOD GROUPING AND Rh TYPING PURPOSE To determine the blood group and Rh type in human. This test is used in the identification of patient’s ABO and Rh blood type prior to surgery or other procedures in which blood loss is a threat and for stored blood. It is used in the determination of ABO and Rh compatibility of donor and recipient’s blood. It is also used in the identification of maternal and infant ABO and Rh blood types to predict potential hemolytic disease of the newborn.


202 PRINCIPLE

This test is based on the principle of direct hemagglutination. The erythrocytes of a person contain antigens on the surface of the membrane. When these antigens are allowed to react with the corresponding antibodies, antigen-antibody reactions are produced. Normal erythrocytes will clump or agglutinate when mixed with Anti A/Anti B/ Anti A and B, if they possess A/B/AB antigens respectively. PERFORMANCE SPECIFICATIONS • Presence of immune antibodies adsorbed on to the surface of the red cells carrying the corresponding antigens will not produce agglutination. These blood group antigens can later be detected by the reaction with anti human gamma globulin reagent. • Visible agglutination reactions require proper proportion of antigen and antibody. PRIMARY SAMPLE • • • •

Use whole blood as specimen. Collect 2 mL of blood in 3.6 mg K2EDTA vacutainer. Process the sample within 1 hour of collection. If a delay in testing is expected or unavoidable, red cells from clotted samples, EDTA/Heparinized anticoagulant samples should be separated from the plasma, washed and stored in a red preservative solution at 2–8°C for no longer than 35 days. • Do not use insufficient sample and sample without an ID number. CONSUMABLES/REAGENTS • • • • • • • •

Anti A, blood grouping reagent (Commercially available) Anti B, blood grouping reagent (Commercially available) Anti AB, blood grouping reagent (Commercially available) Anti D, blood grouping reagent (Commercially available) Anti A1 lectin (Commercially available) Glass slides Applicator sticks 0.9% Isotonic saline

EQUIPMENT Not applicable


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PROCEDURE Slide Agglutination Method • Take a clean, grease free microscope slide. • Draw a line in the middle of this slide using a wax glass marking pencil and label the left portion Anti-A and the right as Anti-B. • Add a drop of Anti-A to the portion marked as Anti-A, and add a drop of Anti-B to the portion marked as Anti-B • Add one drop of well mixed 3–5% cell suspension to each side • With a tooth pick, separate for each side, mix the cells and anti-sera well. • Gently rotate the slide for mixing. • Place the slide against a white background • After 2 minutes examine both macroscopically and microscopically for agglutination. Tube Agglutination Method • • • • • • • • • • • • •

• • •

Switch on the water bath and adjust the temperature to 37°C. Take two tubes of size 12 × 100 mm. Mark one tube as anti A and the other as anti B. Add two drops of anti-A to the tube labelled as anti-A and two drops of anti-B to the tube labelled anti-B Add two drops of 3–5% cell suspension to each tube. Mix well and centrifuge both tubes at 1500 rpm for 1 minute. Remove the tubes and inspect the button of the cells in the bottom. With the gentle shaking or tapping of the tube, observe the dislocation of the cell button. Report - Positive if agglutination is observed Place the tube/tubes in which no agglutination was observed in 37°C water bath for 30 minutes. Remove the tubes from the water bath and centrifuge once again and observe the cell button by shaking the tube gently. Report as positive if agglutination is observed. Double check the results microscopically by putting one or two drops from each tube on microscopic slides. Prepare a 3–5% suspension of test red cell. All suspensions may be prepared in autologous serum /plasma/saline. Place one drop of the appropriate A,B, AB blood grouping reagent on a clean, dry glass slide at room temperature Add one drop of the prepared 35–45% suspension of red cells to each drop of reagent on the glass slide.

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• Mix the cells and reagent thoroughly over an approximate 20 mm circular area, using a separate, clean applicator stick, one for each reagent - red cell mixture • Rock or rotate slides gently and examine for macroscopic hemagglutination. Agglutination may begin within a few seconds. However observation should not continue beyond 2 minutes. INTERPRETATION OF RESULTS • If agglutination is observed with anti A blood group reagent, then the patient’s blood group is A • If agglutination is observed with anti A1 Lectin reagent, then the patient’s blood group is A1 • If agglutination is observed with anti B blood group reagent, then the patient’s blood group is B • If agglutination is observed with both anti A and Anti B blood group reagent, then the patient’s blood group is AB • If agglutination is not observed with both anti A and Anti B blood group reagent then the patient’s blood group is O • If agglutination is observed with anti D blood group reagent, then the patient’s Rh type is Positive. All Rh negative is reconfirmed with Du testing. PRECAUTIONS • Thoroughly clean and dry the test slides before use. • Ensure reagents and specimens are at room temperature before use. SAFETY PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure. POTENTIAL SOURCE OF VARIABILITY • False negative or unexpectedly weak reactions may occur with blood samples of weak A or B subgroups or with cord blood red cells from newborn infants. • False negative or unexpectedly weak reactions may occur with red cells or anti-sera that have been subject to prolonged storage and/or inappropriate storage conditions.


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DU TESTING Purpose: The Du factor (a variant of D antigen present in the red cells of individuals of Du blood type) reacts with anti D but does not bring about hemagglutination that is not strong enough to be visualized. This test is done to reconfirm all Rh negative blood groups. Principle: Cells with Du antigen are sensitized with anti D by incubating at 37oC for 30 minutes. This results in the adsorption of anti D on the surface of the cell without producing hemagglutination. The presence of reacted antibody on the surface of Du cells is recognized by using antihuman globulin which reacts with coated antibody and brings about hemagglutination. Primary sample • Use whole blood as specimen. • Collect 2 mL of blood in 3.6 mg K2 EDTA vacutainer. Mix well. • Process the sample within 1 hour of collection. • Do not process insufficient samples and sample without an ID number. Consumables/Reagents • Slides • Pipettes and tubes • Anti D (Monoclonal IgM + IgG) • Antihuman globulin • Normal saline. Procedure • Prepare 5% suspension of washed red cells with normal saline. • Take 1 tube and label it as test (T) • Place 2 drops of 5% cell suspension in the tube • Add 1 drop of anti D in the tube labeled T • Place the tube in the water bath at 37oC for 30 minutes. • Remove the tubes from the water bath and wash the cells in normal saline 2–3 times. • Add 2 drops of anti human globulin to both the tubes. Mix gently • Centrifuge the tubes at 1500 rpm for 1 minute and look for agglutination. Interpretation of results • Du positive: If agglutination is present in the tube labeled T • Du negative: If no agglutination seen in the tube T.


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DIRECT AND INDIRECT COOMBS’ TEST PURPOSE Coomb’s test is used to detect sensitization of red cells with immune antibody (IgG) or the complement component, generally CD3 by tube agglutination technique. Direct Coombs’ test is used detect sensitization of red cells by Rh antibodies or other antibodies attached to the red cell surface and occurring in vivo (within the body) as in hemolytic disease of newborn erythroblastosis fetalis, autoimmune hemolytic anemias, transfusion reactions and drug induced red cell sensitization. Indirect Coombs’ test detects sensitization of red cells occurring in vitro i.e in the laboratory and is useful to detect weakly reacting D antigen as in Du testing PRINCIPLE Red blood cells coated with incomplete antibody (IgG) or CD3 component of complement either in vivo as in direct antiglobulin test or in vitro as in indirect antiglobulin test do not agglutinate. When these sensitized red cells come in to contact with antihuman globulin reagent (Coombs’ Reagent) they agglutinate by binding to the antibodies coating the cells. PRIMARY SAMPLE Use only serum as specimen for indirect Coombs’ test. Collect 2 mL of venous blood from a peripheral vein in a plain red topped vacutainer tube /0.1 N HCl washed tube. Collect 2 mL of blood in 3.6 mg K2EDTA vacutainer. Do not use hemolyzed, contaminated sera for testing. Process all samples on the same day within 2 hours of collection. If the sample is to be processed on the next day or after 2 hours separate the serum and store it at 2–8 °C until required, up to a maximum 7 days. CONSUMABLES AND REAGENTS Anti-human Globulin reagent, Group O Rh (D) positive cells (Coomb’s control cells) –prepared by pooling blood of three ‘O’ positive samples and then washing the red cells three times in normal saline, anti-D serum (IgG type). Coombs’ control cells should be free from serum make 5% suspension of these washed cells in normal saline. EQUIPMENT No Special equipment is required for the test. Glass test tubes, Pasteur pipettes, Incubator for maintaining 37°C, Lab centrifuge.


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PROCEDURE Preparation of Coombs’ Control Cells Wash the O Rh (D) positive cells three times in saline. Take 0.5 mL of washed and packed cells in a test tube. Add 2 drops of anti-D (1:4 dilution). Mix and incubate at 37°C for 30 minutes. If there is agglutination, repeat the procedure using more diluted anti-D. Wash the cells 4 times and then make 5% suspension in saline. Take one volume of 5% cell suspension and add 2 volumes of anti-human globulin (AHG) reagent. Mix gently and centrifuge the tube. These sensitized cells can be stored at 4°C for 48 hrs. Direct Coombs’ Test Place 1 drop of 2-4% cell suspension to be tested in a clean test tube. Wash the cells 3 times with saline and decant the final wash completely. Add 1-2 drops of anti-human globulin (Coomb’s serum). Mix and centrifuge at 1000 rpm for 1 minute. Gently shake the tubes and read the results using optical aid. If the result is negative, add 1 drop of (control IgG coated cells). Mix and centrifuge at 1000 rpm for 1 minute and look for agglutination. If no agglutination is seen, the test result is invalid. Repeat the test procedure. Indirect Coombs’ Test Take 2 drops of serum to be tested in a labelled tube. Add one drop of 2–4% suspension of O cells. Incubate at 37°C for 45–60 minutes. Look for hemolysis and agglutination. Agglutination or hemolysis at this stage indicates presence of saline reacting antibody. If no hemolysis or agglutination is seen, wash the cells four times in saline, and decant the last wash completely. Add 1-2 drops of coomb’s reagent to the washed cells and mix. Centrifuge the tubes at 1000 rpm for 1 minute. Gently shake the tubes and look for agglutination using optical aid. If the test is negative, add 1 drop of control (IgG coated cells). Mix and centrifuge at 1000 rpm for 1 minute. Look for agglutination. If no agglutination is seen, the test is invalid and whole test procedure is repeated. INTERPRETATION OF RESULTS Direct Coombs’ test: Agglutination of red cells with the addition of AHG indicates a positive Coombs’ test and indicates that the cells are sensitized within the body. Indirect Coombs’ test: If the red cells do not show agglutination even after the addition of anti- D and AHG, then the red cells are considered as truly Rh negative and Du Negative.

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POTENTIAL SOURCE OF VARIABILITY False negative results: Inadequate washing of cells, delay (or) interruption of washing procedure, failure to add coombs’ serum, prozone effect, Insufficient incubation. False positive results: Agglutination of red cells before adding Coombs’ reagent, dirty glassware, Coombs’ reagent containing antihuman species antibody, cold auto-antibodies in sample, presence of saline reacting antibody may cause agglutination or hemolysis. PRECAUTIONS Ensure the reagents and specimens are brought to room temperature before use. SAFETY PRECAUTIONS Handle all reagents with care and avoid contact with eye, mouth and skin. Handle all samples as potentially infectious. Discard used reagents and sample as per disposal procedure. BIBLIOGRAPHY 1. Practical Hematology. Dacie and Lewis 10th edition, Pg 675.

ACTIVATED PARTIAL THROMBOPLASTIN TIME PURPOSE Determination of aPTT is used to diagnose hemophilia that involves the deficiencies of the factors such as VIII, IX, XI, and XII and X. This test can also detect deficiencies of factors V, X and XII. aPTT is increased in the presence of inhibitors of coagulation and in Disseminated Intravascular Coagulation (DIC). It is also useful in the control of heparin therapy. PRINCIPLE The calcium in the whole blood is bound by the anticoagulant sodium citrate to prevent coagulation. The plasma, the fluid component in the anticoagulated blood, contains all intrinsic coagulation factors except calcium and platelets. After the addition of calcium and a phospholipid substitute for platelets with an activator to the plasma, the time required for the plasma to clot is PTT.


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DEFINITION PTT - Partial Thromboplastin Time; rpm - revolutions per minute. PRIMARY SAMPLE • • • • • •

Use only plasma as specimen for the test. Collect 2.7 mL of venous blood in 3.2% trisodium citrate vacutainer. Mix well by rolling the tube in between the palms. Centrifuge the sample at 4000 rpm for 15 minutes. Separate plasma immediately in a vial and store at 2–6oC until tested. Process the samples within one hour of collection.

CONSUMABLES/REAGENTS • • • • •

Pipettes Plastic centrifuge tubes Autoclaved test tubes aPTT reagent 0.025 M calcium chloride reagent.

EQUIPMENT/INSTRUMENT • • • •

37ºC waterbath Stop watch Thermometer Laboratory centrifuge.

PROCEDURE • Collect and prepare blood specimens according to the directions outlined in the sampling instructions. • Prepare the reagents for use in the procedure according to the reconstitution instructions. • Switch on the waterbath and adjust the temperature to 37ºC. • Prewarm the 0.025 M calcium chloride reagent to 37oC for at least 10 minutes in incubator. • Pipette 0.1 mL of test plasma and 0.1 mL of aPTT reagent into the reaction tube and incubate the aPTT reagent and patient plasma for exactly 3 minutes at 37oC. Exact incubation time is critical. • Then add 0.1 mL of pre-warmed 0.025 M calcium chloride reagent to the plasma and aPTT reagent mixture. Simultaneously start the stopwatch. • Mix the tube immediately after adding the calcium chloride. Keeping the tube to remain in the waterbath gently tilt the tube every 5 seconds. At the end 20 seconds remove the tube from the waterbath. Quickly wipe off the outside of the tube with a clean tissue paper, so that the inner contents are clearly visible.

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• Gently tilt the tube back and forth until clot forms, at which point the timer is stopped. • Repeat the same steps from No. 8.4 to 8.8 in another 12 × 100 mm test tube labeled as T2. • Take the average of T1 and T2 values as the patients PTT value. • Perform the same test on a control sample collected from a normal person, labelling the tubes C, C1, C2. Average of C1, C2 is control PTT Value • Repeat the test for abnormal values. • Record the results • Do the platelet count on the plasma (of all the test samples) and record the results in the platelet poor plasma-checking notebook (platelet count should be less than 10,000 /cu mm). INTERPRETATION OF RESULTS The results of the aPTT test should be reported to the nearest second. PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure POTENTIAL SOURCES OF VARIABILITY • The method of clot detection, temperature, pH, collection technique, type of anticoagulant and time and method of plasma storage influence the results. • Presence of factors I, II and V provide abnormal aPTT tests. • aPTT is sensitive to various specific and non specific circulatory anticoagulants. REFERENCE RANGE 22-34 seconds BIBLIOGRAPHY 1. Human Blood Coagulation, Haemostasis Thrombosis, Edited by R Biggs, 1st Edition 1972. 2. Hoffmann JJML and Neulendijk PN: Thrombos.Haemosta (Stuttgart) 39, 640 (1978) 3. CRC, Handbook Series in Clinical Laboratory Science, section 1: Haematology, Vol III, 1980,CRC Press Inc 4. Practical Hematology. Dacie and Lewis 10th edition, Pg 399.


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PROTHROMBIN TIME PURPOSE Determination of Prothrombin Time is an easy screening test which indicates possible coagulation defect. The PT is prolonged when there is lack of vitamin K absorption in obstruction or lack of synthesis in hepatocellular disease. It is used for the diagnosis of congenital and acquired deficiency of the extrinsic pathway involving factors II, V, VII and X. This test is also used to monitor oral anticoagulant therapy. PRINCIPLE When a mixture of tissue thromboplastin and calcium is added to citrated plasma, stage 2 of the coagulation mechanism is triggered in the presence of factor VII . The time taken for a solid clot to form is recorded. Since factors XII, XI, VIII and platelets are bypassed; the test depends on the activity of factors I, II, V, VII and X. Deficiency of any of these factors may cause prolongation of clot formation in this test. PRIMARY SAMPLE • • • •

Use only plasma as specimen for the test Collect 2.7 mL of venous blood in 3.2% trisodium citrate vacutainer Centrifuge the sample at 4000 rpm for 15 minutes. Separate plasma immediately in an autoclaved test tube and store at 2–6oC until tested. • Process the samples within one hour of collection. CONSUMABLES/REAGENTS • • • •

Pipettes Plastic centrifuge tubes Thromboplastin reagent Autoclaved test tubes (12x 100 mm).

EQUIPMENT/INSTRUMENT • • • •

37ºC Waterbath Laboratory centrifuge Stopwatch Thermometer.

PROCEDURE • Collect and prepare blood specimens according to the directions outlined in the sampling instructions.

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• Prepare the reagents for use in the procedure according to the reconstitution instructions • Allow the instrument to warm up for 5 minutes. • Prewarm the PT reagent to 37o C incubator. • Pipette 0.1 mL of test plasma into the test tube. • After incubation, then add 0.2 mL of pre-warmed thromboplastin reagent to the plasma • Pipette out prepared PT reagent, and add 0.2 mL each of this reagent to two tubes labelled as T1 and T2 and place the tubes in the water bath. • Keep the T tube in the water bath for about 5 minutes. • Add 0.1 mL of plasma from T tube into the test tube marked T1 and simultaneously • Start the stopwatch. • Remove the T1 tube from the water bath at the end of 12 secs and quickly wipe outer surface of the tube with a clean tissue, so that the inner contents of the tube are clearly visible • Gently tilt the tube back and forth till a clot forms at which point the timer is stopped. Note the time. • Repeat steps from 7.5 to 7.11, by using T2 tube • Average of T1 and T2 results is the PT of the sample. • Repeat the entire procedure on any normal plasma labelling as C, C1, and C2. Control PT is the average of C1 and C2. • Repeat the test for abnormal values. • Record the results. • Do a platelet count on the plasma (of all the test samples) and record the results (platelet count should be less than 10,000 /cmm). MNPT AND INR • MNPT is a critical requirement in the derivation of INR • Ideally each laboratory must derive its own MNPT from 10 normal patients for a given PT reagent and lot under use. INTERPRETATION OF RESULTS • Report Prothrombin Time results only as INR (International Normalized Ratio) • The results of the prothrombin test should be reported to the nearest 10th of a second. PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin.


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• Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure. POTENTIAL SOURCES OF VARIABILITY • The method of clot detection, temperature, pH, collection technique, type of anticoagulant and time and method of plasma storage influence the results. • The presence of heparin or EDTA as an in vitro anticoagulant may give invalid • Prothrombin time results • Presence of factors I, II and V provide abnormal PT tests. REFERENCE RANGE 11–15 seconds. BIBLIOGRAPHY 1. Human Blood Coagulation , Haemostasis Thrombosis , Edited by R Biggs , 1st Edition, 1972. 2. Practical Hematology. Dacie and Lewis, 10th edition, Pg 398.

BLEEDING TIME (IVY METHOD) PURPOSE To record the time in minutes which takes for a standardized skin wound to stop bleeding. Determination of bleeding time is useful to detect vascular defect and platelet disorders, prolonged bleeding time is associated with Thrombocytopenia and von Willebrand’s disease (where the platelet count is normal). PRINCIPLE A standard incision is made in the skin of the patient and the length of time required for bleeding to cease is recorded. DEFINITION OF TERMS Nil PERFORMANCE SPECIFICATION Not applicable PRIMARY SAMPLE Blood collected by skin puncture.

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CONSUMABLES/REAGENTS • Sterile disposable lancet (capable of making an incision of 1mm wide and 3 mm deep. • Filter paper • Cetavlon • Sterile gauze cotton pad • Stop watch. EQUIPMENT Sphygmomanometer. PROCEDURE • Perform this procedure in the collection department itself. • Tie a blood pressure cuff on the upper arm and inflate it to 40 mm of Hg and maintain this pressure throughout the period of the test. • Select an area on the volar aspect of the forearm, which is devoid of superficial veins and about 3 finger breadths below the bend of the elbow • Clean this area with cetavlon and allow it to dry. • Stretch the skin laterally between the thumb and forefinger by grasping the under side of the forearm and hold in a taut position. • Make a skin punctures 5–10 cm apart in quick succession (3 mm deep) using the disposable lancet. • Start the stopwatch as soon as the incisions are made and bleeding starts. Gently touch the drop of the blood which forms over the wound, with the edge of a filter paper every 30 seconds. Do not rub or remove the clot. Do not touch the skin. • The filter paper should not touch the incision point at any time. Any disruption of the fibrin formed or clot will prolong the bleeding time. • The Bleeding time is reported when no bloodstain is seen on the filter paper after a gentle touch. When bleeding ceases stop the watch and release the blood pressure cuff. INTERPRETATION OF RESULTS The bleeding time is the time when no blood stain is seen on the filter paper after a gentle touch. It is reported in intervals of 30 seconds. PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure.


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POTENTIAL SOURCES OF VARIABILITY Not Applicable. REFERENCE RANGE 1 – 6 minutes. BIBLIOGRAPHY 1. Practical Haematology JV Dacie and SM Lewis, 4th Edition, Pg. 263-64. 2. Practical Hematology. Dacie and Lewis, 10th edition, Pg. 417-18.

CLOTTING TIME PURPOSE To measure the time taken for whole blood to clot. This test is used as a routine screening test in the diagnosis of clotting disorders. Clotting time is prolonged in Hemophilia, Christmas Disease (Hemophilia B) and Disseminated Intravascular Coagulation. PRINCIPLE Venous blood is collected in a clean glass tube without any anticoagulant. The length of the time it takes for the blood to clot at 37ºC is recorded. PERFORMANCE SPECIFICATION Not Applicable. DEFINITION OF TERMS Nil PRIMARY SAMPLE Collect at least 1 mL of venous blood from a peripheral vein with minimum trauma. CONSUMABLES/REAGENTS • • • • •

Sterile syringe and needle. Cotton and cetavlon Test tubes. Stop watch. Water bath with calibrated thermometer to maintain 37ºC.


216 PROCEDURE • • • • • • • • •

•

Perform clotting time in the collection center itself Before performing clotting time on the patient, set the water bath to 37ºC Label three 12x75 mm test tubes as # 1, # 2, and # 3. Clean the blood collection site with cetavlon and allow it to dry completely. Collect 3.0 mL of venous blood in a disposable syringe and start the stop watch the moment blood enters the syringe Dispense 1 mL of the blood each into 3 clean, dry, glass tubes of size 10 mm × 75 mm as # 3, # 2, # 1. Keep the tubes in water bath maintained at 37ºC After every 30 seconds tilt the tubes one after another without removing them from the water bath and see whether blood has clotted. Then remove the first tube and examine it by tilting it slowly to the horizontal level. Repeat this till clotting has occurred in test tube # 1 such that blood does not flow out even when the tube is tilted to 90 degrees Note this time and note the time with the second tube and third tube also and stop the stop watch.

INTERPRETATION OF RESULTS The time taken for the blood to clot in the third tube is taken as the clotting time. PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Discard used reagents and sample as per disposal procedure. • Handle all samples as potentially infectious. • Wear gloves while performing the test. POTENTIAL SOURCES OF VARIABILITY • If the volume of the blood drawn is less than 1ml then the clotting time will be shortened • Presence of air bubbles, inaccurate incubation temperature and agitation of the specimen lead to inaccurate determination of clotting time. REFERENCE RANGE 5-15 minutes BIBLIOGRAPHY 1. Practical Haematology. JV Dacie and S M Lewis. 4th Edition. Pg. 264-5. 2. Practical Hematology. Dacie and Lewis 10th edition. Pg. 417.


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CLOT RETRACTION PURPOSE To determine the clot retraction time in whole blood sample. The quality of clot retraction reflects on both the number and function of the platelets. This test is used in the diagnosis of hemorrhagic disorders related to platelets. PRINCIPLE When the whole blood coagulation is complete, the clot which forms as the end product normally undergoes contraction, where the serum is expressed from the clot, and the clot becomes denser. Thrombosthenin released by the platelets is responsible for the clot retraction. PERFORMANCE SPECIFICATION Not applicable PRIMARY SAMPLE • Use whole blood as specimen. • Collect 3 mL of whole blood by venipuncture and dispense it in a plain tube. CONSUMABLES/REAGENTS Plain tubes EQUIPMENT/INSTRUMENT • Water Bath PROCEDURE • Switch on the water bath and adjust the temperature to 37°C • Place the sample tube in the water bath and note the time. Leave the tube undisturbed for the blood to clot and its subsequent retraction • At the end of 1, 2, 4 and 24 hours inspect the tube • The clot should be firm and retracted from the sides of the tube. It generally occupies more than half of the original volume. INTERPRETATION OF RESULTS • Good—If clot retraction has occurred at 2–4 hours. • Fair—If retraction occurs after 4 hours but within 24 hours • Poor—If no retraction occurs even at 24 hours.


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PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure POTENTIAL SOURCES OF VARIABILITY • The number of platelets present affect the clot retraction. BIBLIOGRAPHY 1. Cartwright, G.E. Diagnostic Laboratory Haematology Grune and Stratton , Inc. New York - 1963.

FIBRINOGEN ASSAY (CLOT WEIGHT METHOD) PURPOSE Quantitative estimation of fibrinogen in plasma. Low plasma fibrinogen is observed in severe liver disease. Reduced levels of plasma fibrinogen are observed in typhoid fever. Increased levels are observed in acute infections such as tuberculosis and rheumatoid disease and also during pregnancy. PRINCIPLE The clot formed in the plasma after the addition of calcium chloride is proportional to the concentration of fibrinogen. PERFORMANCE SPECIFICATION Not Applicable PRIMARY SAMPLE • • • •

Use citrated plasma as specimen. Collect 2.7 mL of whole blood in 3.2% trisodium citrate vacutainer Mix by rotating the tube in between the palms Centrifuge the blood at 4000 rpm for 15 minutes and separate the plasma.

CONSUMABLES/REAGENTS • 3.2% Sodium citrate • 0.025M Calcium chloride


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• Acetone. • Distilled water EQUIPMENT/INSTRUMENT • Water bath • Hot air oven • Balance PROCEDURE • Remove the supernatant plasma with a pasture pipette and transfer it to a plain tube marked as T for test and keep it in a beaker containing ice • Split the ends of an applicator stick and place it in a test tube (12 × 100 mm) labeled as T1. • Pipette out 1 mL of plasma and transfer it in T1. • Add 1 mL of calcium chloride to the above tube and mix well. • Incubate at 37°C for 15 minutes. • Harvest the clot gently by winding the stick and remove all the serum by pressing the clot over the wall of the tube. • Transfer the stick with the attached clot in a tube containing approximately 2 mL of distilled water • Then remove the stick and slit the sleeve of the clot using a tooth-pick and peel it off onto a filter paper. • Blot dry the clot and transfer it to 5 mL of acetone. • Keep it in acetone for 3 minutes till the clot becomes nail hard. • Remove the clot and leave it in hot air oven 160°C for 2 to 3 minutes for acetone to evaporate. CALCULATION Fibrinogen = Clot weight in mg INTERPRETATION OF RESULTS The clot formed is weighed using a balance which is proportional to the fibrinogen concentration. PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure


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POTENTIAL SOURCES OF VARIABILITY Not Applicable REFERENCE RANGE 250 to 450 mgs/DL BIBLIOGRAPHY 1. Human blood coagulation, Haemostasis and Thrombosis - Rose Mary Biggs. Pg. 647-48. 2. Practical Haematology - Sir John V Dacie, SM Lewis, 6th edition. Churchill Livingstone Edinburgh. Pg. 229. 3. Practical Hematology. Dacie and Lewis 10th edition Pg. 401.

EUGLOBULIN LYSIS TIME TEST PURPOSE To determine the euglobulin clot lysis time for the measurement of fibrinolysis. PRINCIPLE The plasma euglobulins are precipitated with 1 % acetic acid and resuspended in a borate solution. The euglobulins are allowed to clot by the addition of calcium chloride. The clot is incubated, and time of lysis is reported. PERFORMANCE SPECIFICATION Not Applicable PRIMARY SAMPLE • • • • •

Use citrated plasma as specimen. Collect 2.7 mL of venous blood in 3.2% trisodium citrate vacutainer Mix by rotating the tube in between the palms Centrifuge the blood at 4000 rpm for 15 minutes and separate the plasma. Process the sample immediately after separation of plasma.

CONSUMABLES/REAGENTS • • • •

3.2% Sodium citrate 0.025M Calcium chloride 1 % Acetic acid Borate Solution


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• Distilled water • Test tubes EQUIPMENT/INSTRUMENT • Water bath. • Centrifuge PROCEDURE • Pipette out the patient’s plasma into test tube (12 × 100 mm size) marked as T • To another test tube (15 × 125 mm) marked as T1, pipette out 9.0 mL of distilled water, 0.5 mL of patient’s plasma and 0.1 mL of 1% acetic acid and mix well. • Refrigerate the preceding mixture for 30 minutes at 4°C to allow for euglobulin precipitation. • Centrifuge at 2500 rpm for 5 minutes • Pour off the supernatant and invert the tube on filter paper to drain. • When drained well revert the tube and add 0.5 mL of the Borate solution and place in a 37°C water bath. • Stir the mixture gently with a glass rod. • Add 0.5 mL of 0.025 M calcium chloride to the mixture • Incubate the tube in the 37°C water bath and periodically check for the clot lysis. When the clot lysis begins, check the tube every 5 minutes until the lysis is complete • Repeat the procedure on plasma collected from normal person, marking the tube as C and C1. INTERPRETATION OF RESULTS Observe the time of completion of clot lysis. PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure POTENTIAL SOURCES OF VARIABILITY Not Applicable REFERENCE RANGE Normal range: 2–4 hours

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BIBLIOGRAPHY 1. Bucknell, M: The effect of citrate on englobulin methods of estimating fibrinolytic activity. J Clinical Pathology 1958. Pg. 11,403. 2. Practical Hematology. Dacie and Lewis 10th edition. Pg. 455.

FACTOR XIII ASSAY PURPOSE Qualitative estimation of factor XIII in blood. Moderately decreased factor XIII levels are observed in liver disease, sickle cell anemia and in pregnancy. Activated factor XIII levels are increased in acute myocardial infarction. Factor XIII exists in the plasma in an inactive form and is activated by thrombin during the conversion of fibrinogen to fibrin. PRINCIPLE The plasma is allowed to clot by the addition of calcium chloride. 5 M urea is then added to the clot. In the presence of normal amounts of factor XIII this clot remains un-dissolved for 24 hours in 5 M urea solution. PERFORMANCE SPECIFICATION Not applicable PRIMARY SAMPLE Use citrated plasma as specimen. Collect 2.7 mL of venous blood in 3.2% trisodium citrate vacutainer. Mix by rotating the tube in between the palms. Centrifuge the blood at 4000 rpm for 15 minutes and separate the plasma. CONSUMABLES/REAGENTS 1. 3.2% Sodium citrate 2. 5 M urea 3. 0.025 M Calcium Chloride EQUIPMENT/INSTRUMENT Water bath PROCEDURE 1. Switch on the water bath and adjust the temperature to 37°C 2. Pipette 0.5 mL of patient’s plasma into a test tube (12 × 100 mm size) marked as T. 3. Then add 0.5 mL of 0.025 M calcium chloride to it.


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4. Incubate the resulting fibrin clot at 37°C for 30 minutes. 5. Loosen the clot from the sides of the test tubes by gently tapping the sides of the tube 6. Then add 5 mL of 5 M urea and shake the clot in the urea solution 7. Allow the tubes to stand in the water bath at 37°C. 8. Check the clot at the end of 1 hour, 2 hours, 3 hours and 24 hours and note if the clot has dissolved 9. Repeat the steps 7.3–7.8 on normal plasma, labeling the tube as C for control. INTERPRETATION OF RESULTS Normal—The clot remains un-dissolved for 24 hours and remains as firm clot. Abnormal—If the clot dissolves within 24 hours and if the clot has become soft. PRECAUTIONS 1. Handle all reagents with care and avoid contact with eye, mouth and skin. 2. Handle all samples as potentially infectious. 3. Discard used reagents and sample as per disposal procedure POTENTIAL SOURCES OF VARIABILITY Not applicable BIBLIOGRAPHY 1. Practical Haematology, 4th edition, Churchill Livingstone , New York , 1975 Pg 301-2.

EXAMINATION OF MALARIAL PARASITE PURPOSE To prepare a blood film for the examination of the malarial parasite. This test helps in identification of the stage of the parasite in life cycle and also the characteristics of certain species of plasmodium in the diagnosis of malaria. PRINCIPLE The thick and thin blood smear is prepared on a microscopic slide which is dried, fixed and stained. The thin smear is stained with Leishman stain which allows studying the morphological abnormalities of red cells. The

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thick smear is stained with Field stain without fixing in methanol, which removes the red cells by hemolysis and leaves behind the imprints of the parasite as pink dots and other structures. PERFORMANCE SPECIFICATIONS • The film must be smooth at the end. There should be no lines extending across or down through the film and it should not contain holes. PRIMARY SAMPLE • Collect 2 mL blood in K2 EDTA Vacutainer and mix well gently. • Do not use clotted sample for testing. CONSUMABLES/REAGENTS • Leishman stain: It is prepared by dissolving 2g of powdered stain in 1000 mL of (acetone free) methyl alcohol • Giemsa stain: • Preparation of the solution: • Giemsa powder certified (Merck or Sigma) 3.8 g • Glycerol 250 mL • Absolute Methanol (acetone free) 250 mL • Weigh 3.8 g of Giemsa powder and transfer to a mortar. Using dry cylinder measure 10 mL glycerol and add this into the mortar and grind well with Giemsa powder and transfer into a dry brown bottle containing glass beads. Add 240 mL of glycerol and keep the bottle in 60°C water bath for 2 hours and allow cooling at room temperature. Then add 250 mL of absolute methanol slowly with stirring. Keep the bottle in 60° C for water bath for 1 hour. Allow to cool and store at room temperature for 1 week. Decant and remove the sediment. Filter before use. • Preparation of Stock Phosphate buffer (pH7) – Stock A: 0.2 M sodium dihydrogen phosphate 3.12 g in 200 mL of distilled water – Stock B: 0.2 M disodium hydrogen phosphate – 10.8 g in 400 mL of distilled water • After preparation the solution is stored at room temperature in dark bottles • Working solution of buffer: To 14 mL of stock A and 36 mL of Stock B is added and made up to 100 mL with distilled water • Preparation of Giemsa stain: Dilute 5 mL of the Giemsa stain with 37 mL of the working solution of the phosphate buffer and transfer in a Coplin jar in the incubator at 37°C. • Buffered water • Immersion oil


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• Glass slides • Cotton • Slide rack INSTRUMENT • Light microscope. PROCEDURE • Preparation of thick smear: • Arrange 2–3 clean grease free - microscope slides on the work table. • Before dislodging the needle from the syringe in which the blood is collected from the patient, place one large drop of blood on each slide. • Using the corner of another microscope slide carefully spread the drop of blood over an area approximately the size of a 10 paise coin. • Allow the smears to air dry for about 30 minutes. • After drying place two drops of distilled water, so that the RBCs will be lysed. • Fix the smear in methanol for 5 min, and stain with Giemsa stain for 30 min at 37°C. • After 30 min the slide is washed off with distilled water • Examine the smears microscopically using the oil immersion objective to identify the species of malaria. PREPARATION OF THIN SMEAR • Take a clean, dry slide. • Transfer a small drop of blood near the edge of the slide. • Place a smaller piece of glass with a width slightly less than that of the slide called as spreader at an angle of about 45º to the surface of the slide. • Pull back the spreader until it touches the drop of blood. • Put the spreader forward to the end of the slide with a smooth movement. • Dry the blood smear at room temperature. STAINING • Staining of thin smear with Leishman stain: • Place the slide with the smear side up on a staining rack horizontally (without sloping). • Label it with the date with a pencil and place it on a staining rack, making sure that the smeared surface faces upward for staining. • Add Leishman stain to cover the entire slide. Then dilute it with working buffer.

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• Fifteen minutes later drain off the stain and wash the slide under running tap water. • Place it on a rack to dry. Examination of stained smears: The air-dried stained smear is viewed 1st under dry 10X and later under 100X oil immersion objective of a light microscope. Interpretation of results: Look for species of the malarial parasite various stages of the parasite namely mature and immature Trophozoites, Schizonts, and Gametocytes • Immature trophozoites: seen in all infected cells and appear as undivided nucleated cells with blue colored cytoplasm or as a ring of cytoplasm within the red cells • Mature trophozoites: Trophozoites with out ring structure but with compact cytoplasm and amoeboid shape • Schizonts: appear as individual nucleated cells arranged in a circle forming a rosette or distributed through out the cell • Gametocytes Appear as round or oval or elongated filling the entire cell. • Infected red cell: may appear normal, enlarged, oval shaped with jagged edges or with pink dots called Schuffner’s dots • Pigment: Some parasites have pigment granules yellow/ brown or black in their cytoplasm. PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure POTENTIAL SOURCES OF VARIABILITY • Dirty slides do not give an even smear. Smears that are too thick or too thin will not stain well. Ensure the stained smears • Expired stains and old stains may not yield good results • An appropriate size of blood drop should be used. • Jerky movement of the spreader slide and the loss of contact between the spreader and the smear slide will yield poor smears. BIBLIOGRAPHY 1. District Laboratory Manual in Tropical Countries.-Monica Cheesebrough. Pg 246,249,250. 2. Practical Hematology. Dacie and Lewis 10th edition, Pg 71-73.


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EXAMINATION OF MICROFILARIA PURPOSE To prepare a blood film for the examination of the microfilaria. This test helps in the diagnosis of filarial fever by finding the microfilaria or adult worm in the blood. PRINCIPLE The thick blood smear is prepared on a microscopic slide which is dried, fixed and stained with Giemsa staining. PRIMARY SAMPLE • Collect 1 mL of blood in EDTA tube. • Do not use clotted sample for testing. CONSUMABLES/REAGENTS • Leishman stain: It is prepared by dissolving 0.15 g of powdered stain in 100 mL of (acetone free) methanol. • Leishman Buffer. • Sodium citrate. • 2% Formalin. • Immersion oil. • Glass slides. • Cotton. • Slide rack. INSTRUMENT • Light microscope. PROCEDURE Preparation of Fresh Smear • Arrange 2–3 clean grease free slides on the work table. • Before dislodging the needle from the syringe in which the blood is collected from the patient, make one or two wet preparations by placing a drop of blood on each of the slide arranged. • Then place a cover glass over the drop of blood, avoiding air bubbles. • View microscopically under 10X objective with reduced condenser aperture for live MF.

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CONCENTRATION METHOD FOR MF Principle • The formalin solution will cause the lysis of the RBCs and the fixation of the MF • Irrespective of a direct preparation being negative or positive, concentration method is used. • Collect about 5 mL of blood into 0.5 mL of sodium citrate anticoagulant in 15 mL centrifuge tubes. • Fill with 2% formalin solution. • Mix the blood well with the formalin solution and leave it to stand for about 5 minutes. Centrifuge the tube at high speed for about 5 minutes. • Remove the supernatant fluid with a Pasteur pipette and discard. • Place a drop of the deposit on a slide and add the stain. • Examine the preparation for MF with a 10X objective. Staining • Staining of thin smear with Leishman’s stain: • Place the slide with the smear side up on a staining rack horizontally (without sloping). • Label it with a pencil and place it on a staining rack, making sure that the smeared surface faces upward for staining. • Add Leishman stain to cover the entire slide. Then dilute it with working buffer. • Fifteen minutes later drain off the stain and wash the slide under running tap water. • Place it on a rack to dry. Examination of stained smears: The air-dried stained smear is viewed 1st under dry 10X and later under 100X oil immersion objective of a light microscope Interpretation of results: Positive: Actively motile organism are seen Negative: No active motile organisms are seen. PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure


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POTENTIAL SOURCES OF VARIABILITY • Dirty slides do not give an even smear. • Smears that are too thick or too thin will not stain well. Ensure the stained smears are translucent • Expired stains and old stains may not yield good results • An appropriate size of blood drop should be used. • Jerky movement of the spreader slide and the loss of contact between the spreader and the smear slide will yield poor smears. BIBLIOGRAPHY 1. Manual of Basic Techniques for Health Laboratories (WHO), Pg. 207. 2. Practical Hematology. Dacie and Lewis. 10th edition. Pg. 69-71.

LE CELL PREPARATION PURPOSE To demonstrate LE cell by Light Microscopy using Leishman’s stain. LE cell preparation is used to diagnose the Systemic Lupus Erythematosus (SLE), a connective tissue disorder that occurs most frequently in women of child bearing age. PRINCIPLE The leukocytes are traumatized by rotating the anti-coagulated blood with glass beads (since LE factor does not attack healthy living leukocytes). The patient’s serum containing LE factor is then directed towards the nuclear material of leukocytes. The nuclear material after reacting with the LE factor is transformed in to an LE body which attracts the neutrophils and is ingested by one of the leukocytes forming an LE cell. The LE cell takes the Leishman’s stain and is examined under high power objective of the microscope when a smear of the buffy coat is made. PRIMARY SAMPLE • Collect about 10 mL of blood by venepuncture and place in a 25 mL conical flask, which contains a few glass beads. • Mix the blood by shaking it hard until a fibrin clot has formed. This takes about 10 minutes. • Process all samples on the same day as collection. CONSUMABLES/REAGENTS • Wintrobes tube.


230 • • • • • •

Hypodermic needle Slide Glass tubes Leishman’s stain Leishman’s buffer Centrifuge tube

EQUIPMENT • Light microscope • Centrifuge • Incubator PROCEDURE • Keep the conical flask at room temperature or 37°C for 2 hours mixing it at intervals. • Transfer the blood into centrifuge tube and centrifuge at 3,000 rpm for 10 minutes to obtain the buffy coat layer. The buffy coat layer lies between the plasma and the red cells and if the LE cells are formed they will be found in the lower part of this layer. • A more concentrated layer of cells can be obtained by transferring the buffy coat layer, together with a few red cells and a small quantity of the serum, charging into a Wintrobe‘s tube and centrifuging again. • Discard the supernatent plasma by a hypodermic needle attached to a 2 mL syringe, make 4 smears from the buffy coat and stain with Leishman‘s stain (follow staining instructions as for Differential count). • Examine the smears with 40 × and 100 × objective. • A smear reported as positive must contain several definite and characteristic LE cells. INTERPRETATION OF RESULTS The presence of an altered nuclear material in the Neutrophilshomogeneous smudgy mass signifies the presence of LE cells. PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure. POTENTIAL SOURCES OF VARIABILITY • The “tart” cells retain their nuclear structure and should not be confused with LE cells.


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• Severe leukopenia and adrenocorticosteroid therapy may give false negative results. REFERENCE RANGE The finding of at least 4 typical LE cells in 15 to 20 Minutes of viewing (100X oil immersion) is considered as Positive LE cell test. BIBLIOGRAPHY 1. A Laboratory manual for rural tropical hospitals - Monica Cheesbrough and John McArthur, Pg. 48. 2. Practical Hematology. Dacie and Lewis. 10th edition. Pg. 602.

SICKLE CELL PREPARATION PURPOSE To examine the presence of sickling phenomenon in blood sample. This is used as a screening test for sickle cell anemia. PRINCIPLE When whole blood is mixed with a strong reducing agent such as sodium metabisulfite, deoxygenation of RBC does occur. If the red cells contain sickle hemoglobin (HbS), they become sickle shaped. PERFORMANCE SPECIFICATION Not applicable PRIMARY SAMPLE • Use whole blood collected in K2 EDTA tube as specimen. CONSUMABLES/REAGENTS • 2% Sodium Metabisulfite • DPX Mountant • Glass slide EQUIPMENT/INSTRUMENT • Microscope PROCEDURE • Arrange a clean grease-free microscope slide on the work table. • Place one drop of whole anticoagulated blood form on the slide.

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• Add one to two drops of 2% sodium metabisulfite. • Mix well with an applicator stick. • Cover the mixture with a cover glass and press it down lightly to remove any air bubbles. • Using an applicator stick gently add DPX Mountant on the edges of the cover glass to seal the preparation. • After 30 minutes examine the preparation for the presence of sickle cells - “holly-leaf” form of RBCs under 40 X. • Allow the preparation to stand at room temperature for 24 hours and re-examine for sickling phenomena. INTERPRETATION OF RESULTS • Positive—Presence of sickling • Negative—Absence of sickling PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure. POTENTIAL SOURCES OF VARIABILITY Not applicable BIBLIOGRAPHY 1. Daland GA, Castle WB: A simple and rapid method for demonstrating sickling of the red blood cells: The use of reducing agents, J Lab Clin Med 33, 1082, 1948. 2. Practical Hematology. Dacie and Lewis. 10th edition Pg. 96,97.


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Part II: Clinical Pathology STOOL EXAMINATION PURPOSE Examination of stool specimen for • Gross and physical examination of color, consistency, mucus, blood by visual observation • Chemical examination to determine the pH and presence of occult blood and reducing substance. • Direct microscopic examination to detect the parasites, erythrocytes and pus cells. PERFORMANCE SPECIFICATIONS • The thickness of stool smear should be such that newsprint can be read through it. • The stool should be fairly fluid and diluted sufficiently so that the various structures gets separated and can be examined under the microscope. PRIMARY SAMPLE • Use stool as specimen for the test • Instruct patients to collect stool samples in a clean, dry plastic container (preferably collected from the laboratory). • Instruct patients that urine should not be passed simultaneously into the collection container. • Keep the faeces covered after collection to prevent from drying up • Examine the feces within 3 hour after collection. CONSUMABLES/REAGENTS • • • • • • • • •

Glass slides Litmus paper Mixing sticks Test tubes Normal saline Lugols iodine Iodine 1 gm Potassium iodide – 2 gm Distilled water – 100 mL.

EQUIPMENT/INSTRUMENT Light microscope


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• Macroscopic examination : Observe the specimen for the following: • Color: Normal: Light to dark brown or yellow Abnormal color

Interpretation of result

Black

Bleeding in the upper gastrointestinal tract

Bright red

Bleeding at the lower level of gastrointestinal tract. Bleeding piles, contamination with menstrual blood

Fresh blood, mucus

Amebic dysentery

Clay colored

Post-hepatic jaundice, obstruction to the flow of bile to intestine

White

After barium meal

• Consistency : Normal: Well formed Abnormal consistency

Interpretation of result

Pale, bulky, frothy

Steatorrhea (poor fat digestion)

Hard

Constipation

Flattened and ribbon like

Obstruction in the lumen of the bowel

Semisolid

Mild diarrhea, after taking laxative digestive upsets

Watery

Bacterial infection, Purgative

Rice water stools (copious, thin with white flakes)

Cholera

• Mucus/Blood: Presence of mucus and blood. CHEMICAL EXAMINATION • • • • •

pH determination: Stool specimens are mostly acidic The reaction of stool can be tested by using simple litmus paper Blue litmus turns red—acidic. Red litmus turns blue—alkaline.

MICROSCOPIC EXAMINATION Procedure Direct Smear Examination Saline Preparation 1. Use clean, dry glass slide and label it with a glass marking pencil on one side


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2. Take the material from the surface of the faeces and from the central portion. 3. Make a thin emulsion of stool in few drops of normal saline. 4. Place a cover slip over it and look under low and high power microscope. 5. Report the presence of Pus cells, RBCs, free-living amebae, flagellates and ciliates, ova of parasites and cysts, undigested starch cells, fat globules and yeast cells. Iodine Preparation: Routine for detection of cysts 1. Use clean, dry glass slide and label with patient ID number with a glass marking pencil on one side 2. Take the material from the surface of the feces and from the central portion. 3. Make a thin emulsion of stool in few drops of normal saline. 4. To a thin saline emulsion of fecal add one drop of Lugol’s iodine which will stain the nuclear structure of the cysts. 5. Place a cover slip over it and look under low and high power microscope 6. Report the presence of cysts. • Concentration Method: Routine for detection for ova PRINCIPLE Also called as enrichment technique, with this technique it is possible to examine a larger quantity of stools in lesser volume and to detect parasites even if present in very small numbers. PROCEDURE 1. In a test tube [12 × 100 mm] add approximately 10 mL of NS. 2. Using an applicator or wire loop take a small portion of stool and mix well with the NS in the test tube. 3. Centrifuge the test tube at 3,000 rpm for 2 minutes. 4. Decant the supernatant fluid and place the sediments on a clear slide. 5. Place a cover slip avoiding air bubbles and view under the microscope using 40 X. PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure


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Potential Sources of Variability • False positive results may be obtained if the container is contaminated with detergents or bleach. • Formation of air bubbles while placing the cover slip. • Opaque slide preparation due to excess of stool. • Iodine should never replace saline for routine use, as it kills living material which makes it impossible to detect motility of flagellates, ciliates, larvae and vegetative amebae. BIBLIOGRAPHY 1. Manual of Basic Techniques for a Health Laboratory 1980. Pg. 116-7,175-6. 2. Textbook of Medical Laboratory Technology: 2nd Edn. Praful B Godkar. Pg. 918-37.

DETECTION OF OCCULT BLOOD IN STOOL PURPOSE Qualitative detection of free hemoglobin from lysed RBC and free myoglobin in urine by chemical examination (Benzidine test). Presence of free hemoglobin indicates neoplastic diseases of the GI tract, bleeding ulcers of stomach duodenum or small intestine, amoebiasis and shigellosis. PRINCIPLE The peroxidase activity of the heme portion of the hemoglobin molecule present in urine results in the liberation of active nascent oxygen from hydrogen peroxide. The liberated oxygen oxidizes benzidine in acidic medium to form a green–blue colored complex. PRIMARY SAMPLE • Use stool as specimen for the test • Instruct patients to collect stool samples in a clean, dry plastic container (preferably collected from the laboratory). • Instruct patients that urine should not be passed simultaneously into the collection container. • Instruct patients to refrain from a meat free and vitamin C free diet for at least 2 days prior to the test. • Keep the faeces covered after collection to prevent from drying up • Examine the feces within 3 hour after collection.


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REAGENTS/CONSUMABLES • • • •

Benzidine powder Glacial acetic acid Hydrogen peroxide Glass tubes.

EQUIPMENT Not applicable PROCEDURE • In a test tube 12 × 100 size make a suspension by adding about ¼ teaspoon of bezindine powder with a spatula in 0.5 mL glacial acetic acid. • Take a portion of stool sample in a tube 18×150 mm test tube and add 7 mL of distilled water and mix well. • To this add the benzidine and acetic acid suspension. • Then add about 2 to 3 drops of hydrogen peroxide solution. • Let stand for 1 minute. A blue-green color within 30 seconds means that occult blood is present and the test is reported as occult blood positive. • No blue-green color means that occult blood is not present and the test is reported as occult blood negative. INTERPRETATION OF RESULTS • No blue or green color indicates absence of Blood/free Hb/free Mb • Blue or green color indicates presence of Blood/free Hb/free Mb PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure POTENTIAL SOURCES OF VARIABILITY • False positive results may be obtained if the container is contaminated with detergents or bleach. • Interfering substances of dietary origin (e.g iron, meat, fat etc.) have peroxidase activity and may give false positive results. BIBLIOGRAPHY 1. Manual of Basic Techniques for a Health Laboratory 1980, Pg. 116-7,175-6. 2. Textbook of Medical Laboratory Technology: 2nd Edn. Praful B Godkar Pg. 936.


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URINE EXAMINATION—COMPLETE AUTOMATED METHOD PURPOSE For Qualitative identification and semi-quantitative estimation of color, pH, specific gravity, protein, glucose, ketone, bilirubin, blood, urobilinogen, nitrites, leukocytes and erythrocytes in urine by automated method using the principle of reflectance photometry. The nature and amount of substance present in urine reflect ongoing physiological process in health and disease status. PRINCIPLE This instrument has an optical system of different wave lengths and a light emitting diode for each of the test areas detects the color change. When the urine is allowed to react with test areas, a color change occurs in the test area which is detected and measured by the optical system and LED. The specific wave lengths for the evaluation of the reacted test areas are: pH This test is based on the double indicator principle that gives a broad range of colors covering the entire urinary pH range. The test strip contains a combination of methyl-red (pH range 4.4–6.2.) and bromothymol- blue (pH range 8.0–9.6) as indicators. The color gradations extend form orange via green to blue. Depending upon the pH of urine the color of the reaction area changes from orange/yellow/green/blue. SPECIFIC GRAVITY In the presence of an indicator the polyelectrolyte present in urine give colors ranging from deep blue green in urine of low ionic concentration through green to yellow green in urine of increasing ionic concentration. The test zone of the test strip is provided with an ion exchanger (Polyelectrolyte) and a pH indicator (Bromothymol Blue). The test is based on the pKa change of pretreated poly electrolytes in the reagent area in relation to ionic concentration of urine. The color of the test area ranges from deep blue-green to yellow–orange depending upon the ionic concentration /SG of urine. TOTAL PROTEIN It is based on the protein error of a pH indicator. At a constant pH any color change that happens to an indicator is due to protein. The test area of the reagent strip is impregnated with an indicator, tetrabromophenol blue,


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buffered to acidic pH 3.0. At this pH it is yellow in the absence of protein. In the presence of protein, it forms a complex with the dye turning the color of the dye from yellow (no protein ) to light green, green, bluish green, blue (presence of protein) depending upon the concentration of protein in the urine. Glucose: The test area is impregnated with glucose-oxidase/peroxidase together with potassium iodide and a blue background dye. The oxygen liberated in the final reaction binds with the dye to produce a series of color changes 30 seconds after wetting the strip with urine. Depending upon the concentration of glucose in urine the color of test area changes from blue (no glucose) to green, yellow, orange or red (presence of glucose) Ketone: The test area contains sodium nitroprusside and glycine and a buffer. Acetoacetic acid and acetone react with sodium nitroprusside and glycine in an alkaline medium to give a violet dye complex (Legal’s test).This test is more sensitive to acetoacetic acid than acetone. It does not react with α hydroxybutyric acid. L-Dopa may give a false positive result. Bilirubin: The test for bilirubin is based on the coupling of bilirubin with a stable diazonium salt (2-4 dichloroanilinediazonium)in the acid environment of the test area of the strip. The product is a red-violet azo dye that effects a color change from buff to tan/tannish purple/pink/violet. Urobilinogen: A stable diazonium salt in dimethyl aminobenzaldehyde reacts immediately with urobilinogen in the strong acid environment of the test to effect a change from orange to brown/red azo dye. The intensity of the brown/red color produced is a measure of the concentration of the urobilinogen present. Nitrite: The sulfanilamide contained in the test area reacts in the presence of an acidic buffer with nitrite to form a diazonium compound. Together with a coupling component, this diazonium compound produces a red azo dye. Leukocyte: The test is for granulocytic leukocytes. The test area for granulocytic leukocytes contains an indoxylcarboxylic ester and a buffer ester, which is hydrolyzed by granulocytic esterases. The indoxyl ester thus liberated then reacts with a diazonium salt to produce a blue-purple color. Blood (erythrocytes, hemoglobin): The test is based on the fact that peroxidase present in hemoglobin and myoglobin catalyze the oxidation of a color indicator 3, 5, 3’5’ Tetra methyl Benzenidine by an organic hydroperoxide to a blue-green dye that appears green on the original yellow test area of the strip.

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PERFORMANCE SPECIFICATIONS pH • The pH test area permits quantitative differentiation of pH values to one unit within the range of 5–9. pH reading is not affected by variation in the urinary buffer concentration. • If proper procedure is not followed and excess urine remains on the strip, a phenomenon known as “running over” may occur, in which the acid buffer from the protein reagent area run onto the pH area, causing a false lowering in the pH result. Specific Gravity • The specific gravity test permits determination of urine specific gravity between 1.000 and 1.030. In general, the specific gravity test correlates within 0.005 with values obtained with the reflective index method. • Highly buffered alkaline urine may cause low readings relative to other methods. • Elevated specific gravity readings may be obtained in the presence of moderate quantities (100–750 mg/dl) of protein. • The chemical nature of the specific gravity test may cause slightly different results from those obtained with the specific gravity methods when elevated amounts of certain urine constituents are present. Protein • The test area is sensitive to 15–30 mg albumin /dl urine • A color matching any color block greater than trace indicates significant proteinuria • The test area is more sensitive to albumin than to globulin, hemoglobin, Bence-Jones protein, and mucoprotein; therefore a negative result does not rule out the presence of these other proteins. • For urine with high specific gravity, the test area may most closely match the trace color block even though only normal concentrations of protein are present. Glucose • The measurement range for glucose is 75–125 mg of glucose/dl of urine and is specific for glucose. • Presence of ascorbic acid in urine (most likely to be present in large amounts in the urine of pregnant women and those taking multivitamin medications) will cause a false-positive result. • This test does not detect other reducing sugars, fructose, galactose, etc. and other non reducing substances in the urine as it is specific for glucose.


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Ketones • The minimum detection limit of ketones by this method is 5–10 mg/dl for Acetoacetic acid and 40–70 mg/dl for acetone and the maximum detection limit is 160 mg of acetoacetic acid/dl of urine. • False Positive results occur in patients receiving Levodopa or with urine containing MESNA or large amounts of phenylketones • This test does not detect the ketone body, beta hydroxyl butyric acid Bilirubin • The test has a sensitivity of 0.4–0.8 mg /dl of bilirubin in urine and is specific for bilirubin only • False positive results may obtained with the urine of patients receiving large doses of Chlorpromazine. • False negative results will be obtained when urine contains large amount of Ascorbic acid and Nitrite and when bilirubin is oxidized to Biliverdin. Urobilinogen • This minimum detection limit of urobilinogen is 0.2 EU/dl in urine. • The absence of urobilinogen cannot be determined with this test. • The test area will react with interfering substances known to react with Ehrlich’s reagent, such as porphobilinogen and p-aminosalicyclic acid. • This test is not a reliable method for the detection of porphobilinogen • Drugs containing azo-dyes (e.g. Azo Gantrisin) may give a masking golden color. Nitrites • This test is specific for nitrite and will not react with substances normally excreted in the urine • This test has sensitivity to sodium nitrite of 0.075 mg/dl. Comparison of the reacted reagent area on a white background may aid in the detection of low levels of nitrite ion, which may otherwise be missed. • The pink color is not quantitative in relation to the number of bacteria present. Any degree of pink coloration should be interpreted as a positive nitrite test suggestive of 105 or more organisms/mL. If the infection is caused by bacteria which do not contain reductase (to convert nitrate to nitrite) the test results will be negative although bacteria are present • Morning specimen of urine is preferred for Nitrite test as only an incubated sample of urine will contain high bacterial content

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Occult Blood • The test has a sensitivity to free hemoglobin of 0.015 mg/dl or 5–10 intact red cells/ul urine. The minimum detection limit for RBC is about 5 RBCs/ul and free Hb equivalent to 10 RBCs/ul urine. • This test is slightly more sensitive to free hemoglobin and myoglobin than to intact erythrocytes. • False positive results may be obtained in the presence of hypochlorite or when the urine has high bacterial count (as in urinary tract infections) as bacteria contain peroxidase • The sensitivity of the blood test is reduced in urine with high specific gravity and/or high ascorbic acid content. Leukocytes • This test can detect as low as 10–15 WBC/mL. and will not react with erythrocytes or bacteria common in urine. • Highly colored urine and the presence of the drugs cephalexin and gentamicin interfere with the test results. • High urinary protein of 500 mg/dl or above diminishes the intensity of the reaction color. • Elevated glucose concentration or high specific gravity may cause decreased results. PRIMARY SAMPLE • Instruct patients to collect a minimum 15 mL of a random sample of urine in a clean dry container (preferably collected from the laboratory). • Instruct patients to void directly into the container and while collecting allow the first portion of urine to escape. • Use fresh well mixed uncentrifuged urine as specimen for the test. • Process the samples within 1 hour of collection. If delay is anticipated instruct patients to store the sample at 2-8 °C in the refrigerator for a maximum of up to 1 hour. • Do not accept if insufficient sample is given and samples without an ID number. Additive/Preservative: No additive or preservative need to be added to the specimen. If delay in transport is anticipated instruct patients to store the sample at 2–8 °C in the refrigerator for a maximum of up to 1 hour. Instrument: Clinitek 500 Urine analyzer, Bayer. Consumables: Multistix 10SG


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TEST PROCEDURE • Ensure the urine sample is at room temperature and mix the urine sample thoroughly before testing. • Take a test strip from the pack and close it again immediately. • Briefly, no longer than 1 sec, dip the test strip into the urine making sure that all the test areas are moistened. If the strip is dipped for a longer time reagents from the test areas of the strip will dissolve in the urine. • When removing the strip, wipe the edge of the strip against the rim of the specimen container to remove excess urine or dab the side edge of the strip on a clean absorbent surface. • Place the test strip with the pads upwards, onto the tray so that its leading edge is held by the clip at the insertion slot. The retaining bar must be open about 2 mm of strip must be held under the clip. • Press the start button. An acknowledging beep will sound. The tray will advance slightly, the retaining bar will close and the grey reference pad on the tray will be read. • Ensure that the retaining bar is locked into place and that the test strip is in the correct position. If the test strip is not correctly located in the middle of the tray, move it gently to the side until it properly aligned, be careful not to move the tray. • Press the ‘Start’ button. • Exactly 55 seconds after you pressed the start button, the first test pad will be measured, followed by the others. After that the tray will return to the start position and the retaining bat will open. • Remove and dispose of the test strip. Wipe any urine residues from the tray with a lint-free cloth. • The result will be printed out and the next sample number will be displayed. The next test strip can be dipped, wiped off, placed in the tray and read by pressing the start button. PRECAUTIONS AND POTENTIAL SOURCES OF VARIABILITY • Carry out the examination in a fresh sample with in 2 hours of collection, as delay will cause leukocytes to be destroyed, casts to decompose and urea will be broken-down due to bacterial action and make the urine alkaline affect test results. • Perform manual methods only for cross-reference for confirmation or whenever there is a failure of the automated system. • Observe the following precautions while using urine dip sticks: – Do not touch the edges of the strip where the reagent patch is present. – Tap excess urine from the strip by stroking along the rim of the container after dipping the strip into the sample.


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Do not expose the strip to the atmospheric air when not in use and keep the bottle closed. Protect the strips from moisture and excessive heat but do not refrigerate. Replace the top on the storage container immediately after removing a strip. Darkening of the enzyme-coated area of the strip indicates loss of sensitivity. Hence do not use discolored strips. Contamination of glassware with Sodium Hypochlorite and Bleaching powder and detergents like sodium phosphate will oxidize and change the color of chromogen in Dipstick. Hence ensure the glassware is free of these chemicals.

REFERENCE RANGE • pH Newborn: 5–7 thereafter: 4.5–8 average: 6. SG of random urine ranges from 1.003–1.030. • Specific gravity • Nitrite : Normally no detectable amount of nitrite is present in urine. • Protein : Normally no detectable amount of protein is present in urine. • Ketone : Normally, no ketones are present in urine. • Glucose : Normally no detectable amount of glucose present in urine. • Bilirubin : Normally, no bilirubin is detectable in urine. • Urobilinogen : Present in normal amounts. • Leukocytes : Normal urine yields negative results. • Pathological leucocyte concentration > 20 leukocytes/mL • Erythrocytes : < 3 erythrocytes/mL • Blood: Normally no blood is present in urine. Blood may be present in the urine of menstruating females. INTERPRETATION OF RESULTS • pH—Compare the color that develops on the reagent strip with that given on the reagent bottle (after 30 seconds) by the manufacturer of the urine strip. • >7—alkaline • <7—acidic • Specific gravity–Compare the color that develops on the reagent strip with that given on the reagent bottle (after 45 seconds) by the manufacturer of the urine strip. • Glucose—Negative: no change in color. • Trace: light green and slightly cloudy. • 1+: green (0.5%) • 2+: yellow (1.0%)


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• 3+: orange (1.5%) • 4+: Brick red (2.0%) • Protein—Negative, Trace , 1+ , 2+, 3+ , 4+ • Ketone bodies Compare the color developed on the test area with the corresponding color chart provided on the bottle by the manufacturer of the strip at exactly the time specified (after 40 seconds) and grade the result accordingly. Depending on the intensity of the color, grade the result as trace (+), (++), (+++) and (++++). Normal urine (in healthy individuals) does not contain any of the ketone bodies. Repeat all positive test results by Ketodiastix strip method. • Urobilinogen, Leukocyte, Nitrite, occult blood—Compare reagent areas to corresponding color chart on the bottle label at the specified time. Hold strip close to color blocks and match carefully. SAFETY PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure. MANUFACTURER Bayer Diagnostics.

QUALITATIVE IDENTIFICATION OF REDUCING SUGAR IN URINE BY BENEDICT’S TEST METHOD Qualitative identification of reducing sugar in urine by - Benedict’s test. This test is useful in the diagnosis of diabetes mellitus, renal glycosuria and glycosuria due to other causes and in monitoring the response to treatment with Insulin or oral hypoglycemic agents. PRINCIPLE Reducing sugars containing free aldehyde group/free keto group form 1,2 enediols in weak alkaline medium that reduce cupric ions to cuprous ions and form a precipitate of cuprous oxide, the color of which varies depending upon the concentration of the reducing sugar in the sample of urine. PRIMARY SAMPLE • Use urine as specimen for the test • Collect a minimum 15 mL of a random sample of urine


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• Instruct patients to collect urine samples in a clean container (preferably collected from the laboratory). • Instruct patients to void urine directly into the container and while collecting, allow the first portion of urine to escape. • Use fresh and un centrifuged samples for the test. • Process the samples within 1 hour of collection. If delay is anticipated, instruct patients to store the sample at 2–8 °C in the refrigerator for a maximum of up to 4 hours till transport to the laboratory. • Do not accept if the sample volume is inadequate/or samples without an ID number. Consumables/Reagents: • Benedict’s qualitative reagent • Bunsen Burner • Test tubes • Pasteur pipette EQUIPMENT Not applicable. PROCEDURE Take 5 mL of Benedict’s qualitative solution in a test tube and boil it. To it add 8 drops (0.5 mL) of urine sample and then boil directly over the flame of a burner for 2–3 minutes and allow the test tube to cool in air. Observe the color of the precipitate formed. INTERPRETATION OF RESULTS • • • • • •

Negative: no change in color. Trace: pale green and slightly cloudy. 1+: definite cloudy green 2+: yellow to orange 3+: orange to red precipitate 4+: Brick red precipitate

PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure Potential Sources of Variability • This test is not specific for glucose as all reducing sugars (fructose, lactose, and galactose) answer this test.


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• Non-carbohydrate reducing substances like Ascorbic acid, Asprin, Uric acid, Homogentisic acid and glucuronic acid also answers this test. • The sugar present in the urine of normal healthy individuals cannot be detected by this method. BIBLIOGRAPHY 1. Practical Clinical Biochemistry-Harold Varley, 4th Edition 1969;Pg 369. 2. Textbook of Medical Laboratory Technology, 2nd Edn. Praful B Godkar Pg. 889.

QUALITATIVE IDENTIFICATION OF PROTEIN IN URINE BY HEAT AND ACETIC ACID METHOD PURPOSE Qualitative identification of protein in urine by precipitation by heat and acetic acid method. This test helps in the diagnosis of persistent proteinuria indicates renal disease, either renal failure due to glomerular or tubular damage and urinary tract infection. PRINCIPLE Sulpho-salicyclic acid is an anion precipitant that neutralizes the cationic protein and causes precipitation of the protein irrespective of the type of protein, whether Albumin or Bence Jones protein. PRIMARY SAMPLE • Use urine as specimen for the test. • Collect a minimum 15 mL of a random sample of urine. • Instruct patients to collect urine samples in a clean dry container (preferably collected from the laboratory). • Instruct patients to void urine directly into the container and while collecting allow the first portion of urine to escape. • Process the samples within 1 hour of collection. If delay is anticipated instruct patients to store the sample at 2-8 °C in the refrigerator for a maximum of up to 4 hours till transport to the laboratory. • Do not accept if the sample volume is inadequate/or samples without an ID number. CONSUMABLES/REAGENTS • 3% Acetic acid. • Bunsen burner


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• Test tubes • Pasteur pipette/urine dropper. EQUIPMENT Not applicable. PROCEDURE Boiling Test: Manual Method • Pour about 4 to 5 mL, of clear urine into a 12 × 100 mm test tube. • If the urine is alkaline add a few drops of 3% acetic acid before carrying out the test. • The protein present is coagulated and can be seen by comparing with the unboiled urine lower down the tube. INTERPRETATION OF RESULTS • • • • • •

Negative: No cloudiness. Trace: Cloudiness just visible against dark background (10 mg/dl) 1+ :- dense cloudiness (10-30 mg/dl) 2+:- cloudiness with granules and definite flocculations (50–200 mg/dl) 3+ :- cloudiness with heavy flocculation (200–500 mg/dl) 4+:- cloudiness with flocculation and precipitation ( more than 0.5 gm/dl).

SAFETY PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure. POTENTIAL SOURCES OF VARIABILITY • This test can detect 5–10 mg/dl of protein in urine specimen and cannot differentiate between albumin and Bence Jones protein. A positive test for protein has to be correlated with the microscopic examination of urinary sediment for pus cells, bacteria and casts. • Normal urine (in healthy individuals) contains only a small amount (less than 150 mg/24 hours) of low molecular (< 68000) weight protein which cannot be detected by this method. BIBLIOGRAPHY 1. Practical Clinical Biochemistry-Harold Varley: 4th Edition. 1969;369. 2. Textbook of Medical Laboratory Technology: 2nd Edn. Praful B Godkar. Pg. 887.


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QUALITATIVE IDENTIFICATION OF KETONE BODIES IN URINE PURPOSE Qualitative identification of ketone bodies in urine by manual method. Presence of ketone bodies indicates serious metabolic imbalance due to uncontrolled diabetes mellitus (diabetic ketoacidosis) and carbohydrate deficiency (starvation, prolonged vomiting). PRINCIPLE Modified Rothera’s Nitroprusside test. This test is based on the principle that ketone bodies, Acetone and Acetoacetic acid form a purple colored complex with sodium nitroprusside in an alkaline medium provided by liquor ammonia. PERFORMANCE SPECIFICATIONS • The ketone bodies include Acetoacetic acid, acetone and α–hydroxy butyric acid. But this test detects only acetone and acetoacetic acid but not α - hydroxyl butyric acid and also the test is more sensitive to acetoacetic acid than acetone. PRIMARY SAMPLE • Use urine as specimen for the test. • Collect a minimum 15 mL of a random sample of urine. • Instruct patients to collect urine samples in a clean container (preferably collected from the laboratory). • Instruct patients to void urine directly into the container and while collecting allow the first portion of urine to escape. • Use fresh and un centrifuged samples for the test. • Process the samples within 1 hour of collection. If delay is anticipated instruct patients to store the sample at 2- 8°C in the refrigerator for a maximum of upto 4 hours till transport to the laboratory. • Do not accept if the sample volume is inadequate/or samples without an ID number. REAGENTS AND CONSUMABLES • Take a few crystals of sodium nitroprusside in a test tube (18 × 150 mm) Add 5 mL of distilled water. Shake well until the crystals are almost dissolved, not all of the crystals are expected to dissolve as the solution is saturated. Use freshly prepared sodium nitroprusside solution for testing ketone bodies in the urine. This solution along with concentrated acetic acid and ammonia makes Rothera’s mixture. • Glass test tubes

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EQUIPMENT Not applicable PROCEDURE • Take 10 mL of clear urine in a test tube. • Add 10 drops of acetic acid to the urine. • Add 10 drops of freshly prepared sodium nitroprusside solution and mix well. • Then holding the tip of a Pasteur pipette against the side of the test tube let 20 drops (1 mL) of ammonia solution to flow onto the surface of the liquid. • Wait for 5 minutes. • Observe for pink-purple colored ring at the junction of the 2 layers. INTERPRETATION OF RESULTS • No pink-purple colored ring – Absence of ketone bodies • Appearance of pink-purple colored ring – Presence of ketone bodies • Depending on the thickness of the ring, it can be graded as trace (+), (++), (+++) and (++++). • Normal urine (in healthy individuals) does not contain any of the ketone bodies. PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure. POTENTIAL SOURCES OF VARIABILITY • If a patient is taking L-Dopa, a false positive result may be obtained. Since normally all the 3 ketone bodies are excreted in urine in DKA, a positive test is generally sufficient for the diagnosis of ketonuria. BIBLIOGRAPHY 1. A Laboratory Manual for Rural Tropical Hospitals - Monica Cheesbrough, John McArthur: Pg. 145. 2. Textbook of Medical Laboratory Technology. 2nd edition. Praful B Godkar. Pg 891-2.


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QUALITATIVE DETECTION OF BILE PIGMENTS BY FOUCHET’S METHOD PURPOSE Qualitative detection of bile pigments (bilirubin and biliverdin) by Fouchet’s test presence of bile pigments in urine is diagnostic of hepatic jaundice or obstructive jaundice. Presence of bilirubin in urine is detected before the clinical symptoms of jaundice (change of skin color) are recognized. PRINCIPLE Barium chloride reacts with sulfate radicals present in urine to form a precipitate of barium sulfate. If bile pigments are present in the urine sample, they adhere to the precipitate and are detected by the oxidation of bilirubin (yellow-colored) to biliverdin (green-colored) on treatment with Ferric Chloride (Fouchet’s reagent) in the presence of trichloroacetic acid. PRIMARY SAMPLE • Use urine as specimen for the test. • Collect a minimum 15 mL of a random sample of urine. • Instruct patients to collect urine samples in a clean container (preferably collected from the laboratory). • Instruct patients to void urine directly into the container and while collecting allow the first portion of urine to escape. • Use fresh and uncentrifuged samples for the test. • Process the samples within 1 hour of collection. If delay is anticipated instruct patients to store the sample in the refrigerator at 2 – 8 °C for a maximum of up to 4 hours till transported to the laboratory. • Do not accept if the sample volume is inadequate/or samples without an ID number. CONSUMABLES/REAGENTS • • • • • •

Fouchet’s reagent prepared by Trichloroacetic acid 25 g 10 % Ferric chloride 10 mL Distilled water 50 mL (Make up to 100 mL in a volumetric flask) 10 % Barium chloride solution Test tubes.

EQUIPMENT Not applicable.


252 PROCEDURE • • • •

Take 2.5 mL of urine into 12 × 100 mm test tube. Add 2.5 mL of barium chloride and mix well. Centrifuge the mixture in a centrifuge at 1500 rpm for 5 minutes. Discard the supernatent and to the deposit add 2 to 4 drops of Fouchet’s reagent.

INTERPRETATION OF RESULTS No color change - bile pigments: Nil [negative] Blue - green color - bile pigments present [positive] PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure. POTENTIAL SOURCES OF VARIABILITY • If the urine is not tested fresh, bilirubin present in urine is oxidized to biliverdin which will not answer the test. Hence, the urine has to be tested fresh for bilirubin. • Also when urine containing bilirubin is exposed to light it is oxidized to biliverdin and will be negative for when tested for bilirubin. • Drugs like asprin and pigmented metabolites of certain drugs may give an atypical color reaction and mask the normal positive color due to the presence of bilirubin. BIBLIOGRAPHY 1. District Laboratory Practice in Tropical Countries Monica Cheesbrough Part-1. Pg. 378,379.

QUALITATIVE DETECTION OF BILE SALTS BY HAY’S TEST PURPOSE Qualitative detection of bile salts by Hay’s test. This test is useful in the differential diagnosis of jaundice. Presence of Bile salts in urine indicates obstruction to the biliary passages which may be either intrahepatic or extra hepatic (due to gallstones any where in the bile passages, carcinoma of head of pancreas and stricture of common bile duct).


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PRINCIPLE Bile salts, when present in urine, lower the surface tension of urine. When sulfur powder is added to the surface of urine, the sulfur particles sink to the bottom of the test tube. In normal urine the sulfur powder floats on the surface. PRIMARY SAMPLE • Use urine as specimen for the test. • Collect a minimum 15 mL of a random sample of urine. • Instruct patients to collect urine samples in a clean container (preferably collected from the laboratory). • Instruct patients to void urine directly into the container and while collecting allow the first portion of urine to escape. • Use fresh and uncentrifuged samples for the test. • Process the samples within 1 hour of collection. If delay is anticipated instruct patients to store the sample in the refrigerator at 2 – 8 °C for a maximum of up to 4 hours till transported to the laboratory. • Do not accept if the sample volume is inadequate/or samples without an ID number. CONSUMABLES/REAGENTS • Sulfur powder • Glass test tubes EQUIPMENT Not applicable PROCEDURE • Take 10 mL of clear urine in a test tube. • Sprinkle a little dry sulfur powder on to the surface of the urine. • Observe the sulfur particles. INTERPRETATION OF RESULTS • Sulfur powder sinks to the bottom of the test tube – indicates the presence of bile salts • Sulfur powder do not sink to the bottom of the test tube but floats on the surface of urine–indicates the absence of bile salts. PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin.


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• Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure. POTENTIAL SOURCES OF VARIABILITY • Even a slight air current will cause the sulfur powder to sink. Hence ensure that the fan is switched off while performing the test BIBLIOGRAPHY 1. Pratical Clinical Biochemistry–Harold Varley, 4th edition. Pg. 369.

QUALITATIVE IDENTIFICATION OF UROBILINOGEN IN URINE BY EHRLICH ALDEHYDE TEST PURPOSE Qualitative identification of urobilinogen in urine by Ehrlich aldehyde test. Normal urine indicates the presence of trace amounts of urobilinogen. Increased urobilinogen discharged through the urine is diagnostic of hemolytic jaundice. Absence of urobilinogen indicates hepatic jaundice, obstructive jaundice, or disturbance in the intestinal micro flora following antibiotic treatment. PRINCIPLE Urobilinogen reacts with Ehrlich aldehyde reagent (p–dimethyl amino benzaldehyde) in acid medium to form a pink colored compound. PRIMARY SAMPLE • Use urine as specimen for the test. • Collect a minimum 15 mL of a random sample of urine. • Instruct patients to collect urine samples in a clean container (preferably collected from the laboratory). • Instruct patients to void urine directly into the container and while collecting allow the first portion of urine to escape. • Use fresh and uncentrifuged samples for the test. • Process the samples within 1 hour of collection. If delay is anticipated, instruct patients to store the sample at 2-8 °C in the refrigerator for a maximum of up to 4 hours till transport to the laboratory.


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• Do not accept if the sample volume is inadequate/or samples without an ID number. CONSUMABLES/REAGENTS • Ehrlich aldehyde reagent: Conc. HCl – 20 mL; Dist.Water - 80 mL. Mix well and add p- Dimethyl Amino Benzaldehyde- 2.0 g and stir well • Glass test tubes EQUIPMENT Not applicable PROCEDURE • Pipette 2.5 mL of urine into a test tube. • Add 5 drops of Ehrlich‘s reagent and allow to stand for 5 minutes. • Note the color change. INTERPRETATION OF RESULTS • Development of pale pink color – Presence of urobilinogen in normal amounts. • Development of dark pink/cherry red color – Presence of urobilinogen in increased amounts PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure. POTENTIAL SOURCES OF VARIABILITY • Use a fresh sample of urine when testing for urobilinogen, since Urobilinogen is unstable and gets immediately oxidized to urobilin, which does not answer this test. • Samples should be tested within 2 hours of collection. • Porphobilinogen in urine also answers this test. Hence this test is considered as a screening test only. BIBLIOGRAPHY 1. District Laboratory Pratice in Tropical Countries Monica Cheebrough Part 1 Pg. 379, 380.


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QUALITATIVE DETECTION OF FREE HEMOGLOBIN BY BENZIDINE TEST PURPOSE Qualitative detection of free hemoglobin from lysed RBC and free myoglobin in urine by chemical examination (Benzidine test). Presence of free hemoglobin indicates hemoglobinuria, myoglobinuria, renal diseases like acute infections, toxic damage to kidneys, renal calculi, renal infarction, renal tuberculosis, and trauma to kidneys, Bleeding disorders and excessive anti coagulant therapy. PRINCIPLE The peroxidase activity of the heme portion of the hemoglobin molecule present in urine results in the liberation of active nascent oxygen from hydrogen peroxide. The liberated oxygen oxidizes benzidine in acidic medium to form a green–blue colored complex. PERFORMANCE SPECIFICATIONS • Do not use alkaline urine as it can cause lysis of RBCs and release of Hb from RBCs. PRIMARY SAMPLE • Use urine as specimen for the test. • Collect a minimum 15 mL of a random sample of urine. • Instruct patients to collect urine samples in a clean container (preferably collected from the laboratory). • Instruct patients to void urine directly in to the container and while collecting allow the first portion of urine to escape. • Use fresh and uncentrifuged samples for the test. • Process the samples within 1 hour of collection. If delay is anticipated instruct patients to store the sample at 2–8°C in the refrigerator for a maximum of up to 4 hours till transport to the laboratory. • Do not accept if the sample volume is inadequate/or samples without an ID number Reagents/Consumables: • Benzidine powder • Glacial acetic acid • 3 % w/v Hydrogen peroxide freshly prepared and stored in amber colored bottles • Glass test tubes


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EQUIPMENT Not applicable PROCEDURE • • • • •

Place a pinch of Benzidine powder in a test tube. Add 2 -3 drops of glacial acetic acid and mix well Add 2 -3 mL of Hydrogen peroxide solution and mix well Add 0.5 mL of previously boiled and cooled urine and mix well Observe the color of the mixture after 5 minutes

INTERPRETATION OF RESULTS • No blue or green color indicates absence of blood/free Hb/free Mb • Blue or green color indicates presence of blood/free Hb/free Mb. PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure. POTENTIAL SOURCES OF VARIABILITY • False positive results may be obtained if the container is contaminated with detergents or bleach. • Positive test result always has to be correlated with the presence of more than 2 RBCs/HPF on microscopic examination of centrifuged urine sediment. • Although normal centrifuged urine sediment does not contain RBCs, the finding of 1–2 RBC per HPF should not be considered as abnormal. BIBLIOGRAPHY 1. Manual of Basic Technique for a Health Laboratory 1980;116-7,175-6.

DETECTION OF BENCE JONES PROTEIN (BJP) PURPOSE Qualitative detection of the presence of Bence Jones Protein (BJP) by Heat Test. This test is used in the diagnosis of multiple myeloma,Waldenström’s macroglobulinemia, some lymphomas and leukemias, osteogenic sarcoma,


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cryoglobulinemia, malignant B-cell disease, amyloidosis, light chain disease, and cancer that has spread to bone. PRINCIPLE Bence Jones protein coagulates when heated to 40°C - 60°C and redissolves partially or wholly on boiling. PERFORMANCE SPECIFICATION • Centrifuge the urine specimen, filter the supernatant if it is turbid, and check the pH with litmus paper. Add a few drops of dilute acetic acid, if necessary to make the specimen slightly acidic. • Start heating from the top to observe for cloudiness. PRIMARY SAMPLE • Use urine as specimen for the test. • Collect a minimum 15 mL of a random sample of urine. • Instruct patients to collect urine samples in a clean container (preferably collected from the laboratory). • Instruct patients to void urine directly into the container and while collecting allow the first portion of urine to escape. • Use fresh and uncentrifuged samples for the test. • Process the samples within 1 hour of collection. If delay is anticipated instruct patients to store the sample at 2–8°C in the refrigerator for a maximum of up to 4 hours till transport to the laboratory. • Do not accept if the sample volume is inadequate/or samples without an ID number. REAGENTS/CONSUMABLES • • • •

Test tubes Beaker Bunsen burner Thermometer.

EQUIPMENT Not applicable PROCEDURE • Take 5 mL of the urine specimen in a test tube. • Place a thermometer inside the test tube and immerse the tube in a beaker of cold water.


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• • • •

Heat the beaker slowly and raise the temperature of the water. Carefully observe the temperature and any signs of precipitation. Heat the water to boiling. Filter the urine while hot. True albumin, which coagulates above 60°C, will be removed. • Allow the water to cool. INTERPRETATION OF RESULTS If Bence Jones protein is present, cloudiness will appear between 40°C and 60°C and will dissolve at about 100°C. On cooling the urine specimen, in presence of BJP will cloud up again between 40°C and 60°C. PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure. POTENTIAL SOURCES OF VARIABILITY • If the water is not stirred, a uniform temperature will not be maintained. BIBLIOGRAPHY 1. Practical Clinical Biochemistry-Harold Varley, 4th Edition. 1969;139-40. 2. Textbook of Medical Laboratory Technology: 2nd Edn. Praful B Godkar. Pg. 818-900.

ANALYSIS OF CSF PURPOSE Quantitative estimation of total WBC and different types of white blood cells in human CSF by manual method. This test is useful in the diagnosis of diseases of Central Nervous System, due to inflammation of meninges, (meningitis due to various causes, bacteria, viruses and fungi) and to diagnose obstruction to CSF circulation. PRINCIPLE • Total WBC count: CSF diluting fluid removes the RBCs and stains only the WBCs. The CSF specimen is diluted 1:20 with a pipette with the diluting fluid and the cells are counted under low power of the microscope using a Neubauer counting chamber.

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• Differential count: The blood smear is prepared on a microscopic slide, which is dried, fixed and stained. The fixative does not allow any further change in the cells and make them adhere to the glass slide. The polychromic staining solutions containing acidic and basic dyes induce multiple colors when applied to cells. Basic components of the cells are stained by acidic dyes, acidic components by basic dyes and the neutral components by both the dyes. Stained cellular components are then viewed under the microscope. PRIMARY SAMPLE • If only cell count is requested, instruct patients to collect at least 2–3 mL of CSF in a clean dry sterile test tube preferably obtained from the laboratory. • If biochemical analysis and culture and sensitivity tests are requested, instruct to collect at least 5 mL of CSF in two separate sterile test tubes • Process the sample within 30 minutes of collection. • Never refrigerate the CSF specimen especially when culture and sensitivity tests are to be performed. • For total WBC count and differential count and culture, use uncentrifuged CSF specimen. CONSUMABLES/REAGENTS • Turk’s fluid. Use CSF diluting fluid only if the CSF is cloudy indicating possibility of increased leukocytes • Leishman stain • Leishman buffer EQUIPMENT/INSTRUMENT • Neubauer Counting chamber • Light microscope PROCEDURE • Appearance: Note whether the uncentrifuged CSF specimen is clear, turbid, cloudy, and bloody or xanthochromic. Observe the uncentrifuged specimen of CSF for clot formation by leaving the CSF in another test tube for 10 minutes and again after 24 hours to observe for clot formation or appearance of Cob–Web/or leave the supernatant of the centrifuged CSF specimen and observe for clot formation for 24 hours (after processing the sediment for cell count). • Interpretation of results: – Normal CSF is crystal clear and colorless. – Purulent/Turbid/cloudy – TB or Bacterial pyogenic meningitis

Hematology and Clinical Pathology –

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Pink/red/ Uniformly bloody – Subarachnoid bleed/intracerebral bleed. – Xanthochromic/yellowish – Old hemorrhage, severe jaundice spinal cord obstruction or obstruction to the flow of CSF. – Normal CSF does not clot or coagulate. – Clot formation is always abnormal and indicates increased protein concentration (mainly due to fibrinogen which is converted to fibrin to form a clot) and is seen in tuberculous or pyogenic meningitis and spinal cord obstruction. - Delicate and fine clot – TB meningitis - Large clot – Pyogenic meningitis - Complete and spontaneous clot – Spinal cord obstruction • Microscopic examination (Cell count-Using Neubauer Chamber) – Total WBC count: - Use uncentrifuged specimen for total WBC count - If the CSF is clear, use it undiluted: If the CSF is turbid /cloudy make 1:20 dilution by using a pipette: Draw CSF up to 0.05 mL and dilute with 0.95 mL CSF diluting fluid. - Mix well and allow to stand for 5 minutes. - Mix the CSF specimen well. - Load the Neubauer chamber with the sample and leave the chamber undisturbed for 5 minutes to allow the cells to settle on the floor of the ruled area of the chamber. - Place the chamber under the stage of the microscope. - Count the WBCs under low power objective (x 10 ) in 4 squares area counted is 4 sq.mm. CALCULATION Total WBC/cu mm =

No. of cells counted × dilution × depth Area counted

dilution 1 /1 , depth 0.1 mm, area 4 sqmm Undiluted CSF = Diluted CSF (1: 20) =

No. of cells counted × 10 4

No. of cells counted × 10 × 20 4

DIFFERENTIAL COUNT • If the CSF contains few cells : <5 cells/mm3 – Place the CSF in the cup of cytocentrifuge chamber.


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• • • • • • •

Centrifuge the CSF at 200-1000 rpm for 10 min, the fluid passes through the tube in the chamber towards a glass slide and is deposited in a concenterated button about 6 mm in diameter. Fix with methanol and stain with Leishman stain and Leishman buffer. Count the cells under 100x oil immersion objective. If the CSF contains many cells: >5 cells/mm3 place 1 drop of mixed, uncentrifuged CSF specimen on a clean glass slide. Make a thin smear and leave to dry. Fix with methanol and stain with Leishman stain and Leishman buffer. Count the cells under 100 x oil immersion objective.

INTERPRETATION OF RESULTS • Normal CSF contains small mature lymphocytes 0-5 lymphocytes/cu mm • Normal CSF does not contain neutrophils. PRECAUTIONS • Ensure the counting chamber is clean and dry. • Take care to ensure air bubbles do not enter while drawing blood or the diluting fluid into the pipette. • Count after allowing the cells to settle for 2–3 minutes and include all cells lying within the square and also those cells lying on the lines or touching the lines. • Use clean slides to get an even smear. SAFETY PRECAUTIONS • Handle all reagents with care and avoid contact with eye, mouth and skin. • Handle all samples as potentially infectious. • Discard used reagents and sample as per disposal procedure. POTENTIAL SOURCES OF VARIABILITY • The CSF specimen should be fresh as the blood cells and offending organisms sought for diagnosis rapidly lyse on standing while the bacteria multiply with delay in analysis resulting in changes in the chemical composition. • The CSF specimen used for culture should not be refrigerated as the pathogens are killed on exposure to cold. • Jerky movement of the spreader slide and the loss of contact between the spreader and the smear slide will yield poor smears. • Dirt, dust particles and yeast cells in CSF diluting fluid may cause errors in counting as it may lead to falsely elevated WBC counts. Hence, take extra care during preparation and storage of CSF diluting fluid.


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BIBLIOGRAPHY 1. Manual of Basic Techniques for a Health Laboratory (WHO),1980:343. 2. Anne Stein Martin—Clinical Hematology, Principles, Procedures, Correlations 2nd edition Pg. 407.

MANTOUX TEST PURPOSE To screen the in vivo sensitization by Mycobacterium tuberculosis either due to active infection or past inoculation and also to check the prophylaxis and efficacy of BCG vaccination. PRINCIPLE When the purified protein derivative of tuberculin is injected intradermally it produces induration and erythema around the point of injection. This is due to the sensitization of the skin by Mycobacterium tuberculosis. It is a qualitative test to detect infection with Mycobacterium tuberculosis by injecting intradermally PPD /Tuberculin using tuberculin syringe. PRIMARY SAMPLE Not applicable CONSUMABLES/REAGENTS • Tuberculin syringe • Tuberculin or PPD (5 TU/0.1 mL) PROCEDURE • The preferred site for injection is the flexor surface of the forearm. • Disinfect the site of injection with cetavlon and allow to dry. • Disinfect the tuberculin PPD vial stopper with cetavlon; allow drying and then drawing 0.1 mL of PPD by using tuberculin syringe. • Using 26 g needle to inject the PPD at the site which is already disinfected. • Inject the PPD intradermally to make the deposition wheel, in the diameter of 6 to 8 mm which will rise up to the point of needle. • Mark the area of injection with indicator. • Read the result after 48 hrs. INTERPRETATION OF RESULTS • Positive test—Induration of 10 mm or more • Negative test—No induration or any induration less than 10 mm


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The test is interpreted as positive, if the diameter of the induration in the transverse direction is more than 10 mm; the test is interpreted as negative if the diameter of the induration in the transverse direction is less than 10 mm. PRECAUTIONS • Do not inject subcutaneously. • Instruct patients not to rub the area, not to apply soap, oil and water till the results are read. • Use only tuberculin syringe and needles. • If a Mantoux test has recently been done in the left arm (within the past 3 months), use the right arm. POTENTIAL SOURCES OF VARIABILITY • Wrong results may be obtained if the tuberculin is injected subcutaneously instead of intradermally. • Acute viral infections like measles, mumps, and polio may hinder the activity of tuberculin. • The activity of the tuberculin is decreased if the tuberculin is not stored at 2–8ºC. • Positive skin test reaction if they: – have had tuberculosis before and have been cured. – have been exposed to the tuberculosis bacteria or have been immunized for tuberculosis (BCG). – have tuberculosis. – negative result may actually be incorrect (false negative) if your child: - is taking medicine to lower your immunity, e.g. steroids or chemotherapy drugs. - has recently been vaccinated with live viruses.

MICROSCOPIC EXAMINATION OF URINE PURPOSE For qualitative identification for microscopic examination of urine: • Centrifuge about 5 to 10 mL of urine at 1500 rpm in a 12 × 100 mm test tube to obtain a deposit. • Discard the supernatant fluid and transfer the deposit onto a prearranged clean grease free microscopic slide.


a. b. c. d. e. f. g. h. i. j. k. l. m. n. o.

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Fig. 3.6: Urine deposit examination Granular casts Hyaline casts Waxy casts WBC casts RBC casts RBCs Leukocytes Squamous epithelial cell Transitional epithelial cell Triple phosphate (coffin lid shaped) Calcium oxalate crystals (envelope shaped) Tyrosine needles Cystine crystals Uric acid crystals Amorphous phosphate

• Place a cover slip avoiding air bubbles. • Examine microscopically using 10x and 40x objectives, with a reduced condenser aperture. The following may be found: 1. White Blood Corpuscles Normal urine - few cells Male - 1–2 cells/hpf Female - 4–5 cells/hpf Abnormal Urine Male - 2–4 cells/hpf Female - 8–10 cells/hpf If found Urine culture is suggested.


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2. Red Blood Corpuscles Normal urine

Abnormal

-

-

in Male Nil; In female may be found if the urine is contaminated with the menstrual blood. if found in male and in non-menstrual period in female.

3. Epithelial Cells Normal urine - Few cells [5–10 cells]/hpf Abnormal - When in large nos. [20–25 cells]/hpf 4. Casts Normal urine : Nil Abnormal urine : Casts present The different types of casts are (i) Granular casts (ii) Hyaline casts (iii) Cellular casts. 5. Crystals: The most common crystals are calcium oxalate and triple phosphate. Other than this Uric acid crystal, Cysteine crystals. 6. Parasite: Trichomonas vaginalis 7. Fungus: Candida albicans BIBLIOGRAPHY 1. A Laboratory manual for Rural Tropical Hospitals - Monica Cheesbrough, John McArthur: Pg: 142-49. 2. Practical clinical Biochemistry-Harold Varley 4th Edition. 1969; Pg. 369. 3. meded.ucsd.edu/isp/1994/im-quiz/urine.htm 4. www.aafp.org/afp/20050315/1153.html 5. Textbook of Medical Laboratory Technology: 2nd Edn. Praful B Godkar. Pg. 900.

UNIT 4

Microbiology and Serology

HN Madhavan K Lily Therese J Malathi B Mahalakshmi

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Part I: Staining Techniques GRAM STAIN PURPOSE To demonstrate the presence of Gram positive and Gram-negative bacteria in clinical specimens based on their morphology and Grams staining characteristics. This test is useful to establish the presence of potential pathogenic bacteria in clinical specimens based on their morphology and to demonstrate the purity of cultures, helps to identify the presence of gonococci, pneumococci. Grams stain is essential in evaluation of all suspected cases of bacterial meningitis. Grams stained smears are also scanned for the presence or absence of pus cells and squamous epithelial cells. PRINCIPLE Gram’s staining procedure requires application of four reagents in a sequential manner namely, a basic dye crystal violet (primary stain), an aqueous solution of iodine, a decolorizing solvent, acetone and ethyl alcohol and counter stain, safranin or dilute carbol fuchsin. The crystal violet dye and iodine combine to form a dark purple compound in the bacterial protoplasm and cell wall. This compound is dissociable in the decolorizer, ethyl alcohol, which dissolves and removes the primary stain from the cell wall, the removal being much slower from Gram positive than from Gramnegative bacteria because the gram positive organisms have thicker and dense peptidoglycan layers in their cell walls, which makes them more permeable to the stain, than the Gram-negative bacteria. The iodine acts as a mordant and has a critical role in enhancing this difference. Gram - positive bacteria retain the crystal violet dye after decolorization with alcohol and appear violet or purplish blue. Gram-negative bacteria are not capable of retaining the crystal violet dye after decolorization and take up the red color of the counter stain dilute carbol fuchsin. PERFORMANCE SPECIFICATION Gram staining helps to identify whether the bacteria are Gram positive or Gram negative but does not help to identify the exact genus and species. Background contamination of the specimen by normal flora may interfere with the result. Certain organisms cannot be stained/differentiated with Grams stain (Acid fast organisms and Legionella pneumophilia). Gram’s stain is not reliable for diagnosis of cervical, rectal, pharyngeal or asymptomatic urethral gonococcal infection.


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PRIMARY SAMPLE Any clinical specimen, tissue biopsy, body fluids received for bacterial etiology and investigation. CONSUMABLES Clean, dry, grease free microslides/slide marking pencil Sterile normal saline (0.85% or 0.9%), Methanol (for fixing) Diamond marker/glass marker Tissue papers Gram’s stain reagent Crystal violet stock Crystal violet (90% dye content) – 2.5 grams Distilled water – 500 mL Working solution : Crystal violet (0.5%) 1 in 10 working solution is used Gram’s iodine stock Iodine crystal – 5 gm Potassium iodide – 20 gm Distilled water – 500 mL Working solution : Gram’s Iodine (1%) 1 in 10 working solution is used Decolorizer Acetone – 50 mL Methyl alcohol – 50 mL Dilute Carbol fuchsin Carbol fuchsin—if 0.5 or 1% – 10 mL Distilled water – 90 mL Cedar wood oil/Immersion oil EQUIPMENT Bunsen Burner Light Microscope - 100 × magnification STEP BY STEP PROCEDURE Mark the area of the smear as a circle on the underside of the slide. Label the slide with the specific lab no. or name of the organism. Fix the smear by gentle heat by passing the slide 3 or 4 times through the flame of bunsen burner so that the smear does not get washed off during the staining procedure or fix the smear in Methanol (in a coplin jar) for 3 minutes.

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GRAM’S STAINING PROCEDURE • • • • • • • • •

Cover the smear with crystal violet and leave it for 1 minute. Wash the slide with distilled water. Cover the slide with Gram’s iodine and leave it for 1 minute. Wash the slide with distilled water (for few seconds). Decolorize quickly with decolorizer until no further violet color comes out Wash with distilled water immediately. Counter stain with dilute carbol fuchsin for 1 minute. Wash with distilled water and blot dry. Observe the stained smear under the oil immersion (100 X) objective.

INTERPRETATION OF RESULTS • Gram positive bacteria appear violet or purplish blue and Gram-negative bacteria appear red or pink. • Indicate the morphology and arrangement specifically, especially for Gram-positive cocci. • Mention the coccobacillary forms with regard to gram-negative bacilli if seen. • Yeast cells appear dark purple in color. • Pus cells appear pink in color. • Epithelial cells appear pale pink in color. QUALITY CONTROL PROCEDURE Check the reliability of stains with known bacteria/ATCC strains of bacteria whenever new batches of stains are used. Staphylococcus aureus purple in color — for Gram positive, Escherichia coli pink in color — for Gram negative bacilli. SAFETY PRECAUTIONS • Observe all precautions as for handling any biological fluid. • Wear facemask and disposable gloves while transferring and handling the specimen. POTENTIAL SOURCES OF VARIABILITY • Process specimens before they dry up • Occasionally Gram positive organisms lose their ability to retain the crystal violet stain and appear Gram negative due to over decolorization or old Gram’s iodine or old cultures or due to over heat fixation. BIBLIOGRAPHY 1. Collee, Dugid, Fraser, Marmion. Laboratory strategy in the diagnosis of infective syndromes. 13th Edition Mackie and McCartney. Practical Medical Microbiology 1989;600-50.


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KOH-CALCOFLUOR WHITE STAIN PURPOSE To detect the presence of fungal elements and cysts of Acanthamoeba in the clinical specimen. PRINCIPLE The alkali potassium hydroxide (10% KOH) helps in digestion of tissue debris and acts as a clearing agent and disinfectant. With the clearing of the debris, the fungal elements become prominent. PERFORMANCE SPECIFICATION Calcofluor stains the cell wall of fungi (filamentous fungi and yeasts) also the cysts of Acanthamoeba. The yield may be reduced by prolonged storage of specimen. Therefore a fresh specimen is preferred always for observation. PRIMARY SAMPLE Clinical specimens—Corneal scrapings, Vitreous aspirate, Aqueous aspirate, Pus material, Eviscerated material, Corneal button, Skin scrapings, Sputum, Nail clippings, Hair or any biopsy specimen. Store the specimen at 2–8 °C if staining is not done immediately. TYPE OF CONTAINERS OR ADDITIVES Collect in a sterile container. REAGENTS AND CONSUMABLES Clinical specimen Micro slides Cover slip 4 mm Nichrome wire-loop Sterile Pasteur pipette. Solution A (10% Potassium Hydroxide) 1 gm of potassium hydroxide dissolved in 10 mL of distilled water and is stored in a plastic dropper bottle. Solution B Calcofluor White - 0.01 gm (0.1%) Evans Blue - 0.005 gms (0.05%) Distilled water - 10 mL Dissolve by mixing well, filter and place in a dropper bottle.


272 EQUIPMENT

Biological safety Cabinet (Class II) Fluorescence microscope with violet filter. Cytospin instrument PROCEDURE Label the slide with the specific lab no. and mark the area of the smear. The smear is prepared by any of the following methods: cytospin, bacteriological loop, by drop method using syringe and needle or Pasteur pipette, depending on type of clinical specimen. The slide is then covered with a drop of 10 % KOH (solution A) and one drop of Calcofluor stain (solution B) and a cover slip is placed. Slide is left as such for 5 minutes for digestion of tissues at room temperature. Observe under fluorescence microscope using the violet filter at 410 nm. Initially at 10 X, then gradually the magnification is increased to 20X, 40X, dry objectives. INTERPRETATION OF RESULT The fungi (filamentous and yeast) take-up the stain and fluoresce bluish white under the fluorescence microscope. Acanthamoeba are seen as double walled, hexagonal or polygonal shaped bluish white cysts. Pneumocystis carinii cysts, Microsporidia spores Cryptosporidium oocysts and Cryptococcus species also appear bluish white in color. QUALITY CONTROL PROCEDURE Check the reliability of stains with known Fungi/Acanthamoeba/ATCC strains of Yeast whenever new batches of stain are used. SAFETY PRECAUTION • Observe all precautions as for handling a biological fluid. • Process the specimen inside the biosafety cabinet only. POTENTIAL SOURCE OF VARIABILITY Process specimens before they dry up. BIBLIOGRAPHY 1. Forbes B, Sahm D F, Weissfeld AS. Laboratory Methods in Basic Mycology. In Bailey Scott’s Diagnostic Microbiology 1998;10:958.


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ZIEHL-NEELSEN STAIN PURPOSE This test is used for the differentiation of bacteria that are acid fast and based on their appearance on staining with acid-fast stain with 20% sulphuric acid (Ziehl-Neelsen stain) as decolorizer PRINCIPLE Bacteria such as mycobacteria are extremely difficult to stain by ordinary methods because of the high lipid contents of the cell wall. Initial staining is done with Carbol fuchsin combined with phenol and heat. The stain binds to the mycolic acid of the cell wall. After staining, an acid decolorizing solution is applied. This removes the red dye from the background cell tissues and other organisms except Mycobacteria and other acid fast group of organisms like Nocardia, which retain the dye. Following decolorization, the smear is counter stained with malachite green or methylene blue which stains the background material, providing a contrast color against which the red acid fast bacilli can be seen. Nocardia, Cryptosporidium, Microsporidium are also partially acid fast, resisting 0.5–1% staining. PERFORMANCE SPECIFICATION Ziehl-Neelsen stain cannot determine the exact species of Mycobacteria. It can only differentiate whether the bacteria is acid fast or not. Further tests must be performed to identify the species of Mycobacteria. PRIMARY SAMPLE USED Sputum: Instruct the patients to collect expectorated sputum or sputum obtained by deep cough, in a sterile wide-mouthed container after rinsing the mouth with water. Instruct patients to collect sputum obtained by deep cough on three consecutive days and to avoid saliva contamination. On the request of clinician, investigations can be carried out on sputum samples collected on a single day. Label the container with date, name of the patient. Urine: Instruct the patients to collect first morning sample of urine on three successive days in a clean, dry and sterile container provided by the laboratory. While collecting urine, instruct patients to clean the genitals and surrounding area with moist cotton/copious amounts of water to avoid contamination by normal flora. Instruct patients to collect mid stream sample of urine for acid fast bacilli (AFB) culture.


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REAGENTS AND CONSUMABLES Clean, dry glass slides Carbol fuchsin stain (10%) Decolorizer Acid - 20% H2SO4 , 5% or 1% or 0.5% H2SO4 (for modified Ziehl-Neelsen stain) Loeffler’s Methylene blue – Methylene blue - 0.3 g – Ethanol - 30 ml – Distilled water - 100 ml EQUIPMENT Light microscope Bio safety cabinet Type III.

CONCENTRATION METHOD FOR DETECTION OF MYCOBACTERIUM PURPOSE Many samples for mycobacterial culture are heavily contaminated with commensal flora. It is important to remove these, as they may grow more rapidly than the mycobacteria and over grow the culture. PRINCIPLE Destructions of contaminants is dependent on exposure of the sample to a chemical agent that will strictly kill them without inhibiting the growth of Mycobacteria. Most decontamination procedures use either acids such as sulfuric, hydrochloric or oxalic acid or alkalis such as Sodium hydroxide or tri-sodium phosphate. Sodium hydroxide (4%) is a useful decontaminating agent for sputum, biopsies, body fluids, etc. followed by washing with sterile Milli Q water. PERFORMANCE SPECIFICATION As all the decontaminants have some inhibitory effect on mycobacteria, a balance has to be struck between effective decontamination following washing and reliable isolation. PRIMARY SAMPLES Sputum, urine, biopsy, other body fluids which are likely to be contaminated with commensal flora.


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EQUIPMENT Laminar flow hood (Class III) Light microscope Shaker MATERIALS REQUIRED Test specimen Dehydrated media (Lowenstein Jensen (LJ) 4 % Sodium hydroxide-sterilize by autoclaving use-within a week. Sterile Milli Q water Other lab wares SAMPLE HANDLING AND INSTRUCTIONS Sputum: Instruct the patients to collect deeply coughed, expectorated, sputum in sterile wide-mouthed red top plastic container after rinsing the mouth with water. Urine: Instruct the patients to collect the first voided (early morning) samples in sterile container provided by the laboratory. While collecting, urine instruct to clean the genitals and surrounding area with moist cotton/ copious amount of water to avoid contamination by normal flora. Label the specimen indicating the identification number of the patient and nature of specimen. PROCEDURE Petroff’s method of sputum/urine concentration The processing of the specimen has to be carried out in the biological safety cabinet level III. With a sterile loop, inoculate the sputum/clinical specimen over the surface of the LJ medium. Close the slopes tightly, incubate at 37°C and label the slope appropriately. Make a smear from the specimen and stain by ZN for AFB. Centrifuge the entire amount of urine sample and inoculate the deposit on the LJ medium. Make smears from the deposit (for urine) before decontaminating it with sodium hydroxide and stain by Ziehl-Neelsen method. To each volume of remaining sputum/urine/other specimens, add two volumes of sterile 4% sodium hydroxide, taking care to avoid contact between the specimen bottle rim and the sodium hydroxide flask. Shake the centrifuge tubes by hand for one minute. Place in a rack on the shaking machine (wrist action shaker) and leave to shake for 20 minutes.

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Remove the specimen from the shaker. Centrifuge for 15 minutes at 3000 rpm. After removal from the centrifuge carefully pour off the supernatant into the disinfectant jar. Fill the centrifuge tubes to approximately 3/4th with sterile Milli Q water, shake by hand to mix the deposit, centrifuge at 4000 rpm for 15 minutes and pour of the supernatant as before. Repeat the washing with sterile Milli Q water two more times. Finally inoculate sediment with a loop onto two previously labeled Lowenstein-Jensen slopes. Place the inoculated L J slants in the 37°C incubator. Prepare a thin smear with the deposit. Allow it to dry inside the hood and stain by Ziehl-Neelsen method. Examine the slopes weekly for the appearance of growth. Continue incubating for 6-8 weeks before generating negative results. STEP BY STEP PROCEDURE FOR PREPARATION OF SMEAR Prepare a thin smear of the specimen before and after decontamination step, only for sputum samples. Dry in air, and fix the smear by gentle heating over a Bunsen burner flame for 2 minutes. Flood the heat fixed smear with carbol fuchsin slide and heat from below the slide till fumes arise and leave it for 5-7 minutes. Do not allow the stain to boil. Wash off the stain with distilled water. Cover the smear with 20 % sulfuric acid for 3 minutes. Wash well with distilled water. Decolorization is repeated until the smear appears pale pink. Cover the slide with methylene blue for 2 minutes. Wash with distilled water, dry and examine the slide under oil immersion microscope. PRECAUTIONS • Follow the usual precautions as for handling any biological fluid • Wear disposable gloves and face mask while doing the procedure • Temperature: 25 to 30°C. INTERPRETATION OF RESULTS If any definite bacilli (pink/red rods) are seen, report the smear as acid fast bacilli (AFB) positive and give an indication of the number of bacteria present as follows: More than 10 AFB/field: Report as 3 + (plenty) 1–10 AFB/ field: Report as 2+ (many) 10–100 AFB/100 fields: Report + (moderate) 1–9 AFB/100 fields: (few)


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BIBLIOGRAPHY 1. Laboratory strategy in the diagnosis of infective syndromes. Mackie & McCartney. Practical Medical Microbiology. Edited by Collee, Dugid, Fraser, Marmion. 1989;14. 600-50.

MODIFIED ZIEHL-NEELSEN STAIN PURPOSE To detect the presence of Nocardia species, Cryptosporidium and Microsporidium spores in clinical specimens. PRINCIPLE Organisms such as Mycobacteria are extremely difficult to stain by ordinary methods because of the lipid contents of the cell wall. Initial staining is done with Carbol Fuchsin combined with phenol. The stain binds to the mycolic acid of the cell wall. After staining, an acid decolorizing solution is applied. This removes the red dye from the background cells and other organisms except Mycobacterium species, Nocardia species, Cryptosporidium and Microsporidium which retains the dye. Following decolorization, the smear is counter stained with malachite green or methylene blue which stains the background material providing contrast color against which the red AFB can be seen. Staining can also differentiate Nocardia depending on percentage of H2SO4 (1%) and Mycobacterium leprae (5%) (for skin scrapings) and 0.5% for Microsporidial spores. PERFORMANCE SPECIFICATION This test cannot find out the exact species of Mycobacteria. It can only differentiate whether the bacteria are AFB positive or not. Further tests must be performed to confirm the species of Mycobacteria and Nocardia. PRIMARY SAMPLE Sputum: Instruct the patients to collect deeply coughed, expectorated, sputum in a sterile wide-mouthed red top plastic container after rinsing the mouth with water. Lacrimal pus, Canalicular pus, Wound swab, Corneal scraping, or pus FNAB or any other clinical specimen. TYPE OF CONTAINER OR ADDITIVES Collect in a sterile leak proof container.


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REAGENTS AND CONSUMABLES Clean, dry glass slides Carbol fuchsin stain Acid alcohol - 5% or 1% H2SO4 Methylene blue Decolorizer EQUIPMENT Light microscope Bisafety hood Type III PROCEDURE Preparation of Smear • Take a clean glass slide and label the slide with a glass marking pencil. • Sterilize the inoculating loop on the flame of a Bunsen burner. • Place a loopful of purulent portion of the sputum on the slide for direct and concentrated smears. • Make a crushed smear preparation with lacrimal/canalicular material. • Spread the suspension with an oval spiral movement of the loop from the center. • Re-sterilize the loop. Allow the smear to dry in the air. • Heat fix the smear by passing the slide 3 to 4 times through the flame very quickly with the smear side facing up. • Cool the slide before staining. Staining • Flood the heat fixed film with carbol fuchsin and heat till fumes arise and leave it for 5 -7 minutes. Do not allow the stain to boil. • Wash with distilled water • Decolorize with 5% sulfuric acid for Mycobacterium leprae and 1% for Nocardia and 0.5% H2SO4 for Microsporidial spores. • Wash with distilled water • Counter stain with Loeffler’s methylene blue for 3 minutes. • Wash with distilled water, drain and blot dry. RESULTS Nocardia species are seen as pink colored delicate branching filamentous bacilli and the microsporidial spores are seen as pink colored round to oval spores measuring about 2-3 μm in size. The Mycobacterium leprae appear as cigar-shaped thin bacilli in bundles.


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QUALITY CONTROL PROCEDURE Check the reliability of stains with known bacteria/ATCC strains of Mycobacterial strains bacteria whenever new batches of stain are used. SAFETY PRECAUTIONS • Follow the usual precautions as for handling any biological fluid • Wear disposable gloves and face mask while doing the procedure. BIBLIOGRAPHY 1. Laboratory strategy in the diagnosis of infective syndromes. Mackie & McCartney Practical Medical Microbiology. Edited by Collee, Dugid, Fraser, Marmion. 1989;13:600-50.

GIEMSA STAIN PURPOSE To demonstrate the presence of inflammatory cells—pus cells, lymphocytes, eosinophils and inclusion bodies of viruses/Chlamydia spp and presence of bacteria (coccal/bacillary forms) in clinical specimen based on their morphology and staining characteristics. Giemsa staining is used for the differentiation in the types of inflammatory cells. In microbiology, it is used for detecting Acanthamoeba cysts also. PRINCIPLE Examination of Giemsa stained clinical specimen allows an assessment of the types of inflammatory cells present. The Giemsa stain may also be useful to enhance microscopic details. PERFORMANCE SPECIFICATION Should be carried with freshly diluted stain. Staining with previously diluted stain may interfere with reading. PRIMARY SAMPLE Blood, corneal scrapings, corneal button, conjunctival swab, conjunctival scraping, any biopsy/clinical specimen and intraocular specimens. TYPE OF CONTAINERS OR ADDITIVES Collect in a sterile leak proof container.


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REAGENTS AND CONSUMABLES Giemsa stain Giemsa powder certified (Merck or Sigma) 3.8 gm Glycerol 98% 250 mL Absolute Methanol (Acetone free) 250 mL Weigh 3.8 gm of Giemsa powder and transfer to a mortar. Using dry cylinder measure 10 mL of glycerol and add this into the mortar and grind well with Geimsa powder and transfer into a dry brown bottle containing glass beads. Add 240 mL of glycerol and keep the bottle in 60°C water bath for 2 hours and allow to cool at room temperature. Then add 250 mL of absolute methanol slowly with stirring. Keep the bottle in 60°C water both for another 1 hour. Allow to cool and store at room temperature for 1 week. Decant and remove the sediment. Filter before use. Giemsa Buffer Preparation of phosphate buffer (pH-7.0). Stock A: 0.2M sodium dihydrogen phosphate 3.12 gm in 200 mL of distilled water. Stock B: 0.2M disodium hydrogen phosphate. 2.33 gm in 400 mL of distilled water. After preparation store the solution at room temperature in dark bottles. Working solution of buffer: To 14 mL of stock A add 36 mL of stock B and make up to 100 mL with distilled water. Preparation of working Geimsa stain: Mix 5 mL of freshly, filtered Giemsa stain with 37 mL of the working solution of the phosphate buffer and transfer to a coplin jar. Distilled water Glass slides Methanol EQUIPMENT Light microscope Biosafety cabinet (level II). PREPARATION OF SMEAR Smears are made by ophthalmologist for corneal scraping, conjunctival scrapings. For intraocular specimens. [Anterior Chamber (AC) tap and vitreous aspirate], cytospin smears are made in the laboratory. 1. Smears of pus material is made by spreading the material with an inoculation loop over the microscopic glass slide.


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2. Prepare cytospin smear for body fluids including intraocular specimens like AC tap and vitreous aspirate. PROCEDURE Fix the smear in methanol for 5 minutes. Stain the slides with diluted Giemsa stain in a coplin jar for 30 minutes at 37°C (incubator). Wash with distilled water, dry and observe under light microscope and report. SAFETY PRECAUTIONS • Follow the universal precautions as for handling any biological fluids • Wear disposable gloves while handling the clinical specimen. INTERPRETATION OF RESULTS The interpretation is based on the number of polymorphs, lymphocytes, inclusion bodies, eosinophils present in the specimen. An increase in the number of eosinophils indicates an allergic condition. BIBLIOGRAPHY 1. Laboratory strategy in the diagnosis of infective syndromes. Mackie & McCartney Practical Medical Microbiology, Edited by Collee, Dugid, Fraser, Marmion 1990;14:600-50.

NIGROSIN STAIN PURPOSE To demonstrate the presence of Cryptococcus neoformans in Cerebrospinal fluid (CSF) based on their morphology and staining characteristics. This test is useful to establish the presence of Cryptococcus neoformans in CSF in the diagnosis of Cryptococcal meningitis. PRINCIPLE Negative staining is an indirect staining process that uses negatively charged dyes to stain the background of the slide. Nigrosin is used because the negatively charged polysaccharides in the capsule of the yeast repel it. This repulsion forces the dye to make a halo around the yeast, therefore showing the contrast of the cell surface. Negative staining allows one to detect the presence of capsule. It also reduces the amount of shrinkage during the staining process. This is helpful in determining the morphology of the organism.


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PERFORMANCE SPECIFICATION • Do not heat fix the smear to avoid shrinking effect on capsules. • Negative staining provides only information about the micro topography of the specimen. PRIMARY SAMPLE CSF: Instruct to collect the specimen in a sterile well-capped tube. REAGENTS AND CONSUMABLES • • • • •

Clean, dry, grease free micro slides Sterile normal saline Diamond marker pencil Tissue paper Nigrosin Stain 10% .

EQUIPMENT • Light microscope - 100 × magnification. STEP BY STEP PROCEDURE • Place a drop of CSF (If Cryptococcus is suspected, CSF should not be centrifuged) on the slide. • Add a drop of nigrosin stain and spread it over the entire slide or make a smear as for blood smear. • Examine under the high dry and the oil immersion objective with reduced light. INTERPRETATION OF RESULTS Capsule of Cryptococcus neoformans appears as a budding yeast with a broad capsule against black background. The capsule is best visible in Nigrosin preparation. QUALITY CONTROL PROCEDURE Check the reliability of stain with known ATCC strain of bacteria that are capsulated (Streptococcus pneumoniae). SAFETY PRECAUTIONS • Observe all precautions as for handling any biological fluid. • Wear facemask and disposable gloves while transferring and handling the specimen.


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BIBLIOGRAPHY 1. Laboratory strategy in the diagnosis of infective syndromes. In Mackie & McCartney Practical Medical Microbiology. Edited by Collee, Dugid, Fraser, Marmion. 1990;14:600-50.

LACTOPHENOL COTTON BLUE STAIN PURPOSE To identify the fungus grown on culture from any clinical material. PRINCIPLE Fungus grown on ‘slide culture technique’ or culture media are stained by lactophenol cotton blue and the preparation is observed under the bright field microscope. Based on colony morphology on culture medium and microscopic morphology by ‘slide culture technique’ the fungus is identified. PERFORMANCE SPECIFICATION Lactophenol cotton blue stain should be performed with slide culture preparation showing sporulation to study the microscopic characteristics, useful in the identification of a particular fungus. REAGENTS AND CONSUMABLES • • • •

Filamentous fungal isolate Micro slides Cover slip “4 mm” Nichrome wire loop

Lactophenol cotton blue stain: Ingredients: • Phenol crystals 20 g • Lactic acid 20 mL • Glycerol 40 mL • Cotton blue 0.05 g • Distilled water 20 mL The ingredients are dissolved by heating in a water bath. Then 0.05 g of Cotton Blue is added. EQUIPMENT • Biological safety cabinet (Class II) • Optical light microscope


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• Slide culture is made by inoculating the fungus grown on culture medium onto 4 corners of a piece of SDA medium cut into 3x3 cm size and a clean cover slip is placed over the medium. • The inoculated bit is placed over a microscopic glass slide. The preparation is incubated at 25°C incubator after keeping the slide culture inside a moist chamber. • The preparation is observed everyday for the production of spores by the fungus. • Once spores have formed, a clean microscopic glass slide is taken to which a drop of lactophenol cotton blue is added. • The slide culture cover slip is then removed from the agar, placed onto the slide and observed under the microscope using 40X objective. INTERPRETATION OF RESULT • The fungi take up the stain and appear blue in color. Microscopic morphology is appreciated well. • Based on colony morphology and microscopic morphology the fungus grown from clinical specimen is identified and reported. SAFETY PRECAUTIONS • Observe all precautions as for handling a biological fluid. • Wear facemask and disposable gloves while transferring and handling the specimen. • Process the specimen under the biosafety hood only. BIBLIOGRAPHY Fungi. In Mackie & McCartney Practical Medical Microbiology. Year 1990, 14th edition Edited by Collee, Dugid, Fraser, Marmion. Pg. 600-50.

HANGING DROP METHOD FOR MOTILITY PURPOSE This test is useful to establish the presence of live motile bacteria (e.g. Vibrio cholerae) in stool specimens based on their motility. This test is useful to presumptively diagnose cholera, and also for characterization (whether motile or not) of any bacilli isolated from any clinical specimens. PRINCIPLE Live Vibrio cholerae and flagellated bacteria are actively motile in fresh stool specimen and the motility of these organisms is demonstrated by mounting a fresh stool sample microscopically.


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PERFORMANCE SPECIFICATION • Brownian movement, jumping and dancing movements exhibited by other bacteria (that have flagella) in the stool sample may mimic ‘true motility’ movement exhibited by Vibrio cholerae. • Confirmatory culture and biochemical tests have to be performed to confirm the presence of Vibrio cholerae and other motile bacteria in the stool. PRIMARY SAMPLE • Use only fresh stool as specimen for the test, as motility cannot be demonstrated in dried up specimen. • Use the liquid portion of the stool specimen for the test. • Use 4-hour pure broth culture of bacteria isolated from culture of any clinical material. CONSUMABLES • • • •

Cover slip Petroleum jelly Concavity slide Light microscope (10 X, 40 × magnification)

PROCEDURE • Place a coverslip on a small piece of paper on the table and place small amount petroleum jelly at the four corners of the cover slip. • Place aseptically loopful of the watery portion of the stool specimen/ broth culture at the middle of the coverslip. Spread it out a bit taking care not to bring it into contact with petroleum jelly. • Invert a cavity slide over the coverslip such that the concave portion of the slide is over the specimen and press gently to allow the jelly to spread and form a seal. • Invert the whole preparation so that the coverslip is on the top and the stool/four hour old broth culture is hanging freely inside. • Examine the suspension, focusing first on the edge of the drop with low power and then under high power. Adjust the light partially closing the iris diaphragm and lowering the condenser. • Observe for motility (It can be detected by the presence of directional motion)- Vibrio cholerae exhibit darting motility. • Look at one organism for about 1 minute and see if it moves out of its original position. Note: Jumping, walking or oscillating organisms in the same plane are not necessarily true motility, but these movements may be due to Brownian


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movement. True motility movement is from one place to another within the same microscopic field. INTERPRETATION OF RESULT • Positive: Motile bacteria seen. • Negative: No motile organisms seen. SAFETY PRECAUTIONS • Observe all precautions as for handling any biological fluid. • Wear facemask and disposable gloves while transferring and handling the specimen. POTENTIAL SOURCES OF VARIABILITY • Process specimens before they dry up. • Brownian movement, jumping and dancing movements exhibited by other bacteria (that have flagella) in the stool sample may mimic ‘true motility’- movement exhibited by Vibrio cholerae and other motile bacteria). BIBLIOGRAPHY 1. Laboratory strategy in the diagnosis of infective syndromes. Mackie & McCartney Practical Medical Microbiology. 14th edition Edited by Collee, Dugid, Fraser, Marmion. 1990;600-50.

IMMUNOFLUORESCENCE STAINING— CHLAMYDIA TRACHOMATIS PURPOSE To demonstrate the presence of Chlamydia trachomatis antigen in clinical specimen based on their morphology (elementary bodies/reticulate bodies) by carrying out direct immunofluorescence staining technique. PRINCIPLE Examination of Immunofluorescence stained conjunctival/corneal/ endocervical/urethral smears nasopharyngeal aspirate allows an assessment of the presence of Chlamydia trachomatis infected cells. PERFORMANCE SPECIFICATION Artifacts non-specifically take up fluorescence stain. A trained microbiologist is needed to interpret the results.


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PRIMARY SAMPLE Blood/Endocervical/urethral /Conjunctival/Corneal smear TYPE OF CONTAINERS OR ADDITIVES Collect sample in a sterile leak proof container with transport medium (2 molar sucrose phosphate buffer with 3% fetal calf serum). REAGENTS AND CONSUMABLES • • • • • • •

Fluorescein isothiocyanate (FITC) conjugated monoclonal antiserum Phosphate buffered saline-Tween 20 (PBST) 90% Glycerol saline Glass slides Microfuge tubes Blotting paper/tissue paper Glass marking pens/pencil/Diamond marker.

EQUIPMENT • Fluorescene microscope with blue filter • Biosafety hood Type II. STEP BY STEP PROCEDURE (COMMERCIAL SOURCE) Reconstituting the reagent • To reconstitute lyophilized reagent, (antiserum) remove the metal seal and rubber stopper from the reagent vial. • Add 2.0 mL of the provided reconstitution diluent and then discard the remaining diluents. • Record the reconstitution date on the reagent vial label. After reconstitution, mix and aliquot the antiserum. • Store the reagents at 2–8°C when not in use. Do not freeze or expose to temperatures above 32°C. • Do not expose to strong light. The reagent is aliquoted in 50 μL amounts in sterile, plastic 1.5 mL microfuge tubes eppendorf vials and stored at 2–8°C and the vials are taken out for use one by one as they get exhausted. PROCEDURE • The smear of the clinical specimen is fixed either in methanol (for 5 minutes) or in cold acetone for 3 minutes. • Add 15-20 μL of FITC monoclonal antibodies (Dako Denmark) reagent to the fixed smear, making sure the entire area of the smear is covered. Return the reagent to refrigeration immediately after this procedure.

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• • • • • • • •

• •

Manual of Medical Laboratory Techniques Note: If necessary, spread the reagent over the smear with the pipette tip, taking care not to disturb the fixed specimen. After spreading, always change pipette tips. Incubate the slides with smears covered by antiserum for 15 minutes at room temperature in a well-humidified chamber. Do not allow the antibodies to dry on the specimen. To prevent drying, periodically inspect the chamber for adequate humidification and remove from incubation promptly at 15 minutes. Rinse the slides by gently agitating them in Phosphate buffer saline Tween 20 (PBST) for 10 seconds. Gently shake off excess PBST and wipe the remaining moisture from the edge of each slide with blotting/tissue paper. Allow them to air dry. Add a drop of mounting fluid (90% glycerol saline) to the center of each slide well. Place a coverslip on top of the drop without introducing air bubbles but if bubbles form, remove the air bubbles by gently pressing the coverslip and remove all air bubbles. Examine the slides under fluorescence microscope using blue filter at 495 nm with barrier filter at 520 nm. For optimum clarity, use oil objectives 100X (for elementary bodies) and use a 20X and 40X for inclusions bodies. Note : If it is not possible to read the slides immediately after staining, store them in the dark at 2–8°C and read them within 24 hours for best results. Allow the slides to reach room temperature before reading or condensation will occur, obscuring the specimens.

SAFETY PRECAUTIONS • Follow the usual precaution as for handling biological fluids. • Wear disposable gloves while doing the procedure. • Temperature range for carrying out this test is 25 to 30°C. INTERPRETATION OF RESULTS Reported as positive if fluorescing elementary/reticulate bodies of C. trachomatis is detected. BIBLIOGRAPHY 1. Clinical Virology Manual. Editors Steva Spector, Richard L. Honinka, Stephen A. Young. 2000, 27-53. 2. Diagnostic Procedures for Viral, Rickettsial and Chlamydial infections. Year 1989, 6th Edition, by Schmidt, Pg. 51-101.


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IMMUNOFLUORESCENCE STAINING— FOR VIRUSES (HSV, CMV, VZV, ADENOVIRUS) PURPOSE To demonstrate the presence of viral antigen in clinical specimens by carrying out indirect immunofluorescence staining technique. PRINCIPLE Examination of immunofluorescence stained conjunctival/corneal/ intraocular fluids CSF/endocervical fluids in urethral smears allows an assessment of the presence of virus infected cells. PERFORMANCE SPECIFICATION Artifacts non-specifically take up fluorescence stain. A trained microbiologist is needed to interpret the results. PRIMARY SAMPLE Conjunctival/endocervical/urethral/corneal smear/blood /intraocular fluids/ skin scrapings. TYPE OF CONTAINERS OR ADDITIVES Collect sample in a sterile leak proof container. REAGENTS AND CONSUMABLES • Monoclonal antiserum stain raised against the specific viral agent (HSV, CMV, VZV, Adenovirus). • Fluorescein Isothiocyanate conjugated secondary antiserum. • Phosphate buffered saline-Tween 20 (PBST) • Glycerol saline • Glass slides • Eppendorf tubes. EQUIPMENT • Fluorescence microscope • Biosafety hood Type II. STEP BY STEP PROCEDURE Reconstituting the reagent: • To reconstitute lyophilized reagent, (antiserum) remove the metal seal and rubber stopper from the reagent vial.

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• Add 2.0 mL of the provided reconstitution diluent and then discard the remaining diluents. • Record the reconstitution date on the reagent vial label. After reconstitution, mix and aliquot the primary and secondary antiserum. • Store the reagent at 2–8°C when not in use. Do not freeze or expose to temperatures above 32°C. • Do not expose to strong light. • The reagent is aliquoted in 100 μL amounts in Eppendorf vials and stored at 2–8°C and the vials are taken out for use one by one as they get exhausted. PROCEDURE • Add 15–20 μL of reagent to each methanol/cold acetone fixed smear, making sure the entire area of the smear is covered. Return the reagent to refrigeration immediately after this procedure. Note : If necessary, spread the reagent over the smear with the pipette tip, taking care not to disturb the fixed specimen. After spreading, always change pipette tips. • Incubate the slides with smears covered by the appropriate antiserum for 45 minutes at room temperature in a well-humidified chamber. • Do not allow the antibodies to dry onto the specimen. • To prevent drying, periodically inspect the chamber for adequate humidification and remove from incubation promptly at 45 minutes. • Rinse the slides by gently agitating them in deionized or distilled water for 10 seconds. • Cover the smear with secondary antiserum and incubate for 45 minutes and wash with PBST thrice. • Gently shake off excess PBST and wick the remaining moisture from the edge of each slide with blotting/tissue paper. • Allow them to air dry. • Add a drop of 1.0% Evan’s blue and leave it for 1 minute and wash with PBST. • Place a drop of mounting fluid (90% glycerol saline) to the center of each slide well. • Place a coverslip on top of the drop and remove all air bubbles. • Examine the slides under fluorescence microscope using blue filter at 495 nm with barrier filter at 520 nm. Note : If it is not possible to read the slides immediately after staining, store them in the dark at 2–8°C and read them within 24 hours for best results. Allow the slides to reach room temperature before reading or condensation will occur, obscuring the visibility of the smear.


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SAFETY PRECAUTIONS • Follow the usual precaution as for handling the biological fluids. • Wear disposable gloves while performing the test. • Temperature range for carrying out this test is 25 to 30° C. INTERPRETATION OF RESULTS Reported as positive if apple green fluorescing cells are observed and as negative if cells appear red. BIBLIOGRAPHY 1. Clinical Virology Manual. Editors Steva Spector, Richard L Honinka, Stephen A .Young, 2000; 27-53. 2. Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections. Year 6th Edition, by Schmidt, 1989; 51-101.

Part II: Culture Methods BACTERIAL AND FUNGAL CULTURECONJUNCTIVAL SWAB SEMIQUANTITATIVE METHOD PURPOSE This test is used for the isolation and identification of bacterial/fungal pathogens present in the conjunctiva causing inflammation of conjunctiva, their nature and the drugs to which they are sensitive. By knowing the pattern of drug sensitivity, the patient can be treated. PRINCIPLE Pathogenic organisms present in the specimen will grow well by providing suitable culture media and can be identified based on the colony morphology, biochemical reactions and appropriate staining procedures. PERFORMANCE SPECIFICATION Culture reports have to be interpreted with caution based on the clinical findings and correlated with the clinical status of the patients. Culture may be negative for growth if the patient is already taking medications for the disease.

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PRIMARY SAMPLE • Collect eye swab (conjunctival swab) specimen by swabbing the lower inferior tarsal conjunctival fornix avoiding the eyelid border and lashes, with a sterile cotton swab moistened with Hanks’ Balanced Salt Solution (HBSS). • Two such swabs are collected, one for culture and the other for smear. • Extreme care must be taken to handle the swab while collecting the discharges from eye. • The specimen must be cultured as soon as possible since the natural secretions of the eye contain anti-bacterial enzymes (lyzozymes). • Swabs after collection is placed in 1 mL of Hank’s Balanced Salt Solution (HBSS) and with another swab smears are made on clean microscopic glass slides for staining. EQUIPMENT • Autoclave • Incubator 37ºC, 37ºC CO2 Incubator, anaerobic work station; incubator 25°C • Biosafety hood (Class II) • Microscopes • Cyclomixer CONSUMABLES • Dehydrated media (Blood agar - BA, Brucella blood agar - BBA, Chocolate agar - CA, Mueller Hinton agar – MHA, Blood Mueller Hinton agar – BMHA, Peptone water – PW) • HBSS (Hank’s balanced salt solution) • Nichrome loop • Micro slide • Sterile cotton swab STEP BY STEP PROCEDURE • Vortex the transport medium (HBSS) with swab using the cyclomixer and discard the swab after squeezing it against the wall of the container. • Inoculate 100 μL of specimen onto BA, CA, and BBA. Spread the inoculum and after drying a streak line of Staphylococcus aureus (for isolation of Haemophilus species) is made in the center of inoculum on BA. • Incubate the BA plates at 37oC aerobically, CA at CO2 incubator and BBA at anaerobic work station and look for growth. • Read methanol fixed direct smear after Gram’s staining and Giemsa staining under oil immersion objective. • Count number of colonies grown after overnight incubation in each plate.


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• Perform Gram’s staining with the colonies grown. • Inoculate the organism grown in culture plate, into peptone water. Incubate for 4 hrs. • Inoculate the broth culture onto MHA by lawn culture method. • For Streptococci and Haemophilus species, the antibiogram tests are carried out on Blood Mueller Hinton agar and Chocolate agar respectively • Place required antibiotic discs over it. Inoculate onto respective biochemicals depending on the organism for identification. • Incubate at 37oC for 24 hrs. • Measure the sensitivity zones and identify the organism. • Depending on the number of colonies and the type of bacterium, the bacterial growth is interpretated as significant or insignificant. • For anaerobic culture, incubate under anaerobic atmosphere for 12 days and give final report. If growth is observed, identify the bacteria by performing the required biochemical reactions. Propionibacterium acnes is the most common anaerobic bacterium isolated from conjunctival swab. • If growth of yeast is observed identify the yeast based on colony morphology and biochemical reactions, if there is no growth incubate for 12 days and give final report, as on anaerobic organism isolated in cultural. INTERPRETATION OF RESULTS • Innumerable colonies of Gram positive bacilli - (Diptheroides/Bacillus) Significant. – Significant. • One colony of Gram negative bacilli • > 50 colonies of Staphylococcus epidermidis – Significant. • > 100 colonies of Staphylococcus aureus – Significant. DATA RECORDING AND VERIFICATION • Enter results of the test in workbook. • Correlate the type of organisms with the nature of specimen. Compare the results of culture with Gram’s stain report of the smear. • Results from the BA and other media plates must be recorded as number of colonies which later can be confirmed with biochemical tests. • Similarly the sensitivity zones (measured in millimeter) must be registered in diameter. PRECAUTIONS • Observe all precautions as for handling any biological fluid. • Inoculate the specimen under laminar airflow chamber only.


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POTENTIAL SOURCES OF VARIABILITY Delay in processing the specimen may give higher colony count. BIBLIOGRAPHY 1. Alan T, Folkens, Barry A. Schlech, Gale A Cupp, Zuzana Wilmer: Quantitative Ocular Microbiology: Infections of the eye. Khalid F. Tabbara, Robert A Hyndiuk. Little Brown company, Boston, 2nd edition, 1996, 85-93.

BACTERIAL AND FUNGAL CULTURE— CONJUNCTIVAL SCRAPING PURPOSE This test is used for the identification and isolation of pathogens present in the conjunctiva causing inflammation of conjunctiva , their nature and the drugs to which they are sensitive. By knowing the pattern of drug sensitivity, the pathogens can be eradicated. PRINCIPLE Pathogenic organisms present in the specimen will grow well by providing selective culture media and can be identified based on the colony morphology, biochemical reactions and appropriate staining procedure. PERFORMANCE SPECIFICATION Culture reports have to be interpreted with caution based on the clinical findings and correlated with the clinical status of the patients. Culture may be negative for growth if the patient is already taking medication for the disease. PRIMARY SAMPLE • Conjunctival scrapings should be collected by ‘Ophthalmologists’ only. • The specimen must be cultured as soon as possible since the natural secretions of the eye contain anti-bacterial enzymes (lysozymes). • Scrapings after collection is inoculated onto solid media and liquid medium. • Smears are made on clean microscopic glass slides for staining. EQUIPMENT • Autoclave • Aerobic Incubator 37ºC, CO2 incubator 37ºC, anaerobic work station 37ºC; aerobic incubator – 25°C


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• Biosafety hood (Class II) • Microscopes CONSUMABLES • Dehydrated media (Blood agar - BA, Brucella blood agar - BBA, Chocolate agar - CA, MacConkey agar - MA, Sabouraud’s Dextrose agar - SDA, Mueller Hinton agar – MHA, Blood Mueller Hinton agar – BMHA, Peptone water – PW) • Liquid media (Brain heart infusion broth and Thioglycollate medium) • Kimura spatula/disposable sterile surgical blade. • Streaking accessories • Microslide and coverslip • Syringe and needles • Sterile cotton swab SPECIMEN COLLECTION The ophthalmologist under the slit-lamp biomicroscope carries out this procedure.Topical anesthetic eye drop is applied and after 2-3 minutes using sterile Kimura spatula/sterile surgical blade the scrapings are taken, from the everted eyelid and the material on the spatula blade is inoculated onto BA, CA, MAC, BBA, SDA, BHIB directly and MEM for Chlamydia and viruses. Material taken by the spatula second time is used to make 4 smears. Scrapings should be taken from the representative part of conjunctiva in both upper and lower eyelids. The blunt edge of the blade can be used for scraping, bleeding should be avoided. STEP BY STEP PROCEDURE • Transfer 1.0 mL material from inoculated Brain Heart Infusion broth onto Thioglycollate medium. • Incubate the plates BA, Mac, BHIB and Thioglycollate media at 37oC aerobically, CA at CO2 incubator & BBA at anaerobic work station, Sabouraud’s Dextrose Agar (SDA) culture plate at 25°C and look for growth. • Put up culture for Acanthamoeba by inoculating the material onto NNA medium. • Observe for bacterial/fungal colonies grown after overnight incubation. • Perform Gram’s staining with the growth. • Read methanol fixed direct smear after Gram’s staining under oil immersion objective. • Read KOH-Calcofluor white preparation for fungus and Acanthamoeba under fluorescence microscope using violet filter.

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Immunofluorescence staining for detection of Chlamydia trachomatis Adenovirus or any specific viral agent • Perform Immunofluorescence staining if requested using appropriate antisera (For if Chlamydia - procedure on page 286). • Inoculate the organism grown in culture plate onto peptone water. Incubate for 4 hrs. • Inoculate the broth culture on to MHA by lawn culture method. • For Streptococci and Haemophilus species the identification and antibiogram tests are carried out directly from culture plate on Blood Mueller Hinton agar and chocolate agar respectively. • Place required antibiotic discs over it. Inoculate in respective biochemicals depending on the organism for identification. • Incubate at 37°C for 24 hrs and measure the sensitivity zones. • Identify the bacteria based on smear, colony morphology and biochemical reactions. • For anaerobic culture, incubate under anaerobic atmosphere for 12 days and give final report. • If growth of fungus or yeast is observed identify the fungi based on colony morphology and lactophenol cotton blue preparation with slide culture, if there is no growth incubate for 12 days and give final report. INTERPRETATION OF RESULTS Presence of eosinophils in the Geimsa smear indicates allergic conjunctivitis. Culture positive result : Presence or absence of organism in Gram staining and growth in more than one culture media in smear negative case and growth in one medium in smear positive case. Report the organism grown with antibiogram. DATA RECORDING AND VERIFICATION • Enter results of the test in workbook. • Correlate the type of organisms with the nature of specimen. Compare the results of culture with Gram’s stain report of the growth. • Results from the MAC, BA and other media plates must be recorded. It can be later confirmed with biochemical tests. • Similarly the sensitivity zones in the Muller Hinton agar must be registered in diameter. PRECAUTIONS • Observe all precautions as for handling any biological fluid. • Inoculate the specimen under laminar airflow chamber only.


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BIBLIOGRAPHY 1. Clifton D, Cokington, Robert A Hyndiuk. Bacterial keratitis. In. Infections of the eye. Khalid F Tabbara, Robert A Hyndiuk. Little Brown Company, Boston, 2nd edition, 1996; 323-49. 2. Richard W Yee, Panida Kosrirukvongs, Swaminathan Meenakshi, Khalid F Tabbara. In. Infections of the eye. Khalid F Tabbara, Robert A Hyndiuk. Little Brown Company, Boston, 2nd edition, 1996; 349-61. 3. Robert A Hyndiuk, David B Glasser. Herpes simplex keratitis. In. Infections of the eye. Khalid F. Tabbara, Robert A Hyndiuk. Little Brown Company, Boston, 2nd edition, 1996; 361.

BACTERIAL AND FUNGAL CULTURE— LID MARGIN SWAB PURPOSE This test is used for the identification and isolation of pathogens present in the lid margin (causing blepharitis), their nature and the drugs to which they are sensitive. By knowing the pattern of drug sensitivity, the pathogens can be possibly eradicated. PRINCIPLE Pathogenic organisms present in the specimen will grow well by providing enriched culture media and can be identified based on appropriate staining procedure, the colony morphology and biochemical reactions. PERFORMANCE SPECIFICATION Culture reports have to be interpreted with caution based on the clinical findings and correlated with the clinical status of the patients. Culture may be negative for growth if the patient is already taking medication for the disease. PRIMARY SAMPLE • Collect lid margin swab by swabbing the upper and lower lid margin using a sterile cotton swab moistened with HBSS. • Two such swabs are collected one for smear and other for culture. • The specimen must be cultured as soon as possible since the natural secretions of the eye contain anti-bacterial enzymes (lyzozymes). • One swab is placed in 1.0 mL of HBSS. • With the second swab, smears are made on clean microscopic glass slide for staining.

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EQUIPMENT • Autoclave • Incubator 37ºC, 37ºC CO2 incubator, anaerobic work station; incubator 25°C • Biosafety hood (Class II) • Microscopes • Cyclomixer CONSUMABLES • Dehydrated media (Blood agar - BA, Brucella blood agar - BBA, Chocolate agar - CA, Mueller Hinton agar – MHA Blood Mueller Hinton agar – BMHA, Peptone water – PW) • HBSS (Hank’s balanced salt solution) • Streaking accessories • Micro slide • Sterile cotton swab STEP BY STEP PROCEDURE • Vortex the transport medium (HBSS) with swab in a cyclomixer and discard the swab after squeezing it against the wall of the container. • Inoculate 100 μL of specimen onto BA, CA, and BBA. Spread the inoculum and after drying a streak line of Staphylococcus aureus (for isolation of Haemophilus species) is made in the center of inoculum on BA. • Incubate the BA plates at 37°C aerobically, CA in CO2 incubator and BBA at anaerobic work station and look for growth. • Read methanol fixed direct smear after Gram’s staining and Giemsa staining under oil immersion objective. • Count number of colonies grown after overnight incubation in each plate. • Perform Gram’s staining with the growth. • Inoculate the organism grown in culture plate onto peptone water. Incubate for 4 hrs. • Inoculate the Broth culture on MHA by lawn culture method. • For Streptococci and Haemophilus species the identification and antibiogram tests are carried out directly from culture plate on Blood Mueller Hinton agar and chocolate agar respectively. • Place required antibiotic discs over it for identification. Inoculate in respective biochemicals depending on the organism. • Incubate at 37oC for 24 hrs. • Measure the sensitivity zones and identify the organism. • For anaerobic culture, incubate under anaerobic conditions for 12 days. • If growth of fungus or yeast is observed identify the fungus based on colony morphology and lacto phenol cotton blue preparation with slide culture.


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INTERPRETATION OF RESULTS • Culture results are correlated with the clinical condition depending on the type and number of colonies grown. • Isolation of normal flora of conjunctiva is ignored. DATA RECORDING AND VERIFICATION • Enter results of the test in workbook. • Correlate the type of organisms with the nature of specimen. Compare the results of culture with Gram’s stain report of the growth. • Results from the MacConkey agar, BA and other media plates must be recorded as number of colonies (Colony forming units) and later can be confirmed with biochemical tests. • Similarly, the sensitivity zones in the Muller Hinton agar must be registered in diameter. PRECAUTIONS • Observe all precautions as for handling any biological fluid. • Wear facemask, apron and disposable gloves, while transferring and handling the specimen. • Inoculate the specimen under laminar airflow chamber only. BIBLIOGRAPHY 1. John H. Brinser, Eilen M Burd. Principles of Diagnostic Microbiology. Infections of the eye. Khalid F Tabbara, Robert A Hyndiuk. Little Brown Company, Boston, 2nd edition, 1996; 69-74.

BACTERIAL AND FUNGAL CULTURE— CORNEAL SCRAPING PURPOSE This test is used for the identification and isolation of pathogens present in the cornea, their nature and the drugs to which they are sensitive. By knowing the pattern of drug sensitivity, the pathogens can possibly be eradicated. PRINCIPLE Pathogenic organisms present in the specimen will grow well by providing enriched culture media and can be identified based on appropriate staining procedure, the colony morphology and biochemical reactions.


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PERFORMANCE SPECIFICATION Culture reports have to be interpreted with caution based on the clinical findings and correlated with the clinical status of the patients. Culture may be negative for growth if the patient is already taking medication for the disease. Growth in more than one media is considered for positive culture reporting in smear negative cases, and in smear positive cases growth of microscopically similar organism in one media would suffice. PRIMARY SAMPLE (CORNEAL SCRAPING) • Ophthalmologist collects corneal scraping. • Scraping material is collected for smear and culture. • Scraping is inoculated onto the plates at the site of collection immediately. EQUIPMENT • • • •

Autoclave Incubator 37ºC, CO2 incubator , anaerobic work station; 25°C incubator Biosafety cabinet (Class II) Microscopes

CONSUMABLES • Dehydrated media (Blood agar - BA, Brucella blood agar - BBA, Chocolate agar - CA, MacConkey agar - MA, Sabouraud’s Dextrose agar –SDA, Non-nutrient agar-NNA, Mueller Hinton agar – MHA, Blood Mueller Hinton agar – BMHA, Peptone water – PW) • Liquid media (Brain heart infusion broth and Thioglycollate medium) • Kimura spatula/disposable sterile Bard-parker blade. • Streaking accessories • Microslide and coverslip • Syringe and needles • Sterile cotton swab SPECIMEN COLLECTION This procedure is carried out by the ophthalmologist under the slit lamp biomicroscope. Topical anesthetic eye drop is applied and after 2–3 minutes using sterile Kimura spatula/sterile surgical blade the scrapings are taken, the sterile surgical blade (disposable) also may be used; it is advisable to use the blunt edge of the blade for scraping; bleeding should be avoided. The material on the spatula/blade is inoculated onto BA, CA, BBA, MAC, SDA, NNA in form of “C” curves and brain heart infusion directly and MEM for Chlamydia and viruses, material taken second time is used to


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make smears. Scraping material is smeared on microscopic glass slide for staining. STEP BY STEP PROCEDURE • Transfer 1.0 mL material from inoculated Brain Heart Infusion broth onto Thioglycollate medium. • Incubate the plates BA, Mac, BHIB and Thioglycollate media at 37°C aerobically, CA in CO2 incubator and BBA in anaerobic work station and look for growth after overnight incubation. • Perform Gram’s stain, observe under the microscope and record the results. • Read KOH-Calcofluor white preparation for fungus and Acanthamoeba under fluorescence microscope using violet filter. • Perform immunofluorescence staining if requested for HSV detection using appropriate antisera (procedure 10 and immunofluorescence staining for HSV 11). • Put up for Acanthamoeba culture using material inoculated on to NNA media. • Observe for bacterial/fungal colonies grown after overnight incubation. • Perform Gram’s staining with the colonies. • Inoculate the organism grown in culture plate onto peptone water. Incubate for 4 hrs. • Inoculate the broth culture on to MHA by lawn culture method. • For Streptococci and Haemophilus species the identification and antibiogram tests are carried out directly from culture plate on Blood Mueller Hinton agar and chocolate agar respectively. • Place required antibiotic discs over it and inoculate in respective biochemicals depending on the organism. • Incubate at 37°C for 24 hrs and measure the sensitivity zones. • Identify the bacteria based on smear, colony morphology and biochemical reactions. • For anaerobic culture, incubate under anaerobic atmosphere for 12 days and give the final report. If growth of yeast is observed. identify the yeast by colony morphology, biochemicals and assimilation tests. • If growth of filamentous fungus is observed, identify the fungi based on colony morphology and lactophenol cotton blue preparation with slide culture, if there is no growth incubate for 12 days and give final report. INTERPRETATION OF RESULTS Culture positive result: Presence or absence of organism in Gram staining and growth in more than one culture media in smear negative case and growth in one medium in smear positive case.


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DATA RECORDING AND VERIFICATION • Enter results of the test in worksheet. • Correlate the type of organisms with the nature of specimen. Compare the results of culture with Gram’s stain report of the growth. • Results from the MacConkey agar, BA and other media plates must be recorded, later can be confirmed with biochemical tests. • Similarly, the sensitivity zones in the Muller Hinton agar must be registered in diameter. PRECAUTIONS • Observe all precautions as for handling any biological fluid. • Inoculate the specimen inside the biosafety cabinet (class II) only. BIBLIOGRAPHY 1. Clifton D, Cokington, Robert A Hyndiuk. Bacterial keratitis. In. Infections of the eye. Khalid F Tabbara, Robert A Hyndiuk. Little Brown Company, Boston, 2nd edition, 1996, 323-49. 2. Richard W Yee, Panida Kosrirukvongs, Swaminathan Meenakshi, Khalid F Tabbara. In. Infections of the eye. Khalid F Tabbara, Robert A Hyndiuk. Little Brown Company, Boston, 2nd edition, 1996, 349-61. 3. Robert A Hyndiuk, David B Glasser. Herpes simplex keratitis. In. Infections of the eye. Khalid F Tabbara, Robert A Hyndiuk. Little Brown Company, Boston, 2nd edition, 1996, 361.

BACTERIAL AND FUNGAL CULTURE— INTRAOCULAR SPECIMENS PURPOSE This test is used for the identification of pathogens present in the intraocular specimens, their nature and the drugs to which they are sensitive. By knowing the pattern of drug sensitivity. PRIMARY SAMPLE • Anterior humor (AH) aspirate • Vitreous aspirate (VA) or Vitreous fluid (VF) • Lens aspirate CONSUMABLES • Dehydrated media (Blood agar - BA, Brucella blood agar - BBA, Chocolate agar -CA, MacConkey agar - MAC, Sabouraud’s Dextrose


• • • • • •

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agar - SDA, Mueller Hinton agar – MHA, Blood Mueller Hinton agar – BMHA, Peptone water – PW) Liquid media (Brain heart infusion broth –BHIB and Thioglycollate medium –Thio) Cytospin chambers and cytospin instrument. Streaking accessories Micro slide and coverslips Syringe and needles Sterile cotton swab

EQUIPMENT • • • • •

Autoclave Incubator 37ºC, 37ºC CO2, anaerobic work station; 25°C incubator Biosafety hood (Class II) Cytospin machine Microscopes

COLLECTION OF SPECIMENS Collection of aqueous humor (AH), vitreous aspirate (VA) and lens aspirate (LA) for the preparation of smears and cultures for detection of bacteria, fungus and viruses (Herpes simplex virus, Varicella zoster virus, Cytomegalovirus and Rubella virus). AH, VA and LA are collected by the ophthalmologist in syringe with needle, either as outpatient or operation theater procedure. After collection of sample the air in the syringe is expelled and the needle is fixed into a sterile rubber cork and placed in the test tube container. This is immediately transported to the laboratory. Note: It is essential that inoculation of media is done first because as the number of organisms are likely to be low, every chance is given for them to multiply. Smears are not to be done first. Bacteria, present in few numbers, in specimen are difficult to be made out in smears. Media are chosen according to the requirement. PROCEDURE • One drop of specimen is inoculated onto BA, CA, BBA and MacConkey agar, SDA in the center of the plate and onto BHIB and Thioglycollate medium. Cytospin smears are made for direct microscopy using the cytospin instrument (Shandon USA). The smear is made by cytospin centrifuging at 1000 rpm for 5 minutes using the cytospin.

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• Incubate the BA and MacConkey plates, BHIB at 37o C incubator, BBA at anaerobic incubator, CA at Co2 incubator and SDA at 25º C. • Leave the smear for drying and fix with methanol for 5 minutes. • Stain the fixed smear by Gram’s staining procedure and observe under microscope. • Stain smear by Giemsa stain for observation of cytological study. • Observe KOH/ Calcofluor preparation for fungus and acanthamoeba under fluorescence microscope using violet filter. • Observe for bacterial/fungal colonies grown after overnight incubation in each plate. • Perform Gram’s staining with the growth. • Inoculate the organism grown in culture plate onto peptone water. Incubate for 4 hours. • Inoculate the broth culture on MHA by lawn culture method. • For Streptococci and Haemophilus species the identification and antibiogram tests are carried out directly from culture plate on Blood Mueller Hinton agar and chocolate agar respectively. • Place required antibiotic discs over it and inoculate in respective biochemicals depending on the organism. • Incubate at 37oC for 24 hrs. • Measure the sensitivity zones and identify the organism. • For anaerobic culture, incubate under anaerobic conditions for 12 days. • If growth of fungus or yeast is observed identify the fungi based on colony morphology and lacto phenol cotton blue preparation with slide culture. INTERPRETATION OF RESULTS • Gram positive bacteria appear violet or purplish blue and Gram negative bacteria appear red or pink in gram stain. • Indicate the morphology and arrangement specifically, especially for Gram positive cocci pneumococci with capsule and lanceolate shaped, gonococci kidney shaped diplococci. • Mention the cocco bacillary forms with regards to Gram-negative bacilli if seen. • Budding yeast cells appear dark purple in color. • Pus cells appear pink in color. • Epithelial cells appear pale pink in color, large round cells with capsules can be Cryptococcus. • Growth of organism in more than one plate is considered as positive.


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SAFETY PRECAUTIONS • Observe all precautions as for handling any biological fluid. • Wear facemask and disposable gloves while transferring and handling the specimen. POTENTIAL SOURCES OF VARIABILITY • Process specimens before they dry up. • Occasionally Gram positive organisms lose their ability to retain the crystal violet and stain Gram negatively due to over decolorization or too old Gram’s iodine or too old cultures or due to over heat fixation. BIBLIOGRAPHY 1. Vitreous Aspirate and Anterior Chamber Tap: John H Brinser, Eileen M Burd. Principles of Ocular Microbiology. Infections of the eye. Khalid F Tabbara, Robert A Hyndiuk. Little Brown Company, Boston, 2nd edition, 1996, 423.

BACTERIAL AND FUNGAL CULTURE— OTHER SPECIMENS PURPOSE This test is used for the identification of pathogens present in the body fluids other than CSF and other clinical specimen, their nature and the drugs to which they are sensitive. PRINCIPLE Pathogenic organisms present in the clinical specimen will grow well by providing enriched culture media and can be identified based on the colony morphology, biochemical reactions and appropriate staining procedure PERFORMANCE SPECIFICATION • Specimen has to be inoculated as early as possible to have better isolation of pathogens. • Specimens are collected by medical officers only (or) lab staff all the case may be. • Culture may be negative for growth if the patient is already on medication.

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PRIMARY SAMPLE • Body fluids other than CSF, Pus swabs EQUIPMENT • Autoclave • Incubator 37ºC, 37ºC CO2 incubator, anaerobic work station; incubator 25°C • Biosafety hood (Class II) • Microscopes CONSUMABLES • Dehydrated media (Blood agar - BA, Brucella blood agar - BBA, Chocolate agar –CA, MacConkey agar - MAC, Sabouraud’s Dextrose agar - SDA, Mueller Hinton agar – MHA, Blood Mueller Hinton agar – BMHA, Peptone water – PW) • Liquid media (Brain heart infusion broth and Thioglycollate medium) • Streaking accessories • Microslide and coverslip • Syringe and needles • Sterile cotton swab COLLECTION OF SPECIMENS Specimens are collected by medical officers only (or) lab staff all the case may be. On arrival in the laboratory, the lab staff inspects the specimens. If it is suitable for processing, it is proceeded for inoculation and smear preparation. PROCEDURE • Inoculate the specimen onto BA, CA, MAC, BBA, SDA. Make first primary inoculum and make secondary and tertiary inoculation from the primary inoculum. • If the clinical specimen is a body fluid, process it like that of CSF ( Refer procedure 16). • Inoculate into BHIB and thioglycollate media. • Incubate the BA and MacConkey plates, BHIB, THIO at 37oC incubator, BBA at anaerobic incubator, CA at CO2 incubator and SDA at 25ºC. • The smear of the specimen is made. Leave the smear for drying and fix with methanol for 5 minutes. For body fluids, make smear by using cytospin instrument. • Stain the fixed smear by Gram’s staining procedure and observe under light microscope and record your result.


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• Stain smear by Geimsa stain for observation of cytological study. • Observe KOH/Calcofluor preparation under fluorescence microscope using violet filter for fungus. • Perform AFB staining if requested. • Observe for bacterial/fungal colonies grown after overnight incubation. • Perform Gram’s staining with the growth. • Inoculate the organism grown in culture plate onto peptone water. • Incubate for 4 hours. • Inoculate the Broth culture on MHA by lawn culture method. • For Streptococci and Haemophilus species the identification and antibiogram tests are carried out directly from culture plate on Blood Mueller Hinton agar and chocolate agar respectively. • Place required antibiotic discs over it and inoculate in respective biochemicals depending on the organism. • Incubate at 37oC for 24 hrs and measure the sensitivity zones. • Identify the bacteria based on smear, colony morphology and biochemical reactions. • For anaerobic culture, incubate under anaerobic atmosphere for 12 days and give final report. • If growth of fungus or yeast is observed, identify the fungi based on colony morphology and lactophenol cotton blue preparation with slide culture, if there is no growth incubate for 12 days and give final report. INTERPRETATION OF RESULTS • Gram positive bacteria appear violet or purplish blue and Gram-negative bacteria appear red or pink in Gram stain. • Indicate the morphology and arrangement specifically, especially for Gram positive cocci pneumococci lanceolate shaped, Gonococci kidney shaped diplococci. • Mention the cocco bacillary forms with regard to Gram-negative bacilli if seen. • Yeast cells appear dark purple in color • Pus cells appear pink in color • Epithelial cells appear pale pink in color, large yeast shaped cells with capsular can be Cryptococcus. • Growth of same organism in more than one plate is considered as significant/true pathogen. SAFETY PRECAUTIONS Observe all precautions as for handling any biological fluid.

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POTENTIAL SOURCES OF VARIABILITY • Process specimens before they dry up • Occasionally, Gram positive organisms lose their ability to retain the Crystal Violet and stain Gram negative due to over decolorization or too old Gram’s Iodine or too old cultures or due to over heat fixation. BIBLIOGRAPHY 1. Laboratory strategy in the diagnosis of infective syndromes. In Mackie and McCartney. Practical Medical Microbiology. 13th edition Edited by Collee, Dugid, Fraser, Marmion. 1989;600-50. 2. Textbook for Medical Laboratory Technology. Second Edition. Praful B Godkar, Darshan P Godkar, Pg. 606, 609.

CONVENTIONAL BIOCHEMICAL TESTS FOR IDENTIFICATION OF BACTERIAL ISOLATES The following tests are carried out for identification of bacteria isolated in pure culture from various clinical samples according to procedures described below. CATALASE TEST Purpose: This test is used to differentiate those bacteria that produce the enzyme “Catalase” such as Staphylococci from non-Catalase producing bacteria such as Streptococci species. Principle: Catalase acts as a catalyst in the break down of hydrogen peroxide to water and oxygen. An organism is tested for Catalase by bringing it into contact with 3% hydrogen peroxide. Bubbles of oxygen are released if the organism is a catalase producer. The culture should not be more than 24 hrs old. Procedure: Place a drop of 3% Hydrogen peroxide (1 in 10 dilution from stock hydrogen peroxide 30% solution) solution on a clean glass slide. Using a sterile wooden stick remove a growth of test organism and immerse it in 3% Hydrogen peroxide solution. Look for immediate bubbling. Repeat the same procedure for positive and negative control. Perform the procedure with the positive and negative control and then the test organism. Note: A nichrome wire loop must not be used, because this may give false positive reaction. Growth from a blood agar should not be tested as it might give false positive reaction because RBCs contain catalase enzyme.


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Interpretation Active bubbling: Positive test - Catalase produced. No release of bubble: Negative test - No Catalase produced. Control Positive control: Staphylococcus aureus Negative control: Streptococcus pneumoniae. OXIDASE TEST Purpose: This test is used to help in the identification of the organisms, which produce the enzyme oxidase examples: Pseudomonas aeruginosa, Neisseria, Vibrio and Pasteurella. Principle: A colony of the test organism is smeared on a filter paper which is impregnated with oxidase reagent. If the organism is oxidase producing, the phenylenediamine in the reagent will be oxidized to indophenol blue resulting in deep purple color. Preparation of oxidase reagent Whatman filter paper No 1 is cut into small squares and placed in a petriplate and exposed to UV light for 2 hours. Oxidase reagent (1%) is prepared for 5 mL and poured over the strips to make the reagent get impregnated in the strip. The petri plate is kept for drying at room temperature for 48 hours. After the strips have dried completely, it can be used for performing the oxidase test. The strips are used after testing with Pseudomonas aeruginosa and Escherichia coli. Method Take a part of the colony to be tested by a wooden stick and rub on the oxidase strip. Observe the reaction. Results Blue purple color formation (within 10 seconds)- Positive test. No blue/purple color formation (within 20 seconds) – Negative test. Control Positive control: Pseudomonas aeruginosa Negative control: Escherichia coli. COAGULASE TEST Purpose: This test is used to differentiate coagulase positive Staphylococcus aureus from coagulase negative Staphylococcus sps like Staphylococcus epidermidis and Staphylococcus saprophyticus, etc.

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Principle: Coagulase is an enzyme, which causes clot formation of the plasma by converting soluble fibrinogen to insoluble fibrin. This bound coagulase can rapidly be recognized by slide test and free coagulase can be detected by tube test. Preparation of Reagents Procedure — Tube Coagulase for bound Coagulase enzyme. Preparation of plasma: 5 mL of blood in EDTA vacutainer, separate the plasma following aseptic precautions. Dilute one ml of plasma with 9 ml of sterilesaline (0.9% NaCl) and store at –20°C. Dispense 0.5 mL of diluted sterile human plasma into sterile test tube. Include one tube for positive control (Staphylococcus and one for negative control (Staphylococcus epidermidis) in each batch of test. Add 0.5 mL 4 hour both suspension of the test organism including controls to the respective tubes. – Incubate at 37°C for 4 hours. – At the and of 4 hours, slightly till the tube to look for clot/gel formation. Interpretation Fibrin clot formation– Positive. No fibrin clot – Negative Slide coagulase for free coagulase Place a drop of sterile normal saline on a clean glass slide. Using a sterile wooden stick remove a growth of test organism and emulsify in normal saline, and look whether there is any agglutination. If there is agglutination then coagulase test is negative. If there is no agglutination then add one drop of sterile EDTA human plasma mix well rotate the slide back and forth for 2 minutes and look for agglutination. If there is agglutination then the coagulase test is positive. The test is performed with positive and negative control, then with test organisms. Interpretation Agglutination – Positive No Agglutination – Negative Control Positive control: Staphylococcus aureus Negative control: S.epidermidis or S. saprophyticus BIOCHEMICAL TESTS TO DETECT FERMENTATION REACTIONS OF SUGARS Purpose: This test is used to detect fermentation reaction of bacteria.

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Principle: Certain bacteria ferment specific sugars when they multiply resulting fall in pH and then color change is produced in the sugar medium - acidic pH turns bromothymol blue to yellow. If gas is produced it is seen as bubbles of gas in Durham’s tube in glucose tube. Procedure Carbohydrates used for the tests are 1% of glucose, sucrose, maltose, lactose. Inoculate 4-hour broth culture of bacteria or a loopful of culture into carbohydrate fermentation medium and incubate at 37°C overnight. Interpretation of result Color changes from blue to yellow – acid No color change — No acid Bubbles of gas in Durham’s tube in glucose tube – gas production. Control Positive control: Escherichia coli acid with gas Negative control: Pseudomonas aeruginosa , No acid production OXIDATION — FERMENTATION TEST Purpose: Oxidation and fermentation test is useful in differentiation of oxidation and fermentation reaction of the organism. Principle: Certain bacteria utilize sugar and produce acid and sometimes gas. This acid changes the pH and therefore there is a change in color from green to yellow. Some bacteria utilize this sugar oxidatively in the presence of oxygen while some utilize it fermentatively in the absence of oxygen. Method: Inoculate a loopful of culture from 4-hour broth or a bacterial colony by stabbing into the center of the 2 tubes of oxidation-fermentation test medium. Add sterile paraffin oil over one of the two oxidation– fermentation test medium. Incubate at 37 °C overnight. Result Fermentation is indicated by change of color of the medium to yellow in both the tubes. Oxidized if there is only change of color of the medium to yellow in one tube to which oil is not added. Inert if there is no change in both the tubes. Control E.coli : Fermenter Pseudomonas aeruginosa: Oxidizer Acinetobacter lwoffii: Inert.

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MANNITOL MOTILITY TEST Purpose: To determine whether a particular bacterium ferments mannitol and also to find out whether the organism is motile or non-motile. Principle: Peptic digest provides nitrogenous requirements. Mannitol fermentation is indicated by the change in the color of the medium. Motility is aided by the semi solid nature of the medium. Method Inoculate a loopful of culture from 4 hour broth or a bacterial colony by stabbing in to the centre of the Mannitol motility test medium. Incubate at 37ºC over night. Result Mannitol fermentation is indicated by change of color of the medium to yellow. Motile organisms grow out from the stab throughout the medium. Non-motile organisms grows only along the stab line. Control: Mannitol fermentation Positive control: Escherichia coli Negative control: Pseudomonas aeruginosa Mannitol fermented and non-motile-Klebsiella aerogenes UREASE TEST Purpose: Proteus strains are strong ‘Urease’ producers, Salmonellae and Shigellae do not produce urease. This test helps in differentiating certain species of Enterobacteriaceae. Principle: The test organisms are cultured in Christensen’s Urease broth/ agar. Decomposition of urea by urease enzyme produced by the organism results in the production of ammonia and CO2. The medium becomes alkaline and color of the medium changes to magenta pink. Method Inoculate Christensen’s urea agar with 4 hour broth organism or a bacterial colony. Incubate at 37°C overnight. Examine the medium by looking for a red-pink coloration of the slant. Result: Red- pink medium: Positive test (Urease produced) No red – pink medium: Negative test (No Urease produced) Control: Positive control: Proteus vulgaris Negative control: E.coli.


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CITRATE UTILIZATION TEST Purpose: This test is performed in the identification of Enterobacteriaceae based on their ability to use citrate as the sole carbon source. Principle: This test is based on the ability of the microorganisms to utilize citrate as the sole carbon source and ammonia as the sole source of nitrogen. The citrate utilization is followed by alkaline reaction (medium containing bromothymol blue indicator which changes to blue from green) causes formation of blue color. Method: Streak a loopful of culture from 4-hour broth or a bacterial colony on a slant of citrate agar. Incubate the slant at 37° Covernight. Look for the change in the color of the slant from light green to blue. Result: Formation of blue color – Positive. No growth (same color) – Negative Control: Positive control – Klebsiella pneumoniae Negative control – Escherichia coli TRIPLE SUGAR IRON AGAR TEST Purpose: TSI slants are useful in the identification of Enterobacteriaceae by their specific reactions on the slants. Principle: Alkaline reaction (red color) is shown by organisms that fail to ferment any one of the sugars. Fermentation of the sugars is indicated by yellow color. Gas production (CO2) is indicated by splitting of the agar. Production of hydrogen sulfide imparts black shade to the slant by reacting with ferrous ions. Method Streak a loopful of culture from 4-hour broth or a bacterial colony on the TSI slant with a loop and stab the butt with a straight loop. Incubate at 37°C for over night. Result The various possible reactions observed on the inolated slants are as follows: Color of slant/butt

Gas

H2S

Possible organisms

Yellow/Yellow (A/A) Yellow/Yellow (A/A) Pink /Yellow (K/A)

+ + -

+ -

E. coli, Klebsiella, Enterobacter sp. Proteus vulgaris Shigella sp., Morganella morganii, Contd.

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Contd. Providencia sp. Pink/Yellow (K/A) Pink/Yellow (K/A) Pink/Yellow (K/A) Pink/Yellow (K/A) Pink/ Red (K/Inert)

+ + -

+ + -

Salmonella typhi Other Salmonella sp., Proteus mirabilis Providencia alcalifaciens Do not belong to Enterobacteriaceae Pseudomonas aeruginosa

NITRATE REDUCTION TEST Purpose: This is a test for detection of presence of the enzyme nitrate reductase which causes the reduction of nitrate in the organism. Principle: The microorganisms produce the enzyme, nitrate reductase which reduces nitrate to nitrite which gives a deep dark pink colored compound with solution A and solution B. Preparation of nitrate reagent Nitrate A: Dissolve 8.0 g of sulfanilic acid in 1 lt of acetic acid 5 mol/ lt Nitrate B: Dissolve 5.0 g of α – naphthylamine in 1 lt of acetic acid 5 mol/ lt. Note: Immediately before use, mix equal volume of solution A and solution B to give the test reagent. Procedure: Inoculate 4-hour broth culture of bacteria in the medim and incubate at 37° C over night. Add 0.1 mL of test reagent to the test culture tube and look for color change. Interpretation of result: Formation of a red color: Positive test (presence of nitrate reductase) No red color: Negative test (absence of nitrate reductase) Control Positive citrate control – E.coli. Negative citrate control – Pseudomonas aeruginosa INDOLE TEST Purpose: This test is important in the identification of Enterobacteria such as E. coli, Proteus vulgaris, P. rettgeri, Morganella morganii and Providencia sps. which have the capacity to produce indole. Principle: The microorganisms produce indole by the break down of the amino acid tryptophan.


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The production of indole is detected by the addition of Kovac’s reagent which contains p-dimethylaminobenzaldehyde. This reacts with indole to produce a red colored compound. Preparation of Kovac’s reagent p-dimethylaminobenzaldehyde : 10 g Iso-amylalcohol/iso-butyl alcohol :150 mL Concentrated HCl : 50 mL Procedure Inoculate 4-hour broth culture of bacteria in tryptone water and incubate at 37°C overnight. Add 0.5 mL of Kovac’s reagent along the side of the test tube to form a layer at the top. Interpretation Formation of a red ring at the junction: Positive test (Indole produced) No red ring formation: Negative test (No indole produced) Control Positive control: E.coli Negative control: Enterobacter aerogenes BILE AESCULIN HYDROLYSIS Purpose: This test is important in the identification of Enterococcus faecalis from other Streptococcus species. Principle: The organism is able to resist bile present in the medium and hydrolyze aesculin which is seen as color change to black indicating a positive reaction. Procedure Streak a bacterial colony on the Bile aesculin medium slant with a loop. Incubate at 37° C overnight. Interpretation of result Change of medium to black color : Positive test No change of medium color : Negative test Control Positive control: Enterococcous faecalis Negative control: Streptococcus pneumoniae GROWTH IN 6.5% SODIUM CHLORIDE BROTH Purpose: This test is used in the identification of Enterococcus faecalis from other Streptococci species.

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Principle: The organism is able to withstand 6.5% NaCl and grow in the medium, making turbid indicating positive reaction. Procedure Inoculate a bacterial colony on the 6.5 % NaCl medium with a loop. Incubate at 37°C overnight. Interpretation of result Growth seen in the medium : Positive test No growth seen in the medium: Negative test Control Positive control: Enterococcus faecalis Negative control: Streptococcus pneumoniae LIPASE AND LECITHINASE PRODUCTION USING EGG YOLK AGAR Purpose: This test is important in detecting the production of lipase and lecithinase useful for characterization of Bacillus and Clostridium species. Principle: On solid media containing egg-yolk, lipolysis is shown by the formation of a thin,‘iridescent pearl layer’ overlying the colonies and a confined opalescence in the medium underlying them, seen best when the colonies are scraped off. Lecithinase is shown by wide zones of opalescence around colonies, more intense and larger than the zones caused by lipolysis. Procedure Streak a bacterial colony on the Egg yolk medium plate with a loop. Incubate at 37°C over night. Interpretation of result Presence of pearly layer indicates lipase production, zone of opalescence indicates lecithinase production : Positive test Absence of pearly layer and zone of opalescence : Negative test Control Positive control: Bacillus cereus Negative control: Bacillus subtilis GROWTH AND HYDROLYSIS OF CASEIN, TYROSINE, XANTHINE Purpose: This test is used in the identification of Nocardia species. Principle: If the Nocardia species utilize casein, xanthine, tyrosine present in the medium clear zone of hydrolysis is seen around the colonies of Nocardia at the end of 7-21 days of incubation.


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Procedure Streak a bacterial colony on the casein, xanthine, tyrosine medium plate with a loop. Incubate at 37°C for 7 days. Interpretation of result Clear zone of hydrolysis is seen around the colonies: Positive test No clear zone of hydrolysis is seen around the colonies: Negative test Control Positive control: Nocardia brasiliensis (casein, xanthine) Nocardia caviae (tyrosine) Negative control: Nocardia asteroides. Biochemical Tests for Identification of Different Bacteria Organism

Tests for identification of bacteria

Staphylococcus species

Catalase, oxidase, oxidation-fermentation, mannitol fermentation, slide coagulase and tube coagulase, sensitivity to novobiocin, methicillin Catalase, oxidase, growth in 6.5% NaCl, bile aesculin hydrolysis, sensitivity to optochin and bacitracin Catalase, oxidase, fermentation of glucose, sucrose, maltose, mannitol, urease, nitrate Catalase, oxidase, motility, fermentation of glucose, sucrose, maltose, citrate, nitrate and growth in 6.5% NaCl, lipase and lecithinase production on egg yolk Catalase, oxidase, urease, nitrate, reductase, hydrolysis of casein, xanthine and tyrosine, and fermentation of glucose Catalase, oxidase, motility, Fermentation of glucose, sucrose, maltose, lactose, oxidation and fermentation, mannitol, urease citrate, triple sugar iron agar, nitrate and indole Catalase, oxidase, motility, fermentation of glucose, sucrose, maltose, lactose, oxidation and fermentation, mannitol, urease citrate, triple sugar iron agar, nitrate and indole

Streptococcus species Corynebacterium species Bacillus species

Nocardia species

Gram negative bacilli Enterobacteriaceae and non-fermenters Haemophilus species

BIBLIOGRAPHY 1. JG Collee and RS Miles. Tests for identification of bacteria. In Mackie & McCartney’s Practical Medical Microbiology. Edited by JG Collee, JP Duguid, AG Fraser, BP Marmion. 13th Edition, Churchill Livingstone, 1989, 141-61.


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ANTIBIOTIC SENSITIVITY PURPOSE Antibiotic susceptibility testing has become a very essential step for the proper treatment of infectious diseases. It is used: a) To guide the clinician in selecting the best anti-microbial agent for therapy; b) To accumulate epidemiological information on the resistance of microorganisms of public health importance. PRINCIPLE The antibiotic disc with known concentration of antibiotics placed in the solid medium will diffuse circularly. The microorganism swabbed over the medium will grow and if it is sensitive to the antibiotic, the bacterial growth will be inhibited by the diffused antibiotic. The inhibited growth results in the formation of a clear zone in the petri plate called zone of inhibition. The diameter of zone of inhibition is measured in mm, and interpreted using the standard chart. PERFORMANCE SPECIFICATION Sensitivity: It is based on the concentration of the antibiotic used in the disc. Test only five disc on a 90 mm Petri plate and 6 discs on a 100 mm Petri plate. Specificity: Some organisms are resistant to particular antibiotics and susceptible to other antibiotics. REAGENTS AND CONSUMABLES • • • •

Mueller Hinton Agar, Blood Mueller Hinton Agar Peptone water Petri plate/dishes Antibiotic discs

EQUIPMENT • Biosafety Cabinet Type II • 37°C incubator • 37°C 10% CO2 incubator PROCEDURE Prepare the inoculum from the primary culture plate, by touching the top of the 3-5 colonies of the organism to be tested, with a bacteriological loop and suspending in peptone water. Incubate the broth at 37°C for 4 hours. Dip a sterile swab into the 4-hour-old broth suspension and remove the excess by pressing and rotating the swabs firmly against the side of the tube above the level of the liquid.


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Inoculate the broth culture onto Mueller Hinton Agar by lawn culture method by streaking the swab all over the surface of the medium, rotating the plate through an angle of 60o after each application. Finally, pass the swab round the edge of the agar surface. Leave the inoculum to dry for a few minutes at room temperature with the lid closed. For fastidious bacterial isloates 3-5 colonies are taken and proceeded directly. For example Streptococci and Haemophilus species the identification and antibiogram tests are carried out directly from culture plate on Blood Mueller Hinton agar and chocolate agar respectively. Place the antibiotic discs on the inoculated plates using a sterile needle tip. Place the antibiotic discs as given below in the table, depending upon the specimens and the organisms isolated. Test all Staphylococcus spp for Methicillin resistance by inoculating the Staphylococcus species on Mueller Hinton agar with 5% NaCl and place the methicillin disc. Place the discs at equal distance (approximately 15 mm from the edge of the plate). Each disc should be pressed down gently to ensure even contact with the medium. Incubate MHA plates and Blood Mueller Hinton agar at 37°C and chocolate agar at 37°C 10% CO2 incubator for overnight. Antibiotic discs are placed depending on the specimen and type of organism grown as the list given below. Nocardia species • Cefazolin • Amikacin • Co-trimoxazole • Cefotaxime • Ceftazidime • Vancomycin • Ciprofloxacin Rapid growing mycobacteria (M. fortuitum, M. chelonae M. abscess) • Amikacin • Ciprofloxacin • Ceftazidime • Cephotaxime • Ofloxacin • Tobramycin • Cefuroxime • Cefoperazone • Azithromycin • Ceftriaxone


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Staphylococcus species – Methicillin, Novobiocin Streptococci – Optochin, Bacitracin in additions to the other antibiotics RESULT After overnight incubation, the diameter of each zone (including the diameter of the disc) should be measured and recorded in mm. The results should then be interpreted as sensitive or resistant according to the critical diameters by comparing with standard chart supplied by the manufacturer of the discs. The measurements can be made with a ruler under the surface of the plate without opening the lid. The end-point of inhibition is judged by the naked eye at the edge where the growth starts, but there are three exceptions. With sulfonamides and co-trimoxazole, slight growth occurs within the inhibition zone such growth should be ignored. When β-lactamase producing Staphylococci are tested against penicillin, zones of inhibition are produced with a heaped-up, clearly defined edge; these are readily recognizable when compared with the sensitive control, and regardless of the size of the zone of inhibition, they should be reported as resistant. Certain Proteus spp. may swarm into the area of inhibition around some antibiotics, but the zone of inhibition is usually clearly outlined and the thin layer of swarming growth should be ignored. Note: For vancomycin, take reading after 24 hours of incubation. BIBLIOGRAPHY 1. Scott AC. Laboratory control of antimicrobial therapy. In Mackie & McCartney’s Practical Medical Microbiology. Edited by JG Collee, JP Duguid, AG Fraser, BP Marmion. 13th Edition, Churchill Livingstone, 1989, 161-81.

ACID FAST BACILLI (AFB) CULTURE PURPOSE This test is used for the isolation and identification of Mycobacterium in clinical specimens. PRINCIPLE Mycobacterium tuberculosis and other mycobacteria species will grow well aerobically in a protein-enriched medium. The optimal temperature for growth is 35–37°C. Mycobacterium tuberculosis is slow-growing and pigment is not produced. Several media are available for culturing mycobacterium.


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Lowenstein-Jensen egg medium is the most widely used medium. It contains fresh whole eggs, glycerol, several mineral salts, malachite green, selectively inhibits the growth of contaminants. PERFORMANCE SPECIFICATION This test cannot be used to differentiate the species of mycobacteria. Culture for growth may be negative if the patient is already taking medications for the disease. Hence culture reports have to be interpreted with caution based on the clinical status of the patient and correlated with the clinical findings on the patient. PRIMARY SAMPLE Sputum, Urine and other body fluids can be used as specimens. Sputum: Instruct the patients to collect deeply coughed, expectorated, sputum in sterile wide-mouthed red top plastic container after rinsing the mouth with water. Urine: Instruct the patients to collect early morning first voided sample of urine on three successive days in a clean, dry and sterile container provided by the laboratory. While collecting urine, instruct the patient to clean the external genitals and surrounding area with moist cotton/copious amount of water to avoid contamination by normal flora. Label the container with the ID number of the patient and nature of specimen, date, name of the patient. Process the urine specimen immediately within 1 hour of collection or the urine should be stored in the refrigerated (4-8°C) till processing. CONSUMABLES • • • • •

Dehydrated media (LJ medium) Streaking accessories Centrifuge tube 4% sodium hydroxide Micro slides


Autoclave Incubator 37°C Biosafety hood (type III) Centrifuge Shaker

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PRECAUTIONS • Observe all precautions as for handling any biological fluid. • Wear face mask, apron and disposable gloves, while transferring and handling the specimen. • Inoculate the specimen under biosafety hood only. • Process the urine within 1 hour of collection, or pressure in the refrigerator till further processing. STEP BY STEP PROCEDURE • Petroff’s method of sputum/urine concentration. • The processing of the specimen has to be carried out inside the Biosafety Cabinet (BSC) Type III. • With a sterile loop, inoculate the sputum/clinical specimen over the surface of the LJ medium. Close the slopes tightly, incubate at 37°C. • Centrifuge the entire amount of urine sample using centrifuges with bigger buckets and inoculate the deposit on the LJ medium. • Make smears before decontaminating it with sodium hydroxide and stain by Ziehl-Neelsen method. • To each volume of remaining sputum/urine/other specimens, add two volumes of 4% sodium hydroxide, taking care to avoid contact between the specimen bottle rim and the sodium hydroxide flask. • Shake the centrifuge tubes by hand for one minute. • Place in a rack on the shaking machine and leave to shake for 20 mintues. • Remove the specimen from the shaker. • Centrifuge for 15 minutes at 4000 rpm. • After removal from the centrifuge carefully pour of the supernatant into the disinfectant. • Fill the centrifuge tubes with approximately 3/4 th of sterile Milli Q water, shake by hand to mix the deposit, centrifuge at 4000 rpm for 15 minutes and pour of the supernatant as before. • Finally inoculate sediment with a loop on to two previously numbered Lowenstein-Jensen slopes. • Place the inoculated medium in the 37°C incubator. • Prepare a thin smear with the deposit. Allow it to dry inside the hood and stain by Ziehl-Neelsen method. • Examine the slopes weekly for the appearance of growth. Continue incubating for 6-8 weeks before generating negative results. STAINING METHOD By Ziehl-Neelsen staining procedure (Refere pg 275), the acid fast bacilli are observed by performing the ZN staining given on pg 275.


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INTERPRETATION OF THE RESULTS The cultures are examined every week and observed for the growth of mycobacteria. If growth of Mycobacteria is seen, it is confirmed by performing acid fast staining and final identification is done by doing the required biochemical tests. BIBLIOGRAPHY 1. Manual of Laboratory Methods Bacteriology, 1987, P Venkataraman, C Alexander, Tuberculosis Research Center , Pg. 24 and 25.

FUNGAL CULTURE PURPOSE To isolate the fungus causing infection from clinical specimens. PRINCIPLE In the diagnosis of fungal infections, inoculating the specimen into the suitable media can isolate the pathogenic fungi. PERFORMANCE SPECIFICATION • A single negative culture does not rule out the presence of fungal infection. • The yield may be reduced by prolonged storage or on standing. Therefore a fresh specimen is preferred. • Culture reports have to be interpreted with caution based on the clinical findings and correlated with the clinical status of the patient if provided. PRIMARY SAMPLE • Corneal scraping and other ocular specimens • Nail clippings. CONSUMABLES • • • •

Sterile cotton swabs. Sabouraud’s dextrose agar. Blood agar. Brain heart infusion broth.

EQUIPMENT • Biological safety Cabinet Type II • Incubator 25 ºC.


324 PROCEDURE

• Collect the specimen in a sterile container. Inoculate the specimen in Sabouraud’s dextrose agar, Blood agar, BHIB. (If bacterial contamination is suspected then 50 μg/ml chloramphenicol can be used). • For intraocular specimen one drop of specimen is inoculated in the center of the plate, for scraping the collection is carried out by ophthalmologist and inoculated in the form of “C” streaks, for other specimens first primary streak are made from that secondary and tertiary inoculations made. • Incubate the SDA plates at 25ºC + 2 ºC, blood agar and BHIB at 37ºC. • Observe the plates for growth of fungi everyday. • If fungal growth is noted, perform lactophenol cotton blue preparation identify the fungi based on the colony morphology and microscopic morphology under light microscope. • Incubate plates with negative growth for 12 days if the direct smear report is negative, if the smear report is positive incubate the plates for one month, before discarding the culture as ‘no growth’. PRECAUTIONS UNIVERSAL • Observe precautions as for handling a biological fluid. • Process the specimen inside the biosafety hood only. INTERPRETATION OF RESULT • Growth Positive: Presence of the fungal colonies. • No Growth: Absence of fungal colonies. BIBLIOGRAPHY 1. LJR Milne. Fungi: In Mackie and McCartney’s Practical Medical Microbiology. 13th edition. Eds. Collee JG, Dugid JP, Eraser AG, Marmion. 1989; 675-700.

BACTERIAL AND FUNGAL CULTURE BLOOD CULTURE AND SENSITIVITY PURPOSE Blood culture method is used to identify if pathogenic bacteria are present in blood. If present, their nature and the drugs to which the organism is sensitive can be found.


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PRINCIPLE Pathogenic organisms present in blood grow well when provided with selective and enriched culture media and can be identified from the culture based on Gram’s staining procedure, morphology of the colony, biochemical reactions. PERFORMANCE SPECIFICATION Culture reports are to be interpreted with caution, based on the clinical findings and correlated with the clinical status of the patient. Culture results may be negative if the patient is already under medications for the disease. PRIMARY SAMPLE BLOOD • Wear disposable gloves and sterile disposable syringes and needles while collecting blood. • Clean the skin over the site with surgical spirit before collection. • Collect 10 mL of venous blood following aseptic precautions. • The blood should not be allowed to clot in culture media, because bacteria will get trapped in the clot. • The specimen must be inoculated immediately after collection. • Insert the needle through the rubber liner of the bottle cap after disinfecting with spirit; dispense 5 mL of blood into bottle containing 50 mL of brain heart infusion broth (BHIB) and thioglycollate broth respectively. • Incubate the bottle at 37oC for up to 2 days. In case of neonates, inoculate 2.5 mL of blood onto each bottle. EQUIPMENT • Autoclave • Biosafety hood (Class II) • Incubator 37ºC. CONSUMABLES • Dehydrated media (Brain heat infusion and thioglycollate medium) • Syringe and needle PROCEDURE • Incubate at 37°C for overnight. Look for growth. If no growth is seen incubate the bottle further. • If no growth is observed at the end of 48 hours, subculture onto BA, Mac Conkey from BHIB and on to BBA and BA from thioglycollate and make smear and Gram’s staining from both culture media to look for bacterial growth. The blood culture bottles to be subcultured at the end


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of the 7th and 14th day to look for any growth. If growth of bacteria are not seen incubate the culture for maximum of 21 days. • If growth is seen, perform Gram’s staining and subculture as before. Inoculate the colonies in Peptone water, incubate for 4 hours at 37oC. Inoculate onto biochemicals to identify the bacteria, and on MHA by lawn culture method and place antibiotic discs. Measure the sensitivity zones. • To confirm Salmonella, use “Specific high titer sera” S. typhi, S. paratyphi A, S.paratyphi B by slide aggulation test. • In absence of growth, at the end of 21 days of incubation, subcultures to look for growth and the final report is given. DATA RECORDING AND VERIFICATION • Correlate the type of organisms with nature of specimen isolated • Compare the results of culture with Gram’s staining of the growth. SAFETY PRECAUTIONS • Observe the usual precautions as for handling any biological fluid. • Inoculate the specimen under laminar airflow chamber only. • Process the specimen immediately. BIBLIOGRAPHY 1. Diane M, Citron BS, Martha AC, et al. Microorganisms encountered in the blood. In. Bailey & Scott’s Diagnostic Microbiology. Eds. Ellen Jo Baron, Lance R Peterson, Sydney M. Finegold, 11th edition, Mosby-Year book Inc, 2002, 193-209.

PROCESSING OF CEREBROSPINAL FLUID FOR BACTERIAL AND FUNGAL CULTURE PURPOSE This test is used for the identification of pathogens present in the CSF, their nature and the drugs to which they are sensitive. By knowing the pattern of drug sensitivity, the patient can be treated. PRINCIPLE Pathogenic organisms present in the CSF specimen will grow well by providing enriched culture media and can be identified based on the colony morphology, biochemical reactions and appropriate staining procedure.


327

PERFORMANCE SPECIFICATION • Always perform a Gram stain on the CSF specimen prior to culturing to provide presumptive information and to help interpret culture results. Culture may be negative for growth if the patient is already on medication. • CSF should be stored only at 37°C if there is a time delay of more than one hour between the collection and processing.

PRIMARY SAMPLE Use CSF as specimen for the test. Instruct nursing staff and physicians to collect cerebrospinal fluid in 2 sterile test tubes. EQUIPMENT • Autoclave • Incubator 37ºC, 37ºC CO2 incubator, anaerobic work station; (37°C) incubator maintained at 25°C • Biosafety hood (Class II) • Cytospin machine • Light microscope CONSUMABLES • Dehydrated media (Blood agar - BA, Brucella blood agar - BBA, Chocolate agar - CA, MacConkey agar - MA, Sabouraud’s Dextrose agar, Mueller Hinton agar – MHA, Blood Mueller Hinton agar – BMHA Peptone water – PW) • Liquid media (Brain heart infusion broth and thioglycollate medium) • Cytospin chambers • Streaking accessories • Microslide and coverslip • Syringe and needles • Sterile cotton swab PROCEDURE • Note the macroscopic appearance including volume, turbidity, presence or absence of RBCS, presence or absence of cobweb appearance. With sterile precaution transfer the whole specimen to a sterile centrifuge tube and centrifuge at 5000 rpm for 5 minutes. Transfer the supernatant leaving a deposit of around 500 μL and use the deposit for further processing.

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• The required number of cytospinned smear is made from the deposit. The cytospinned smear is made by centrifuging at 1000 rpm for 5 min. • Inoculate the specimen in BA, CA, MAC, BBA, SDA. Make first primary inoculum with the centrifuged deposit of the CSF and make secondary and tertiary inoculation from the primary inoculum without intermediate heating. Inoculate NNA agar for a acanthamoeba. • Incubate the BA and MacConkey plates, BHIB, Thioglycollate on 37 oC incubator, BBA in anaerobic incubator, CA at CO2 incubator and SDA at 25º C. • Stain the fixed smear by Gram’s staining procedure (Do Ziehl-Neelsen smear if tuberculous meningitis is suspected) observe under microscope and report. Perform Nigrosin stain if cryptococcal infection is suspected. • Observe KOH/ Calcofluor preparation for fungus and Acanthamoeba under fluorescence microscope using violet filter and report. • Observe for bacterial/fungal colonies grown after overnight incubation. • Perform Gram’s staining with the growth. • Inoculate the organism grown in culture plate onto peptone water. Incubate for 4 hrs. • Inoculate the broth culture on MHA by lawn culture method. • For Streptococci and Haemophilus species the identification and antibiogram tests are carried out directly from culture plate on Blood Mueller Hinton agar and Chocolate agar respectively. • Place required antibiotic discs over it. Inoculate in respective biochemicals depending on the organism for identification. • Incubate at 37oC for 24 hrs and measure the sensitivity zones. • Identify the bacteria based on smear, colony morphology and biochemical reactions. • For anaerobic culture, incubate under anaerobic conditions for 12 days. • If growth of yeast is observed identify the yeast based on colony morphology and biochemical reactions, if there is no growth incubate for 12 days. SAFETY PRECAUTIONS • Observe all precautions as for handling any biological fluid. • Wear facemask and disposable gloves while transferring and handling the specimen. POTENTIAL SOURCES OF VARIABILITY • Process specimens before they dry up • Occasionally Gram positive organisms lose their ability to retain the crystal violet and appear Gram negative due to over decolorization or too old Gram’s Iodine or too old cultures or due to over heat fixation.


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BIBLIOGRAPHY 1. Diane M, Citron BS, Martha AC, et al. Microorganisms encountered in the cerebrospinal fluid. In Bailey & Scott’s Diagnostic Microbiology. Eds. Ellen Jo Baron, Lance R Peterson, Sydney M Fine Gold, 11th edition, 2002, Mosby-Year book Inc, Pg. 210-18.

BACTERIAL CULTURE—URINE PURPOSE Urine culture is used for the identification of pathogen causing urinary tract infections, their nature and the drugs to which they are sensitive. By knowing the pattern of drug sensitivity, the patient is treated. PRINCIPLE Pathogenic organisms present in the urinary tract will grow well by providing enriched culture media and can be identified based on appropriate staining procedure, the colony morphology and biochemical reactions. PERFORMANCE SPECIFICATION • Specimen has to be inoculated as early as possible to have better isolation of pathogenic bacteria. • Culture may be negative for growth if the patient is already on medication. PRIMARY SAMPLE Urine—The patient is asked to collect the midstream urine in the given sterile wide mouthed plastic disposable container following aseptic precautions (after cleaning the external genitalia with water). EQUIPMENT • • • •

Autoclave Incubator 37ºC Biosafety cabinet (Class II) Microscopes

CONSUMABLES • Dehydrated media (Blood agar - BA, MacConkey agar - MA, Mueller Hinton agar –MHA, Blood Mueller Hinton agar - BMH, Peptone water – PW)


330 • • • •

Streaking accessories Microslide and coverslip Centrifuge tube Sterile cotton swab

PROCEDURE • One loopful (0.01 mL/10 μL) of uncentrifuged mixed urine sample is taken using sterile nichrome wire loop or disposable calibrated sterile plastic loop (4 mm in diameter) inoculated on to blood agar and MacConkey agar plates. • Primary, secondary and tertiary streaks are made with the same loop with out intermittent heating in order to make the colony count. It is incubated at 37°C overnight. • 7.8 mL of sample is centrifuged at 3,000 rpm for 10 minutes and deposit is observed for pus cells and motile bacteria under bright field microscope by net count and for pus cells and bacteria by Gram stain. • After overnight incubation, count the number of colonies grown on the media. • Perform Gram staining with the grown colonies. • Inoculate the organism grown in culture plate onto peptone water. Incubate for 4 hrs. • Inoculate the broth culture on MHA by lawn culture method. • For Streptococci and Haemophilus species the identification and antibiogram tests are carried out directly from culture plate on Blood Mueller Hinton agar and chocolate agar respectively. • Place required antibiotic discs over it. Inoculate in respective biochemicals depending on the organism for identification. • Incubate at 37oC for 24 hrs and measure the sensitivity zones. • Identify the bacteria based on smear, colony morphology and biochemical reactions. INTERPRETATION OF RESULTS If the colonies can be enumerated, they are counted and multiplied by 100 to get CFU/mL of urine. According to the number of colonies counted it is given as significant i.e. >105 or insignificant i.e < 105 colonies forming units/mL (CFU/mL). Colony count of > 105 CFU/mL is reported as significant and colony count of > 102- < 105 considered probably significant and colony count <102 CFU/mL is considered insignificant. SAFETY PRECAUTIONS Observe all precautions as for handling any biological fluid. Wear disposable gloves while transferring and handling the specimen.


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POTENTIAL SOURCES OF VARIABILITY Process specimens as early as possible as organism in urine may overgrow. Occasionally Gram positive organisms lose their ability to retain the crystal violet and stain Gram negative due to over decolorization or Gram’s Iodine or old cultures or due to overheat fixation. POTENTIAL SOURCES OF VARIABILITY Laboratory strategy in the diagnosis of infective syndromes. BIBLIOGRAPHY 1. In Mackie & McCartney. Practical Medical Microbiology, 14th edition Edited by Collee, Dugid, Fraser, Marmion. 1990; 600-50.

BACTERIAL AND FUNGAL CULTURE— THROAT SWAB PURPOSE This test is used for the identification of pathogens present in the throat swab, their nature and the drugs to which they are sensitive. By knowing the pattern of drug sensitivity, the patient can be treated. PRINCIPLE Pathogenic organisms present in the throat swab specimen will grow well by providing selective culture media and can be identified based on the colony morphology, biochemical reactions and appropriate staining procedure. PERFORMANCE SPECIFICATION Culture reports have to be interpreted with caution based on the clinical findings and correlated with the clinical status of the patients. Culture may be negative for growth if the patient is already taking medication for the disease. PRIMARY SAMPLE • Use sterile tongue depressor to lower the tongue. Have enough light to view the tonsils by using a torch light. Collect the specimen using a dry, sterile cotton swab by swabbing both the tonsils, peri-tonsillar fossae and oropharynx. Avoid contact of the swab with other areas of the oropharynx, mouth, cheek lips and saliva.


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Table 4.1: List of antibiotics to be used for antibiotic susceptibility testing for bacterial isolates based on clinical specimens Gram Negative Bacteria

Gram Positive Bacteria

Intraocular specimens: Vitreous aspirate, Aqueous aspirate, IOL, Eviscerated material, Iris tissue, Blood culture, Ocular pus Gentamicin Cefotaxime Ceftazidime Ciprofloxacin Amikacin Vancomycin

Gentamicin Cefotaxime Ceftazidime Ciprofloxacin Clindamycin Vancomycin Penicillin

External ocular specimens: Conjunctival swab, corneal scraping, corneal button, conjunctival scraping, donor Corneoscleral Rim, contact lens, infected suture Cefazolin Ciprofloxacin Moxifloxacin Norfloxacin Gentamicin Tobramycin Ofloxacin Gatifloxacin

Cefazolin Ciprofloxacin Moxifloxacin Norfloxacin Gentamicin Tobramycin Ofloxacin Gatifloxacin Penicillin, Vancomycin

Exudates including pus, CSF, Sputum and other aspirate fluid Cefotaxime Chloramphenicol Co-trimoxazole Ciprofloxacin Erythromycin Tetracycline Norfloxacin Gentamicin

Cefotaxime Chloramphenicol Co-trimoxazole Ciprofloxacin Erythromycin Tetracycline Norfloxacin Gentamicin Vancomycin Penicillin

Urine Ciprofloxacin Co-trimoxazole Tetracycline Chloramphenicol Gentamicin Norfloxacin Amoxycillin Piperacillin Amikacin

Ciprofloxacin Co-trimoxazole Tetracycline Chloramphenicol Gentamicin Norfloxacin Amoxycillin Piperacillin Amikacin Vancomycin Penicillin


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• Collect two swabs one for making a smear and other for culture. • Process the specimen immediately as otherwise the swab will get dried EQUIPMENT • Autoclave • Incubator 37ºC, 37ºC CO2 incubator, anaerobic work station; incubator 25°C • Biosafety hood (Class II) • Microscopes • Consumables • Dehydrated media (Blood agar - BA, Brucella blood agar - BBA, Chocolate agar –CA, MacConkey agar - MA, Sabouraud’s Dextrose agar, Mueller Hinton agar –MHA, Blood Mueller Hinton agar – BMHA, Peptone water – PW) • Streaking accessories • Microslide and coverslip • Sterile cotton swab PROCEDURE • Inoculate the specimen on to blood agar and MacConkey agar, Brucella blood agar, chocolate agar and on to SDA if fungal culture is recommended. Make first primary inoculum with the swab onto plates and make secondary and tertiary inoculation from the primary inoculum. • Incubate the blood agar, MacConkey agar at 37ºC incubator and chocolate agar plate at CO2 incubator, Brucella blood agar plate in anaerobic work station. • The smear of the specimen is made on glass slide. Leave the smear for drying and fix with methanol for 5 minutes. • Prepare a Gram stain, observe under light microscope and record your result. • Observe KOH/Calcofluor preparation under fluorescence microscope using violet filter for fungus, and record your result. • Observe for bacterial/fungal colonies grown after overnight incubation. • Perform Gram’s staining with the growth. • Inoculate the organism grown in culture plate onto peptone water. Incubate for 4 hrs. • Inoculate the broth culture on MHA by lawn culture method. • For Streptococci and Haemophilus species the identification and antibiogram tests are carried out directly from culture plate on Blood Mueller Hinton agar and Chocolate agar respectively.


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• Place required antibiotic discs over it and inoculate in respective biochemicals depending on the organism. • Incubate at 37oC for 24 hrs and measure the sensitivity zones. • Identify the bacteria based on smear, colony morphology and biochemical reactions. • For anaerobic culture, incubate under anaerobic conditions for 12 days. • If growth of fungus or yeast is observed identify the fungi based on colony morphology and lactophenol cotton blue preparation with slide culture, if there is no growth incubate for 12 days. INTERPRETATION OF TEST RESULTS • Look for colonies suggestive of Streptococcus pyogenes, (beta hemolytic) S. pneumoniae, Haemophilus influenzae and C. diphtheriae • If few colonies of normal flora are grown, they are ignored. • If Candida species are grown in culture it is to be reported with species level identification. SAFETY PRECAUTIONS • Observe all precautions as for handling any biological fluid. • Inoculate the specimen inside Type II laminar airflow chamber only. BIBLIOGRAPHY 1. Medical Laboratory Manual for Tropical Countries, 4th Edition, Volume II, by Monica Cheesbrough, Pg. 108-111. 2. Textbook for Medical Laboratory Technology, Second Edition, Praful B Godkar, Darshan P. Godkar, Pg. 599, 605. 3. Laboratory strategy in the diagnosis of infective syndromes. In Mackie & McCartney Practical Medical Microbiology, 13th edition Edited by Collee, Dugid, Faser, Marmion. 1989; 600-50.

MAINTENANCE OF CELL CULTURES PURPOSE To maintain the continuous cell lines for the isolation of viruses and Chlamydia trachomatis from clinical samples. PRINCIPLE Cells from animal organs and tissues are established and maintained for the purpose of isolation of viruses and Chlamydia trachomatis from clinical samples. The cell cultures are maintained under the laboratory conditions.


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PERFORMANCE SPECIFICATION • Cell culture should be examined everyday under phase contrast microscope. • The process of trypsinization has to be carried out carefully as over trypsinization will destroy cells. CONSUMABLES • Dulbecco’s minimum essential medium supplemented with 10% fetal calf serum. • Trypsin-EDTA solution • Tissue culture flask • Sterile pipettes EQUIPMENT • Biological safety Cabinet Type II • Incubator 37ºC with 10% CO2. PROCEDURE • Pour off or pipette out the culture medium from the tissue culture flask and rinse the cell line with trypsin-EDTA solution. • Add sufficient amount of trypsin-EDTA solution to cover the cell layer. • Incubate the cells in contact with trypsin-EDTA solution at room temperature till the cells show sign of detachment from the bottle surface. • Remove the trypsin solution . • Add required volume of fresh growth medium and suspend the cells. • Distribute the cells onto tubes, shell vials and tissue culture flask according to the need. • Add fresh growth medium to the tissue culture flask. • Incubate at 37ºC CO2 incubator. BIBLIOGRAPHY 1. Clinical Virology Manual. Ed. Steva Spector, Richard L Honinka, Stephen A Young, 2000; 27-53. 2. Diagnostic Procedures for Viral, Rickettsial and Chlamydial infections 1989, 6th Edition, by Schmidt. Pg. 51-101.

ACANTHAMOEBA CULTURE PURPOSE To isolate acanthamoeba causing corneal ulcer from corneal specimens or from cerebrospinal fluid (CSF).


336 PRINCIPLE

In the diagnosis of corneal ulcer infections, the causative infective agent is isolated by inoculating the specimen into the suitable media as this would expose the microorganisms. PERFORMANCE SPECIFICATION The yield may be reduced by prolonged storage. Therefore, a fresh specimen is preferred. Culture reports have to be interpreted with caution based on the clinical findings and correlated with the clinical status of the patient, if provided. PRIMARY SAMPLE Corneal scraping, CSF. CONSUMABLES • • • •

Non-nutrient agar medium (NNA) Escherichia coli culture Petri dishes Kimura spatula/Sterile surgical blade.

EQUIPMENT • • • •

Biological safety Cabinet Type II Incubator 37º C Moist chamber Microscope

PROCEDURE • Corneal scraping is collected by the ophthalmologist using Kimura spatula/sterile surgical blade. • Observe the direct smears under fluorescence microscope using Calcofluor white stain. • The specimen is inoculated in the form of a ‘C’ curve on non-nutrient agar and in the laboratory, 100 micro liters of 24–48 hours old culture of Escherichia coli suspension is added over the inoculum and spread with help of a loop. • Incubate at 35ºC + 2 ºC for 10 days keep the inoculated NNA plate inside a moist chamber. • Observe the plates for presence of trophozoites cysts of acanthamoeba under bright field microscope using 10 × of the bright field microscope everyday upto 10 days.


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INTERPRETATION OF RESULT • Blush white Fluorescing double walled cyst of acanthamoeba measuring around 8-10 μm seen under fluorescence microscope in the direct smear shows positive result. • Growth: Growth of trophozoites cysts in non-nutrient agar seen under bright field microscope. • No Growth: Absence of cyst and trophozoite forms of Acanthamoeba after 10 days of incubation. BIBLIOGRAPHY 1. Donald J. Krogstad, Govinda S Visveswara, Kenneth W. Walls and James W Smith. Blood and tissue protozoa. In manual of clinical microbiology, 4th edition, editors, Edwin H.Lennette, Albert Balows, William J.Hadslaer, Jean Shandomy H. 1985, Pg. 612-31.

CHLAMYDIA TRACHOMATIS CULTURE BY RAPID SHELL VIAL TECHNIQUE PURPOSE This test is used for the isolation and identification of Chlamydia trachomatis present in the conjunctival swab, conjunctival scraping, eye discharges, endocervical, urethral swabs. Identification will help in the institution of the appropriate antibiotic therapy. PRINCIPLE Chlamydia trachomatis present in the clinical sample grows in McCoy cell culture pretreated with cycloheximide and growth is identified by performing Immunofluorescence staining. The isolation of Chlamydia trachomatis from clinical samples is done by rapid shell vial technique. PERFORMANCE SPECIFICATION Culture reports have to be interpreted with caution based on the clinical findings and correlated with the clinical status of the patient. Culture may be negative for growth if the patient is already taking medication for the disease. Process the specimen immediately, if not possible store it at - 80°C until further process.


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PRIMARY SAMPLE • Wipe the discharge from eye by using a dry sterile cotton swab. • Collect conjunctival swab specimen by swabbing the lower inferior tarsal conjunctival fornix avoiding the eyelid border and lashes with a sterile cotton swab. • Ocular Specimens: Conjunctival Scraping should be collected by ophthalmologist only, while conjunctival swab is collected by laboratory staff. Extreme care must be taken to handle the swab while collecting the swab from eye. • The specimen must be cultured as soon as possible since the natural secretions of the eye contain antibacterial enzymes (lyzozymes). • Process the specimen immediately; otherwise the swab will get dried. After collection swab is placed in 1 mL of transport (2 SP/ MEM with out antibiotics) medium and transporte to laboratory for inoculation. In the laboratory, the vial with swab is vortexed and swab is squeezed against wall of the container and discarded. EQUIPMENT • • • • •

Autoclave Incubator 37°C Laminar airflow chamber (Class II) Fluorescence and phase contrast microscope Centrifuge.

CONSUMABLES • Sterile Cotton Swab • Dulbecco’s minimum essential medium with 10% fetal serum (growth medium) • Dulbecco’s minimum essential medium with 10% fetal calf serum and 1 μg/mL concentration of cyclohexamide (maintenance medium). • Phosphate buffer saline-tween 20 (PBST) • Shell vial with coverslip (autoclaved) • Tissue paper • Rubber corks STEP BY STEP PROCEDURE • Grow McCoy cell culture over the coverslip in the shell vials by overnight incubation. • Pretreat the cells by adding 1.0 mL of Dulbecco’s medium with 10% fetal serum and 1μg/mL of cycloheximide after removing growth medium.


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• After 24 hours, use aseptic technique and aspirate the cell culture medium above the McCoy cell monolayer in the shell vial (made available by sub-culturing McCoy cells from the stock) and add 0.2 mL of the patient specimen collected in transport medium to the vial. • Centrifuge the vial for 1 hour at 3000 rpm at ambient temperature in centrifuge. • Add l mL fresh maintenance medium containing l mg/mL Cycloheximide and 10% fetal calf serum. • Recap the vials tightly, and incubate at 35°C–37°C for 48 to 72 hours. • Remove medium from the vials, fix the coverslip with methanol or cold acetone and stain with fluorescein-conjugated monoclonal antibody raised against Chlamydia trachomatis (Trinity). • Examine slides at 200 to 100 × magnifications for apple-green (fluorescent stain) intracytoplasmic inclusions and/or elementary bodies. • Interpretation of results (refer staining procedures). PRECAUTIONS • Observe all precautions as for handling any biological fluid. Wear facemask, apron and disposable gloves, while transferring and handling the specimen. • Inoculate the specimen under laminar airflow chamber only. BIBLIOGRAPHY 1. Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections. 6th Edition, by Schmidt. 1989; 51-101.

VIRUS ISOLATION BY CONVENTIONAL TUBE CULTURE METHOD PURPOSE This test is used for the isolation and identification of viruses from clinical specimens. PRINCIPLE Virus replication in susceptible cell culture produces the degenerative morphological changes referred to as cytopathic effect, which may appear as a rapid extensive rounding of the cells or as a slowly progressive change involving only discrete foci. Certain viruses produce such characteristic


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CPE that a presumptive identification can be made on this basis. Preliminary identification of specific viruses is usually dependent upon the type of CPE, the length of time required for CPE to appear. The isolation of viruses from clinical samples is done by conventional tube culture method. PERFORMANCE SPECIFICATION Non-specific changes of cell cultures may be mistaken for true CPE, therefore, reading should be done only by trained personnel. Process the specimen immediately, if not possible, store it at - 80 °C until further process. PRIMARY SAMPLE Conjunctival scraping/Swab, Corneal scraping Nasopharyngeal aspirate. TYPE OF CONTAINER USED Collect in a sterile, leak proof container, containing viral transport medium with 3% fetal calf serum and antibiotics. REAGENT AND CONSUMABLES • • • • •

Viral transport medium (MEM). Biosafety hood type II Autoclave 37°C Incubator Rocker.

PROCEDURE • Select tube of appropriate cell culture, with confluent or nearly confluent monolayer and remove growth medium. • Inoculate cells with 0.2 mL of the specimen. • Keep for rocking at room temperature for 30 minutes. • Re-feed tube with 1-2 mL of maintenance medium. • Incubate tube at 35-37°C (Medium should cover the monolayer, but should not touch the tube’s cap or neck). • Observe the inoculated culture everyday, for the cytopathic effect(s) of viral replication under phase contrast microscope. • If any CPE is observed, harvest the cells using rubber policemen and stain by indirect immunofluorescence method using appropriate antiserum. • In absence of CPE, incubate tubes for 10 days and harvest the cells on the 11th day and perform immunofluorescence staining for reporting.


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INTERPRETATION • The negative cell contrast should show no CPE and should appear red. • Virus control should show CPE and apple green color fluorescence. • Test sample is considered negative if it appears red and positive if it shows apple green fluorescence. SAFETY PRECAUTIONS • Wear the facemask, apron and disposable gloves while transferring and handling the specimen. • Inoculate the specimen inside bio safety hood only. BIBLIOGRAPHY 1. Clinical Virology Manual. Ed, Steva Spector, Richard L Honinka, StephenA. Young, 2000, 27-53. 2. Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections. 6th Edition, by Schmidt, 1989; 51-101.

Part III: Immunology RHEUMATOID ARTHRITIS TEST PURPOSE Quantitative estimation of Rheumatoid Arthritis (RA) factor in human serum by latex agglutination method. Estimation of RA is useful mainly in the diagnosis, monitoring and follow-up of Rheumatoid factor (RF)-positive Rheumatoid Arthritis. However, it is also detectable sometimes in the serum of patients with Systemic Lupus Erythematosus (SLE) and in certain non rheumatic conditions. PRINCIPLE RF is an IgM class of antibody directed against the antigenic sites in the Fc portion of the IgG molecule. When human serum containing RF is mixed with Inert Polystyrene Latex particles coated with specific human gamma globulin, visible agglutination occurs. PERFORMANCE SPECIFICATIONS Measurement range: This Latex Reagent kit has a measurement range of 10–3000 IU/mL of RF in serum. Sensitivity: The minimum detection limit is 10 IU/mL of Rheumatoid Factor

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Specificity: Increased levels of RF may be observed in some diseases other than Rheumatoid Arthritis such as infectious mononucleosis, sarcoidosis, lupus erythematosus, Sjogren’s syndrome. Certain patients with proven Rheumatoid Arthritis (RF Negative RA) may not have the RF present in their serum. PRIMARY SAMPLE • Use only serum as specimen for the test • Do not use plasma as specimen for the test • Collect 2 mL of venous blood in a plain red topped vacutainer tube/ plain 0.1 N HCl washed tube. Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500–3000 rpm for 5–10 minutes. ADDITIVE No additive/Preservative is needed. REAGENTS/ CONSUMABLES: AS PER PRODUCT INSERT • RF Latex Reagent: A suspension of uniform polystyrene particles coated with IgG (human) in glycine buffer, pH 8.2; • RF Positive Control containing RF of more than 10 IU/mL • RF Negative Control containing RF of less than 10 IU/mL • Glycine-Saline Buffer EQUIPMENT • Applicator Sticks • Serological Pipette/Micropipette of 5-50 μL capacity • Timer/Stop watch with an accuracy of 1 second. CALIBRATION PROCEDURE Perform a positive control for RF and a negative control for RF with every new reagent lot or whenever the results are equivocal. PROCEDURAL STEPS Qualitative Test • • • •

Bring reagents and specimens to room temperature before use. Shake the latex suspension well before use. Thoroughly clean and dry the test slide before use. Using the separate serological pipette place one drop (40 μL) of test samples in the test fields. • Add one drop of RF Latex Reagent to each field.


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• Use separate Applicator sticks/stir sticks to spread reaction mixture over entire area of the particular field. • Tilt the slide back and forth for 2 minutes in a rotary shaker so that the mixture rotates slowly. Observe for agglutination after 2 minutes under bright artificial light. Semi-quantitative test • Prepare serial dilutions of the positive sample using normal saline i.e., 1:2, 1:4, 1:8, 1:16, 1:32. • Add one drop (40 mL) of diluted serum to the circle of the test slide. • Add one drop of RF latex reagent. • Mix with separate sticks and spread the fluid over the entire area of the particular circle. • Tilt the slides back and forth for 2 minutes, so that the mixture rotates slowly inside the circle. • At the end of 2 minutes read results under bright light. RESULTS Read the titre in the last dilution step with visible agglutination and the approximate concentration of the rheumatoid factor can be determined as follows: RF in IU/mL= Sensitivity of latex Gammaglobulin reagent in IU/mL × Titre. QUALITY CONTROL PROCEDURE Run a positive and negative control with each batch of patient’s sample. Validity of results: The results are valid only if the positive control shows agglutination and the negative control - no agglutination. PRECAUTIONS • Ensure the reagents and specimens are at room temperature before use. • Store the kits at 2–8°C.

C-REACTIVE PROTEIN PURPOSE Qualitative detection and semi-quantitative estimation of C-Reactive Protein (CRP) in human serum samples. C-Reactive Protein (CRP), the classic acutephase of human serum, is synthesized by hepatocytes. Normally, it is present only in trace amounts in serum, but it can increase by as much as l000-fold in response to injury or infection. The level of CRP increases significantly after most forms of tissue injuries, bacterial and viral infections

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inflammation and malignant neoplasms. Hence the clinical measurement of CRP in serum, is a valuable screening test for organic disease and a sensitive index of disease activity in inflammatory, infective, and ischemic conditions. PRINCIPLE The C-Reactive-Protein test is based on the immunological reaction between human-C-Reactive Protein in a patient’s serum or control serum and the corresponding antihuman CRP antibodies bound to latex particles. When serum containing CRP is mixed with latex reagent, a distinctly visible agglutination of the latex particles in the test cell of the slide. PERFORMANCE SPECIFICATIONS Sensitivity: The minimum detection limit is 6 mg /liter of serum. PRIMARY SAMPLE • Use only serum as specimen for the test. Do not use plasma as specimen • Collect 2 mL of venous blood in a plain test tube or 0.1 N HCl washed tubes. • Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500–3000 rpm for 5–10 minutes. • Do not use lysed or contaminated serum for the test. • Do not use severely lipemic serum as it interferes with the state of latex particles. • Process the specimen on the same day within 2 hours of collection. • If analysis is not done on the same day of collection, separate the serum and store it at 2–8 °C for up to 3 days. ADDITIVE OR PRESERVATIVE No need to add any anticoagulant or preservative while collecting blood sample. CONSUMABLES AND REAGENTS • • • •

Test slides Test tubes Disposable stirrers CRP Latex reagent : A suspension of uniform polystyrene particles coated with monospecific antihuman CRP (goat) in buffer • CRP Positive control • CRP Negative control


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Rotary shaker Timer Serological pipettes Test tubes Isotonic saline (0.9%)

STEP BY STEP PROCEDURE • Bring CRP latex reagent, controls and the specimen to room temperature before the test. • Using a disposable plastic dropper/serological pipette dispense one drop (50 μL) of CRP patient’s serum into the test circle. • Shake the latex reagent and by using a disposable plastic dropper/serological pipette dispense one drop (50 μL) of suspension into the test circle. • Mix well with applicator sticks used in the previous step and spread mixture over the entire area of the particular cell and then tilt the slide back and forth for 2 minutes, so that the mixture rotates slowly inside the cells or gently rock the slide in a rotary shaker for two (2) minutes and read immediately under direct bright light. INTERPRETATION OF RESULTS Negative result: A negative reaction is indicated by no agglutination. Positive result: A positive reaction is indicated by any observable agglutination in the reaction mixture. SEMI-QUANTITATIVE TEST • Prepare serial dilutions of the positive sample using diluted normal saline, i.e., 1:2, 1:4, 1:8 and 1:16. • Add 50 μL of each dilution of the test sample to individual wells and spread the diluted test serum over the entire area of the test circle using separate applicator sticks. • Gently mix CRP latex suspension for uniform resuspension of the latex particles and add one drop (50 μL) to test field. • Mix well with separate applicator sticks used in the previous step and spread mixture over the entire area of the particular cell and then tilt the slide back and forth for 2 minutes, so that the mixture rotates slowly inside the cells or gently rock the slide in a rotary shaker for two minutes and read immediately under direct bright light.

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QUALITY CONTROL PROCEDURE Include positive and negative controls with each test batch. Acceptable performance is indicated when a uniform milky suspension with no agglutination is observed with the CRP negative control and agglutination in the form of large aggregates is observed with the CRP positive control INTERPRETATION OF RESULTS A positive reaction is indicated by any observable agglutination in the reaction mixture. Record the last dilution showing a positive reaction. Most normal healthy humans have CRP concentrations of less than 6 mg/liter. In patients with high serum concentrations continued monitoring of the CRP levels can give a good indication of patient response to therapy during inflammatory disorders. SAFETY PRECAUTIONS • Handle all samples as potentially infectious. • Handle all reagents with care and avoid contact with eye, mouth and skin. • Do not perform mouth pipetting. • Discard used reagents and sample as per disposal procedure. POTENTIAL SOURCES OF VARIABILITY • CRP latex reagent should be shaken well before use as otherwise erroneous results will be obtained. • Ensure the CRP latex reagent is uniform without visible clumping. When stored refrigerated, a slight sedimentation may occur and should be considered normal. • Do not use the latex reagent or controls if they become contaminated. • Ensure that the sample is free of fibrin/particles by centrifuging. • Reaction time is critical. If reaction time exceeds 3 minutes, drying of the reaction mixture may cause false positive results. • Freezing the CRP latex reagent will result in spontaneous agglutination. • Intensity of agglutination is not necessarily indicative of relative CRP concentration; therefore, screening reactions should not be graded. • A false negative can also be due to prozone phenomena (antigen excess). Therefore, it may be necessary to check even negative sera by retesting at a 1:10 dilution with glycine buffer.


347 SERUM ANTISTREPTOLYSIN ‘O’ (ASO) TITER PURPOSE Qualitative detection and semi-quantitative estimation of antibodies to Streptococcal exoenzyme Streptolysin ‘O’ in human serum. ‘Group A,’ betahemolytic Streptococci produce a number of exotoxins which can act as antigens. One of these exotoxins , Streptolysin ‘O’, causes the production of specific antibodies in infected subjects. This test is useful to establish the presence of both past and present infection due to the Group A betahemolytic streptococci and also the degree of infection. Raised levels of ASO are found in many diseases including acute rheumatic fever, scarlet fever, acute glomerulonephritis and tonsillitis. PRINCIPLE This test is based on an immunological reaction between streptococcal exoenzymes (Anti-Streptolysin ‘O’) bound to biologically inert latex particles and anti-streptolysin ‘O’ antibodies present in the test sample. If Antistreptolysin ‘O’antibodies are present in the serum in a titer of more than 200 IU/mL a visible agglutination of the latex particles occurs without sample dilution. PERFORMANCE SPECIFICATIONS Sensitivity: The minimum detection limit by this ASO latex reagent is 200 IU /mL in serum. Specificity: Following acute streptococcal infection, the ASO titer will usually rise after one week, increasing to a maximum level within 3 to 5 weeks and usually returning to the pre-infection levels in approximately 6 to 12 months. Hence the ASO titer test may be negative in the early stage of streptococcal infection. PRIMARY SAMPLE • Use serum as specimen for the test. Do not use plasma. • Collect 2 mL of venous blood in a plain red topped vacutainer tube or 0.1 N HCl washed tubes. • Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500–3000 rpm for 5–10 minutes. • Do not use lysed or contaminated serum. • Process the specimen on the same day. • If analysis is not done on the same day of collection, separate the serum and store it at 2–8 °C for up to 3 days.


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ADDITIVE OR PRESERVATIVE No need to add any anticoagulant or preservative while collecting blood sample. Collect venous blood in a plain red topped vacutainer tube or acid washed (0.1 N HCl) tube. EQUIPMENT • • • •

Rotary shaker Timer Serological pipettes Glass test tubes

CONSUMABLES AND REAGENTS • • • •

Disposable test slides Disposable stirrers ASO latex reagent ASO positive control containing at least 200 IU/mL of ASO reactive with the test reagent • ASO negative control containing less than 200 IU/mL of ASO reactive with the test reagent PROCEDURE: QUALITATIVE TEST • Bring reagents and specimens to room temperature before use. • Place one drop (50 μL) of ASO Positive Control on field 1 of the reaction slide and one drop (50 μL) of the ASO Negative Control on field 2 of the reaction slide using serological pipette • Deliver 1 drop (50 μL) of undiluted test serum sample to field 3. • Gently mix ASO latex suspension for uniform resuspension of the latex particles and add one drop to each test field. • Mix well with the applicator stick. Gently rock the slide to and fro or in a rotary shaker for two minutes and read immediately under direct light. INTERPRETATION OF RESULTS • Negative reaction: Uniform milky suspension with no agglutination as observed with the ASO Negative Control. • Positive reaction: Any observable agglutination in the reaction mixture. A positive reaction indicates that the concentration of ASO in the specimen is equal or greater than 200 IU/mL. • Perform a semi-quantitative estimation of ASO titer if the qualitative test is positive.


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SEMI-QUANTITATIVE TEST Set up five test tubes: 1:2, 1:4, 1:8, 1:16, 1:32 and dilute samples. • Place one drop of each dilution on successive fields of the reaction slides. • Gently resuspend the ASO latex reagent and add one drop to each test field. • Mix well with the applicator stick. Gently rock the slide to and fro for two (2) minutes and read immediately under direct light. INTERPRETATION OF RESULTS A positive reaction is indicated by any observable agglutination in the reaction mixture within 2 minutes. Record the last dilution showing a positive reaction. Concentration of ASO can be determined by multiplying the last positive dilution factor of the sample with the concentration of the positive control (200 IU/mL). The titer of the serum is the reciprocal of the highest dilution, which exhibits a positive reaction. IU/mL of sample = Conc. of positive control × reciprocal Dilution

Reciprocal

IU/mL

1/2 1/4 1/ 8

2 4 8

400 800 1600

QUALITY CONTROL PROCEDURE Includes positive and negative controls. Acceptable performance is indicated when a uniform milky suspension with no agglutination is observed with the ASO Negative Control and agglutination with large aggregates is observed with the ASO Positive Control. SAFETY PRECAUTIONS • Handle all samples as potentially infectious. • Handle all reagents with care and avoid contact with eye, mouth and skin. • Do not perform mouth pipetting. • Discard used reagents and sample as per disposal procedure. POTENTIAL SOURCES OF VARIABILITY • Read results exactly two minutes after the mixing of the reagent with the specimen or control on the slide. A reading obtained after this period of time may be incorrect.

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• Ensure the ASO latex reagent is uniform without visible clumping. When stored refrigerated, a slight sedimentation may occur and should be considered normal. • Do not use the latex reagent or controls if they become contaminated.

SERUM RAPID PLASMA REAGIN (RPR) TEST PURPOSE It is a qualitative detection and semi-quantitative estimation of Reagin antibodies in human serum or plasma by slide flocculation method. This test is used in the diagnosis of syphilis. PRINCIPLE RPR is a non-treponemal rapid plasma flocculation test for detecting and estimating reagin antibodies in persons with syphilis. The antigen used is a modified VDRL (Cardiolipin) antigen, in which micro particulate charcoal particles are used to enhance the visual difference between positive and negative results. If the specimen contains reagin, flocculation occurs with a coagglutination of carbon particles contained in the antigen suspension, which appears as black clumps. Non-reactive specimens appear as an even light gray homogeneous suspension. The use of carbon particles obviates the requirement for microscope when reading the test results. PERFORMANCE SPECIFICATIONS Specificity and sensitivity: False positive reactions may occur occasionally with the RPR test. Such reactions may occur in narcotic addiction and in diseases such as systemic lupus erythematosus, infectious mononucleosis, leprosy, viral pneumonia, auto-immune diseases and even in pregnancy. Reactive RPR test specimens should be subjected to further confirmation testing, as the diagnosis of syphilis cannot be made on a single reactive result. PRIMARY SAMPLE • Use serum or K2 EDTA plasma as specimen for the test. • If serum is used, collect 2 mL of venous blood in a plain red topped vacutainer tube or 0.1 N HCl washed tubes. • If plasma is used collect in Lavender topped vacutainer tubes containing 5.4 mg of K2EDTA/collect in tubes containing 2 mg of K2EDTA per mL of blood (20 μL of 10 % EDTA per mL of blood).


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• Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500- 3000 rpm for 5-10 minutes. • Do not use lysed or grossly lipemic contaminated serum. • Do not use severly lipemic serum as it interferes with the state of carbon particles. • Process the specimen on the same day. • If analysis is not done on the same day of collection, separate the serum and store it at 2–8 °C for up to 2 days. EQUIPMENT • • • •

Rotary shaker Timer Serological pipettes Test tubes

CONSUMABLES • • • • •

Disposable test slides Disposable stirrers RPR reactive control RPR non reactive control RPR carbon antigen suspension antigen.

STEP BY STEP PROCEDURE • Bring the RPR carbon antigen suspension and controls to room temperature before the test • Prior to opening the bottles of RPR antigen suspension, shake the bottle for 10–15 seconds to resuspend the antigen. • Test reactive RPR control and non-reactive RPR control with each batch of patient samples. QUALITATIVE PROCEDURE • Using a disposable plastic dropper, dispense one drop (50 μL) of serum or plasma onto the circle on the test card. • Spread the sample over the entire area of the test circle using an applicator stick. • Using the reagent dispensing dropper, allow one free falling drop of antigen to drop onto the test specimen. Do not mix the sample and RPR antigen. • Place test card on an automatic rotator and rotate for 8 minutes at 100 rpm. • Immediately after the 8th minute, inspect the results with naked eye in bright light. • Test RPR reactive control and RPR non-reactive control like that of test serum.

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RESULTS If a specimen contains reagin antibodies, flocculation occurs with a plasma coagglutination of carbon particles contained in the antigen suspension, which appears as black clumps. Non-reactive specimens appear as a light gray homogenous suspension. Confirm all reactive specimens by semi quantitative test. • Reactive—Medium and large aggregates. • Non reactive—No visible aggregates or finely dispersed aggregates which are smooth and light gray appearance. Validity of test results: • The test results are valid only if the RPR reactive control shows aggregates and RPR non-reactive control shows no visible aggregates SEMI-QUANTITATIVE TEST • For each specimen to be tested, add 100 μL of 0.9% saline into test tubes numbered 1 to 5. • Add 100 μL of specimen onto test tube 1. • Mix the mixture. Avoid formation of bubbles. • Transfer 100 μL of mixed sample from tube 1 to 2. • Repeat this serial dilution procedure in tube 3 to 4, and then 5. Dispose 100 μL from test tube 5 after mixing. Tubes 1 to 5 now represent a dilution series as follows: Circle 1 2 3 4 5 Dilution 1/2 1/4 1/8 1/16 1/32 • Add 50 μL of the 1:2 diluted specimen to circle 1, 1:4 to circle 2, 1:8 to circle 3, 1:10 to circle 4 and 1:32 to circle 5 of the test cord. Spread that diluted serum in each circle. • Shake the carbon antigen suspension dispensing bottle prior to use. • Place one (1) drop of “free falling” antigen suspension onto each test circle containing the sample. • DO NOT MIX the sample and the antigen. • Place test card on an automatic rotator and rotate for 8 minutes at 100 rpm. • Read results macroscopically under a high intensity incandescent lamp or strong daylight. • The titer of the sample is the reciprocal of last dilution that contains macroscopic aggregates. QUALITY CONTROL PROCEDURE RPR reactive control and RPR non-reactive control. Include the positive and negative controls in each batch of testing.


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SAFETY PRECAUTIONS • • • •

Handle all samples as potentially infections. Handle all reagents with care and avoid contact with eye, mouth and skin. Do not perform mouth pipetting. Discard used reagents and sample as per disposal procedure.

POTENTIAL SOURCES OF VARIABILITY • RPR reagent suspension should be shaken well before use as otherwise erroneous results will be obtained. • The test slide should be clean and dry. • Plasma specimens over 48 hours old may give erroneous results. • Temperature of the reagents and specimens is critical to the test outcome. • Ensure the reagents and specimens are at room temperature before use. Store the kit at 2–8°C. • Reactive RPR test specimens should be subjected to further confirmation testing by specific Treponemal tests as the diagnosis of syphilis cannot be made on a single reactive result.

TREPONEMA PALLIDUM HEMAGGLUTINATION (TPHA) PURPOSE Qualitative detection of specific antibodies to Treponema pallidum in human serum by hemagglutination test. This test is used in the diagnosis of syphilis PRINCIPLE Stabilized fowl/chicken erythrocytes are sensitized with an antigenic extract of Treponema pallidum (Nichols strain). These sensitized erythrocytes will agglutinate with T. pallidum antibodies if present in the patient’s serum. The cells form a characteristic pattern of cells in the bottom of a micro titration plate well. In the absence of antibody they form a compact button in the well. Non-specific reactions are detected by the unsensitized control reagent. Non-pathogenic Treponemal antibodies are absorbed by an extract of Reiter’s treponemes included in the diluent solution. PERFORMANCE SPECIFICATIONS Cross reactions can occur with other species of Treponema. This test cannot discriminate between antibody due to T. pallidum infection and antibody

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due to infection with other pathogenic treponemes, i.e, T. pertenue and T. carateum False positive reactions can occur with samples from patients with infectious mononucleosis, leprosy, autoimmune diseases and drug addiction. Occasionally, in early primary syphilis, the specific antibodies could not be detected by the TPHA technique. Use of samples other than serum or CSF have not been validated in this test. False positive reactions may occasionally occur if plasma is used as specimen. PRIMARY SAMPLE • Use only serum as specimen for the test. Do not use plasma as specimen. • Collect 2 mL of venous blood in a plain test tube or 0.1 N HCl washed tubes. • Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500 to 3000 rpm for 5–10 minutes. • Do not use lysed or grossly lipemic or contaminated serum. • Process the specimen on the same day. • If analysis is not done on the same day of collection, separate the serum and store it at 2–8 °C for up to 5 days or at –20°C for longer periods. ADDITIVE OR PRESERVATIVE No need to add any anticoagulant or preservative while collecting blood sample. Collect venous blood in a plain sterile tube. CONSUMABLES AND REAGENTS Antigen reagent suspension: Suspension of sensitized fowl/chicken erythrocytes. Control reagent suspension: Suspension of unsensitized chicken erythrocytes. Diluent solution: Phosphate buffered saline solution containing soluble components of Reiter Treponemal strain and stabilizing agents. Positive control serum: Immune rabbit serum. Prediluted to 1:20. See exact titer on the vial label. A variation of titer of ± one doubling dilution is accepted. Negative control serum : Normal rabbit serum. Prediluted to 1:20. EQUIPMENT No special equipment required only micro pipettes/cell droppers and microtiter plates are required.


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STEP BY STEP PROCEDURE: QUALITATIVE DETECTION • Allow the reagents and specimen to reach room temperature before the test. • Ensure that antigen and control reagents suspensions are thoroughly resuspended before use. • Dispense diluent into the micro titration plates as follows: In well 1 - 25 μL (1 drop) In well 2 - 100 μL (4 drops) In well 3 & 4 - 25 μL (1 drop) • Dispense 25 μL (1 drop) of each sample into a well in row 1 • Using a micro pipette, mix well 1 and transfer 25 μL volume to well 2. Mix and transfer 25 μL to well 3. Mix and discard 25 μL from well 3. Transfer a 25 μL from well 2 to well 4, mix and discard 25 μL from well 4. • Add three drops (75 μL) of test cells to well 4 using the attached cell dropper and three drops (75 μL) of control cells to well 3 using the attached cell dropper. This results in final serum dilutions in well 3 and 4 to 1/80. • Mix the contents of the wells by tapping the sides of the plate or by using a plate shaker for at least 30 seconds. • Cover and let stand for 45–60 minutes. • Examine for agglutination patterns. INTERPRETATION OF RESULTS Agglutination pattern 4+ : Smooth mat of agglutinated cells covering the entire bottom of the well, sometimes with folded edges. 3+ : Smooth mat of agglutinated cells partially covering the bottom of the well. 2+ : Smooth mat of agglutinated cells surrounded by a ring of cells. 1+ : Smooth mat of agglutinated cells surrounded by a heavy ring of cells. ±: Button of cells with a small central opening. Negative: Button of cells with or without a very small hole in the center. POSITIVE AND NEGATIVE RESULTS • • • • •

4+ to 1+ - is considered as positive ± - is considered as Borderline No agglutination - is considered as a negative test result All reactive samples are tested further to get the end titer. Any agglutination seen in the control well (well 3) indicates the presence of non-specific agglutinins. (In this event this test is not valid and should be repeated with following technique).

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• Dilute the test serum ¼ with control cells (25 μL of test serum + 75 μL of control cells) and allow to stand at room temperature for 45–60 minutes. • Centrifuge the sample for 5 minutes at 1000 rpm (25 μL of 1:4 diluted serum + 100 μL of diluent) • After centrifugation dilute the supernatant 1/5 in diluent. Test this dilution directly. Add 25 μL of diluted treated serum to two cells. Add 75 μL of control wells to well 1 and 75 μL of test cells to well 2 Mix the contents of the wells and let stand for 45–60 mins. • A positive result indicates the presence of antibodies to T. pallidum, resulting from a past or present infection. All positive test samples should be tested with further dilutions of the sample to get end titer. • A negative result indicates the absence of antibodies to T. pallidum. • A borderline may correspond to a low level of antibodies in early stages of syphilis or to residual antibodies in treated syphilis. SEMI-QUANTITATIVE ESTIMATION • 25 μL of the diluent is added to 5 wells in a row. • 25 μL from the second well of the sample showing reactive result is added to 25 μL of diluent in the first well, mixed and transferred to the second well and like-wise proceeded till the fifth well. • 25 μL is discarded from the fifth well. • 75 μL of the test cells are added to all the five wells of the diluted sample • Mix the contents of the wells by tapping the sides of the plate or by using a plate shaker for at least 30 seconds. • Cover and let stand for 45-60 minutes • Examine for agglutination patterns. • The titre of the sample is the reciprocal of the last dilution that shows agglutination. QUALITY CONTROL PROCEDURE Include the positive and negative controls in each series of samples taking into account that the controls are prediluted to 1:20. Add 25 μL of each control directly into wells 3 and 4. Do not add any diluent solution. SAFETY PRECAUTIONS • Handle all samples as potentially infectious. • Handle all reagents with care and avoid contact with eye, mouth and skin. • Do not perform mouth pipetteing. • Discard used reagents and sample as per disposal procedure.


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POTENTIAL SOURCES OF VARIABILITY • False positive reactions may occur if plasma is used as specimen • Specific antibodies may persist for a long period of time, even after successful treatment of the disease. In order to assess the response to treatment, the use of Reagin test (RPR test) is recommended • Any agglutination seen in the control well indicates the presence of non-specific agglutinins.

WIDAL TEST PURPOSE Quantitative estimation of antibodies to Salmonella antigens H and O Salmonella para A `H’ antigens and Salmonella para B `H’ antigen in human serum by tube agglutination test. This test is used in the diagnosis of enteric fever (Typhoid & Paratyphoid fever). Antibodies specific to flagellar antigen ‘H’ of S. typhi, S. para A, S. para B and Somatic Antigen ‘O’ of S.typhi usually become detectable in blood, 6 days after the onset of infection in individuals infected with typhoid or paratyphoid bacilli. PRINCIPLE When the patient serum is mixed with attenuated antigenic suspensions of `O’ antigen of S. typhi and H antigen of S. typhi, S. para A and S. para B if antibodies to ‘H’ and ‘O’ antigens are present in the patient’s sample clearly visible macroscopic agglutination takes place. Positive result is indicated by the presence of agglutination of anti-Salmonella antibodies in the patient’s sample. Absence of agglutination indicates a negative result. PERFORMANCE SPECIFICATIONS Sensitivity and specificity: False positive results may occur in normal healthy individuals who posses low titres of antibodies that react with Salmonella antigens H and O. The test may be positive in individuals who have received typhoid vaccination or in individuals who have had previous S.typhi infection. A negative result cannot rule out typhoid fever if the sample had been taken very early in the course of the illness (earlier than 7 days from the onset of infection). False positive results may also occur in auto immune diseases and rarely in non specific febrile illness especially with respect to H titers and is, therefore, of little value if used alone in the diagnosis of Typhoid fever. There may 2–4 times difference in O titres on the same sample of serum tested with antigen suspensions from different manufacturers.


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Diagnosis of typhoid fever can be more definitively established from increasing titers of H and O antibodies rather than from a single test result of H and O titers. In endemic areas titers of 1 : 100 values may be detected in normal population. PRIMARY SAMPLE • Use only serum as specimen for the test. • Collect 2 mL of venous blood in a plain red topped vacutainer tube or 0.1 N HCl washed tubes. • Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500–3000 rpm for 5–10 minutes. • Do not use lysed or contaminated serum. • Process the specimen on the same day. ADDITIVE OR PRESERVATIVE No need to add any anticoagulant or preservative while collecting blood sample. REAGENTS/ CONSUMABLES • • • • • • • • •

S.typhi “O”Antigen suspension (STO) S.typhi “H” Antigen suspension (STH) S.paratyphi A. “H”Antigen suspension (STAH) S.paratyphi B. “H” Antigen suspension (STBH) Antisera - S. typhi `O’ Antisera - S. typhi `H’ Antisera - S. paratyphi A `H’ Antisera - S. paratyphi B `H’ Normal saline (0.9%)

EQUIPMENT • Incubator - 37°C • Micropipettes • 36 Widal tubes/Kahn tubes for each sample (5 × 50 mm) STEP BY STEP PROCEDURE: QUANTITATIVE DETERMINATION Master dilution of serum • Add 2.3 mL of normal saline to a test tube. Add 0.2 mL of the test serum and mix well to get 1:12.5 diluted serum. Master dilution of antisera • Take 4 eppendorf vials and label them as STO, STH, STAH and STBH • Add 230 μL of normal saline to all the 4 eppendorf vials.


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• Add 20 μL of the respective antisera STO, STH, STAH, STBH to each of the respective vials and mix well. • Take a set of 7 clean dry 10 × 75 mm test tubes for each serum under test • Add 0.2 mL of normal saline in tubes 2 to 7. • Add 0.2 mL of 1:12.5 dilution test serum to the first and second tubes. • Mix and transfer 0.2 mL from the second tube in serial dilutions till the 7 th tube. • Repeat steps 1 to 4 for the other three rows of tubes. • For positive control, add 0.2 mL of 1: 12.5 diluted respective antisera. • For negative control add 0.2 mL of saline. • Add 0.2 mL of STO antigen to all the tubes of row 1 including positive and negative control • To get final two fold dilutions of 1: 25 to 1: 1600 for the test serum. • Add 0.2 mL of STH antigen to all the tubes of row 2 STAH to row 3, STBH to row 4 including positive and negative control. • Mix well and incubate at 37°C in a incubator for 16 to 20 hours. • Examine for agglutination, the antibody titer is the highest dilution of serum showing clear cut agglutination. Note: O antigens shows granular agglutination whereas H antigens show the characteristic floccular appearance. In negative reactions, the appearance of suspension remains unchanged with a minute button of deposit at the bottom of the tube. INTERPRETATION OF RESULTS The titer of the patient serum is the highest dilution of serum sample that gives a visible agglutination. A titer of 1: 100 for ‘O’ agglutinin and 1:100 for ‘H’ agglutinin is considered significant for the diagnosis of typhoid fever. A rise in titre is considered as a definitive evidence of S.typhi infection. Validity of test results: The results are valid only if each of the specific antisera shows agglutination and the negative control shows a cell button. SAFETY PRECAUTIONS • Handle all samples as potentially infectious • Handle all reagents with care and avoid contact with eye, mouth and skin • Do not perform mouth pipetteing • Discard used reagents and sample as per disposal procedure POTENTIAL SOURCES OF VARIABILITY • Shake the antigenic suspensions well before use and ensure the suspension is homogeneous.


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• A moderate rise in titre of all three ‘H’ agglutinins occurring simultaneously is suggestive of recent TAB vaccination • Antibiotic treatment prevents a rise in titer • A negative result cannot rule out typhoid fever if the sample had been taken very early in the course of the illness (earlier than 7 days from the onset of infection). • Diagnosis of typhoid fever can be more definitively established from increasing titers of H and O antibodies in serum sampls collected in 2-4 needs interval rather than from a single test result of H and O titers • Ensure the reagents and specimens are at room temperature before use • Store the reagents at 2–8°C.

BRUCELLA AGGLUTINATION TEST PURPOSE Qualitative detection and semi quantitative estimation of Brucella Antibodies to Brucella spp. by tube agglutination method. This test is useful in the laboratory diagnosis of Brucellosis. Brucella is a strictly aerobic, Gramnegative coccobacillus. This organism is sometimes carried by animals and only causes incidental infections in humans. The four species of this genus that can infect humans are named for the animal which they are most commonly found: B. abortus (cattle), B. suis (swine), B. melitensis (goats), B. canis (dogs). PRINCIPLE Antibodies in serum produced in response to exposure to Brucella abortus/ Brucella melitensis/Brucella suis will agglutinate the Brucella abortus bacterial suspension carrying homologous antigen. The antigen is used for tube agglutination test for diagnosis of Brucellosis. PERFORMANCE SPECIFICATIONS • False positive results may occur due to cross reaction with Yersinia, Francisella, and Vibrio • A false negative result may be obtained in the first few days after infection. A positive test result does not necessarily indicate current infection. Hence a repeat test may have to be performed to demonstrate a rise in titer.


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PRIMARY SAMPLE • Use only serum as specimen for the test • Collect 2 mL of venous blood in a plain red topped vacutainer tube or 0.1 N HCl washed tubes • Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500–3000 rpm for 5–10 minutes • Process all samples within 8 hours of collection • If sample is to be processed on another day or after 8 hours, separate the serum and store it at 2–8°C for up to 8 days or at –20°C up to 3 months • Do not use hemolysed or lipemic samples for testing ADDITIVE OR PRESERVATIVE No need to add any anticoagulant or preservative while collecting blood sample. Collect venous blood in a plain red topped vacutainer tube or acid washed (0.1 N HCl) tube. CONSUMABLES/REAGENTS Brucella abortus plain antigen: Brucella abortus plain antigen is a suspension of a pure smooth culture of Brucella abortus strain 99 in phenol saline. The standard antigen is supplied by the Institute Veterinary Preventive Medicine, Ranipet, North Arcot Dt. Tamil Nadu. Carbol saline (Normal saline containing 0.5% Carbolic acid) EQUIPMENT No special equipment is required. • Incubator at 37°C • Glass test tubes • Micropipettes/serological pipettes • Test tube stand STEP BY STEP PROCEDURE • Place five Felix tubes in the test tubes rack. • Add 0.8 mL of carbol saline in the first tube and 0.5 mL in all the other tubes. • Add 0.2 mL of serum to the first tube, mix well and transfer 0.5 mL to the second tube. Mix thoroughly and transfer 0.5 mL to the third tube. • Continue this process up to the fifth tube and discard 0.5 mL from the last tube, after mixing. • Add 0.5 mL of the standardised Brucella abortus plain antigen to each tube. Mix by rolling in between palms or in a cyclomixer and incubate at 37°C for 16–18 hours (over night). • Perform the same test with a positive control.


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ANTIGEN CONTROL TUBES For each days work, put a set of control tubes, as shown below for comparing the results of the test sample. Antigen Control tubes

0.5% carbol saline

Antigen

Degree of agglutination

Tube 1 Tube 2 Tube 3 Tube 4 Tube 5

1.0 mL 1.25 mL 1.50 mL 1.75 mL 2 mL

1.0 mL 0.75 mL 0.50 mL 0.25 mL nil

No agglutination 25% agglutination + 50% agglutination (+ +) 75% agglutination (+ + +) 100% agglutination (+ + + +)

Incubate the antigen control tubes, at 37°C for 24 hours along with the test samples Each batch of test sample should include a positive control and antigen control tube. INTERPRETATION OF RESULTS Positive: Presence of granular agglutination in any of the tubes indicates a positive test Negative: Absence of agglutination in all tubes indicates a negative test The titer of the serum is the reciprocal highest dilution of the serum which shows agglutination. Results of agglutination should be noted after keeping the tubes for an hour or two on the bench at the room temperature. Examine all the incubated tubes against light and compare the tubes in the test series, with the antigen control tubes. Note the degree of agglutination for each sample of serum as shown under. The degree of agglutination is to be judged by opacity of the supernatant fluid. ++++ (Comparable with tube 5 of the antigen control series) +++ (Comparable with tube 4 of the antigen control series) ++ (Comparable with tube 3 of the antigen control series) + (Comparable with tube 2 of the antigen control series) (No agglutination) (Comparable with tube 1 of the antigen control series) ++ (50% agglutination) should be considered as end-point. REFERENCE RANGE/SIGNIFICANT TITRE A titre of 1: 80 or greater is considered significant. SAFETY PRECAUTIONS • Handle all samples as potentially infectious • Handle all reagents with care and avoid contact with eye, mouth and skin


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• Do not perform mouth pipetting • Discard used reagents and sample as per disposal procedure. POTENTIAL SOURCES OF VARIABILITY Ensure that the sample is free of fibrin/particles by centrifuging as it may interfere with test results As a positive test does not differentiate between a past and current infection, a repeat test may have to be performed on a fresh sample after 10- 12 days to demonstrate a rise in titer.

QUALITATIVE DETERMINATION OF HIV1/2 ANTIBODY PURPOSE Qualitative determination of HIV 1/2 antibody in human serum by rapid screening technique. This test is indicated for the screening of blood and blood products for HIV 1/2 antibodies. PRINCIPLE HIV antigens are immobilized on a porous immunofiltration membrane. Sample and reagents pass through the membrane and absorbed by the underlying absorbent. As the patient’s sample passes through the membrane, HIV antibodies, if present, bind to the immobilized antigens. Conjugate binds to the Fc portion of the HIV antibodies to give the distinct pinkish purple color DOTs against a white background. PRIMARY SAMPLE • • • •

Use only serum as specimen for the test If plasma is used, use only heparinized plasma. Do not use pooled specimens. Collect 2 mL of venous blood in a plain tube/plain 0.1 N HCl washed tube. • Process the sample on the same day with in 2 hours of collection. • If the specimen contains precipitates, it must be centrifuged or filtered. • If the analysis is not done within 2 hours of collection, separate the serum and store it at 2–8°C for up to 3 days or at –20°C if longer storage is required. REAGENTS/ CONSUMABLES • Disposable HIV 1/2 tri dot test device. • Dropper

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• Buffers • Protein A conjugate • Sodium hypochlorite solution (1%) or other suitable disinfectant for treating the specimens before disposal. PROCEDURE • Bring the specimen, buffer and pouch containing the Tri-dot device to room temperature before use. • Add 3 drops of buffer solution to the center of the device. • Hold the dropper vertically and add 1 drop of patient’s serum. Use separate dropper for each specimen to be tested. • Add 5 drops of buffer solution. • Add 2 drops of liquid conjugate directly from the conjugate vial. • Add 5 drops of buffer solution and read the results. RESULT Reactive: If two dots, one for the control and the other for HIV-1 appear, the specimen is reactive for antibodies to HIV-1. If two dots, one for the control and the other for HIV-2 appear, the specimen is reactive for antibodies to HIV-2. Nonreactive: Presence of a single dot, only at the control spot. Wrong result: If two dots are present one in the HIV-1 and other with HIV-2 with no dot in control then test has to be repeated. If no dot appears in either control or HIV-1/HIV-test needs to be repeated. LIMITATIONS • The kit works well with fresh samples. Frozen and thawed samples may block the membrane. • HIV-1 and HIV-2 viruses share many morphological and biological characteristics. It is likely that due to this, their antibodies have a cross reactivity of 30-70%. SAFETY PRECAUTIONS • Handle all samples as potentially infectious • Handle all reagents with care and avoid contact with eye, mouth and skin • Discard used reagents and sample as per disposal procedure.


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RAPID DETECTION OF HEPATITIS B SURFACE ANTIGEN (HBs Ag) BY– IMMUNOFILTRATION METHOD PURPOSE Qualitative determination of antibodies to Hepatitis B Surface antigen (HBsAg) concentration in human serum/plasma by Immunochromatography. This test is indicated for the screening of blood and blood products to be used for transfusion and an aid for the diagnosis of existing or previous hepatitis B infection. PRINCIPLE The test is a one step Immunochromatography, which employs monoclonal antibodies conjugated to colloidal gold conjugate and polyclonal antibodies immobilized on a nitrocellulose strip in a thin line. The strip is coated with monoclonal antibodies specific for HBsAg. The sample flows laterally from the well through an absorbent pad and mixes with the signal reagent. If the sample contains HBsAg, the colloidal gold antibody (mouse) conjugate binds to the antigen forming an antigen antibody complex. The complex then migrates through the nitrocellulose strip by capillary action. Which are stopped by immobilized Ab zone forming a purple band. PERFORMANCE SPECIFICATION Sensitivity: This test can detect hepatitis B antigen in serum or plasma in a concentration as low as 1 ng/mL. PRIMARY SAMPLE • Use only serum/plasma as specimen for the test. • Do not use pooled specimens. • Collect 2 mL of venous blood in a plain tube/plain 0.1 N HCl washed tube. • Process the sample on the same day within 2 hours of collection. • If the specimen contains precipitates, it must be centrifuged or filtered. • If the analysis is not done within 2 hours of collection, separate the serum and store it at 2–8°C for up to 3 days or at –20°C if longer storage is required. REAGENTS/ CONSUMABLES • Disposable HEPA cards • Dropper

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• Sodium hypochlorite solution (1%) or other suitable disinfectant for treating the specimens before disposal. PROCEDURE • Bring the specimen and pouch containing the HEPA card to room temperature before use. • Remove one test card from the pouch and place it on clean flat surface. • Using the dropper provided add 2–3 (70 μL-100 μL) drops of serum sample into the sample well. Avoid overflowing. • Let the reaction to proceed up to 20 minutes. • Read the results after 20 minutes. • Strong positive reactions may be visible in 5 minutes. • If negative or questionable results are obtained, and HBV infection is suspected, the test should be repeated on a fresh serum specimen. RESULT Positive: If a distinct purple line is formed at the test zone marked ‘T’ (Test line) and the control zone marked ‘C’ (control line)— indicating the sample contains Hepatitis B antigen. Negative: If a distinct purple line is formed only at the control zone marked ‘C’ (control line)— indicating the sample is negative. LIMITATIONS This test will indicate only the presence or absence of the Hepatitis B surface antigen in the specimen. SAFETY PRECAUTIONS • Handle all samples as potentially infectious • Handle all reagents with care and avoid contact with eye, mouth and skin • Discard used reagents and sample as per disposal procedure • Potential sources of variability • HBsAg card is used for the detection of HBsAg in human serum or plasma. Based on a single reactive test result, a sample should not be considered HBsAg positive. Further testing, including confirmatory testing, should be performed before a specimen is considered positive for HBsAg. • A non-reactive test result does not exclude the possibility of exposure to hepatitis B virus. Levels of HBsAg may be undetected both in early infection and late after infection. Specimens containing precipitate may give inconsistent test results.


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DETECTION OF HEPATITIS C VIRUS (HCV) ANTIBODIES PURPOSE Qualitative determination of anti HCV antibodies in human serum by TRIDOT technique. This test is indicated for the screening of blood and blood products for HCV antibodies. PRINCIPLE HCV antigens are immobilized on a porous immunofiltration membrane. Sample and reagents pass through the membrane and absorbed by the underlying absorbent. As the patient’s sample passes through the membrane, HCV antibodies, if present, bind to the immobilized antigens. Conjugate binds to the Fc portion of the HCV antibodies to give the distinct pinkish purple color dots against a white background. PRIMARY SAMPLE • Use only serum/plasma as specimen for the test • Do not use pooled specimens • Collect 2 mL of venous blood in a plain tube/plain 0.1 N HCl washed tube. • Process the sample on the same day within 2 hours of collection. • If the analysis is not done within 2 hours of collection, separate the serum and store it at 2–8°C for up to 3 days or at –20°C if longer storage is required • If the specimen contains precipitates, it must be centrifuged and the clear supernatant to be used for test. REAGENTS/ CONSUMABLES • • • • •

Disposable HCV Tri dot test device Dropper Buffers Protein A conjugate, positive and negative control. Sodium hypochlorite solution (1%) or other suitable disinfectant for treating the specimens before disposal.

PROCEDURE • Bring the specimen, buffer and pouch containing the tri-dot device to room temperature before use. • Add 3 drops of buffer solution to the center of the device. • Hold the dropper vertically and add 1 drop (50 μL) of patient’s sample. Use separate dropper for each specimen to be tested.


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• Add 5 drops of buffer solution. • Add 2 drops of protein A conjugate directly from the conjugate vial. • Add 5 drops of buffer solution and read the results. RESULT It can be found by comparing the test strip with the given standard strip. Reactive • Appearance of two dots, one at the control region “C” and other at the test region “T1” indicates that the sample is REACTIVE for antibodies to HCV. • Appearance of two dots, one at the control region “C” and other at the test region “T2” indicates that the sample is REACTIVE for antibodies to HCV. • Appearance of all the three dots one each at “C”, “T1” and “T2” region indicates that the specimen is reactive for antibodies to HCV. • Invalid result: If no dot appears after the completion of test, either with clear background or with complete pinkish/purplish background the test indicates ERROR. Non-reactive: Appearance of dot only at “C” and no dot at ‘T1’ or ‘T2’ LIMITATIONS The kit works well with fresh samples. Frozen and thawed samples may block the membrane. SAFETY PRECAUTIONS • • • •

Handle all samples as potentially infectious Handle all reagents with care and avoid contact with eye, mouth and skin Do not perform mouth pipetting Discard used reagents and samples as per disposal procedure.

ELISA TEST FOR THE DETECTION OF ANTIBODIES TO HIV-1 AND HIV-2 PURPOSE To detect the presence of antibodies to HIV-1 and HIV -2 in serum by ELISA method using commercially available bioelisa HIV 1+2 (rec) (Bio Elisa kit, Barcelona, Spain). PRINCIPLE Bioelisa HIV-1+2 (rec) is a third generation solid phase enzyme immunoassay in which highly purified recombinant antigens are used for


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the combined detection of antibodies to HIV-1, HIV-2 and HIV-1 subtype O. The immunodominant N-terminal parts of the transmembrane proteins gp41 and the highly reactive core antigen p24 of HIV-1 and gp36 of the HIV-2 are produced by means of recombinant technology. The microplate wells are coated with a mixture of these highly purified recombinant antigens. Serum or plasma samples are added to microplate wells. If HIVspecific antibodies are present in the sample, they will form complexes with the antigens on the well. Unspecific antibodies are removed by subsequent washing step. The antigen-antibody complexes formed during the first incubation step are detected with highly purified, peroxidase labeled HIV-1 and HIV-2 recombinant antigens. The enzyme activity of the bound conjugate is then determined by addition of a substrate solution containing a chromogen. This solution will develop a blue color if the sample is positive. The blue color changes to yellow after blocking the reaction with sulphuric acid. The intensity of color is proportional to the concentration of HIV antibodies in the sample. Wells containing negative samples remain colorless. PERFORMANCE SPECIFICATION Patients with recent exposure to EB virus may show false positive result. Performance of this test with other body fluids as specimen is not wellestablished. A sample reactive may be negative nonreactive retesting due to presence of fibrin clots or cellular materials or particulate matter. PRIMARY SAMPLE • Use only serum free from fibrin as specimen for the test • Plasma collected in Lithium Heparin, Sodium Citrate, ACD, CPDA-1, and EDTA tubes may also be used • Collect 2 mL of venous blood from a peripheral vein in a plain redtopped vacutainer tube • Do not use hemolyzed/contaminated samples/serum containing fibrin and particulate matter for testing • Separate serum within 30 minutes of collection by centrifugation • Process the sample on the same day within 3 hours of collection • If the analysis is not done on the same day within 8 hours of collection, separate the serum and store it at 2–8°C for up to 14 days OR at –10°C for up to 12 weeks. REAGENTS/CONSUMABLES As per Product Insert.

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COMPONENTS Microplate: 12 × 8 wells coated with HIV-1 and HIV-2 recombinant antigens. Round bottom. Concentrate conjugate: HIV-1 and HIV-2 recombinant antigens conjugated with peroxidase. Contains stabilizers protein and preservatives. Dilute 1/51 with the conjugate diluent before use. Conjugate diluent: Phosphate buffer containing stabilizers protein and preservatives. Sample diluent: Phosphate buffer with stabilizers protein and preservatives. Ready to use. Washing solution: Concentrate saline buffer (20x) with additives. Dilute 1/20 with distilled or deionized water before use. Substrate buffer: Citrate-acetate buffer saline containing hydrogen peroxide. Chromogen: 3,3’5,5’-Tetramethylbenzidine (TMB) in solution. Dilute 1/100 with the substrate buffer before use. HIV-1 positive control: Diluted and inactivated human serum positive for antibodies to HIV-1. Contains preservatives. Ready to use. HIV-2 positive control: Diluted and inactivated human serum positive for antibodies to HIV-2. Contains preservatives. Ready to use. Negative control: Normal human serum, negative for antibodies to HIV-1 and HIV-2. Contains preservatives. Ready to use. Stopping solution: 1N sulfuric acid. Ready to use. Adhesive seals: To cover plate during incubations. Resealable bag: For storage of unused strips. PROCEDURE • Previous operations • Allow all the reagents to reach room temperature (20–25°C) before running the assay • Gently mix all liquid reagents before use • Dilute the concentrate washing solution 1/20 with distilled or deionized water. For example: 50 mL of concentrate washing solution in 950 mL of water. If the kit is stored for long time crystals may appear in the washing solution. To dissolve them put the bottle in a water bath at 37°C. Homogenize the solution before use. • Dilute the concentrate conjugate with the conjugate diluent according to Table given below. Mix well. It is recommended to dilute the conjugate 10 minutes before use.


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Preparation of working dilution of conjugate (1:5 dilution) Strips required 1 Conjugate diluent mL 1.0 Concentrate conjugate μL 20

2 2.0 40

4 4.0 80

6 6.0 120

8 8.0 160

10 12 10.0 12.0 200 240

ASSAY PROCEDURE Determine the total number of wells needed for the assay. In addition to test samples, it is necessary to include 1 well for the substrate blank, 3 wells for negative control, 2 wells for HIV-1 positive control and 1 well for HIV-2 positive control. Dispense 100 μL of sample diluent in each well except blank well. Dispense 50 μL of each sample in the designated well. Dispense 50 μL of negative control, 50 μL of HIV-1 positive control and 50 μL of HIV-2 positive control in the designated wells. Cover the plate with the adhesive seal, mix gently and incubate at 37°C for 60 minutes. Remove and discard the adhesive seal. Aspirate the content of the wells and fill them completely (approximately 300 μL) with the diluted washing solution. Repeat the process of aspiration and washing 4 more times. Ensure that each column of well soaks for at least 15 seconds before the next aspiration cycle. Transfer 100 μL of diluted conjugate into each well of the microplate except the blank. Prepare the diluted conjugate during the last 10 minutes of step 3. Cover the plate with the adhesive seal and incubate at 37°C for 30 minutes. During the last 15–20 minutes of this incubation prepare the substratechromogen solution. The substrate buffer and the chromogen must be at room temperature, before preparing the working solution. Prepare the required volume according with Table given below. Mix well. The final solution should be colorless; discard if it becomes blue. Preparation of substrate (1:101 dilution) Strips required Substrate buffer mL Chromogen (TMB) μL

1 1.0 10

2 2.0 20

4 4.0 40

6 6.0 60

8 8.0 80

10 12 10.0 12.0 100 120

Remove and discard the adhesive plate cover. Aspirate and wash the plate as in step 4. Add 100 μL of substrate-TMB solution to each well including the blank. Incubate for 30 minutes in the dark at room temperature (20–25°C). Stop the reaction by adding 100 μL of stopping solution in the same sequence and time intervals as for the substrate-TMB.


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Blank the reader at 450 nm with the blank well and read the absorbance of each well, within 30 minutes. It is recommended to read in bichromatic mode using a 620–630 nm reference filter. POTENTIAL SOURCE OF VARIABILITY This test does not detect all potentially infectious individuals/ blood units. A negative test result does not rule out exposure to or infection with HIV 1 and/or HIV 2. This test does not differentiate between HIV 1 and 2 antibody. INTERPRETATION OF RESULTS Calculation The system calculates the cut off rate from the mean rate of two-index calibrator. It replicates and stores the result. The cut off rate is determined by multiplying the mean index calibrator rate by 2.5. Cut off Rate (CO) = INDEX CALIBRATOR MEAN RATE × 2.5 The HIV - /HIV –2 protocol calculates a result based on the ratio of the samples rate to the cut off ratio (CO) for each specimen and control. S/CO = SAMPLE RATE/CUT OFF RATE INTERPRETATION • Specimens with S/CO VALUES < 1.00 are considered NEGATIVE • Specimens with S/CO VALUES > or equal are considered POSITIVE. • Repeat all positive results obtained with MEIA Technique by SANDWICH ELISA technique through freshly collected samples and also confirmed by Western Blot Technique by sending a fresh sample to an approved External Laboratory The Assay will be completed within 40 minutes.

WESTERN BLOTTING TECHNIQUE— CONFIRMATION TEST FOR DETECTION OF HIV–1/2 ANTIBODIES PURPOSE Qualitative determination of HIV 1/2 antibody concentration in human serum by western blot technique. This test is indicated for the confirmation of blood and blood products for HIV 1/2 antibodies.


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PRINCIPLE To perform the assay the strip is incubated with the patient serum/plasma diluted in a buffer. Antibodies to HIV 1 and HIV 2 if present in the serum, binds to the antigens located on the strip. Unbound materials are washed away. Then the strip is incubated with antihuman IgG conjugated to alkaline phosphatase. After washing the unbound conjugate, substrate is added which results in the initiation of the color bands. PRIMARY SAMPLE • • • •

Use only serum as specimen for the test If plasma is used use only heparinized plasma Do not use pooled specimens Collect 2 mL of venous blood in a plain tube/plain 0.1 N HCl washed tube • Process the sample on the same day within 2 hours of collection • If the specimen contains precipitates, it must be centrifuged or filtered • If the analysis is not done within 2 hours of collection, separate the serum and store it at 2–8°C for up to 48 hours or at –20°C if longer storage is required. REAGENTS/ CONSUMABLES • • • • •

Disposable HIV 1/2 Membranes/Test strips Dropper Forceps Buffers Preparation of reagents for one strip

Working eash buffer: Wash buffer concentrate Distilled water

- 1 mL - 19 mL

Working diluent buffer: Diluent buffer concentrate Distilled water No. of spoons of blotting powder

- 0.5 mL - 4.5 mL - 2 spoons

Working conjugate: Working diluent buffer 100X conjugate

- 2 mL - 20 μL

Substrate: Ready to use Centrifuge Sodium hypochlorite solution (2%) or other suitable disinfectant for treating the specimens before disposal.

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PROCEDURE—RAPID METHOD • Bring the specimen, buffer and pouch containing the membrane strips to room temperature before use. • Positive and negative controls should be included with each run of the test • Using forceps remove required number of strips from the pouch and place it on clean flat surface. Ensure that the numbered sides of the strips are facing up. Note down the strip number with respect to samples and control on the worksheet for correct identification. • Proper and gentle shaking of the tray at 50–60 rpm is extremely important. Improper shaking may affect the sensitivity of the test. • Reagents and samples should be added only to the ends of the trays and not to the strips directly. • Use separate tips for all reagents including serum. • Add 2 mL of working wash buffer per strip to each tray and incubate for 5 minutes on rotary shaker and aspirate wash buffer. • Prepare working diluent buffer according to the number of tests and controls to be run. • Add 2 mL of the working buffer and 20 μL serum and controls to appropriate wells. • Incubate for one hour at room temperature (25–30°C) on rotary shaker. The covers of the wells should also be marked to prevent interchange of covers which may lead to cross-contamination. • Carefully remove covers, aspirate solution completely from tray and discard into sodium hypochlorite solution. • Wash 3 times with 2 mL working wash buffer per strip for 5 minutes each on W.blot/rotary shaker. • Prepare working conjugate solution according to the number of strips to be run. • Add 2 mL of working conjugate per strip. • Incubate for 1 hour on W.blot/rotary shaker. • Aspirate conjugate. Wash 4 times with 2 mL working wash buffer per strip for 5 minutes each on W.blot/rotary shaker. • Add 2 mL ready to use substrate per strip. Incubate 0.5–15 min away from the light preferably in dark till bands develop • Continue to observe the reaction till gp160/gp120/gp41 appear and stop the reaction after their appearances. However, in case the above bands do not appear, then continue the reaction up to the point. • Before strong background is formed on the strip. • Up to 15 minutes, whichever is earlier. • Aspirate substrate and add distilled water and wash strips to stop reaction. • Remove strips on paper towels and mount on work sheet keeping number side up.


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INTERPRETATION OF RESULTS Negative control: No HIV-1 and HIV –2 specific bands should be observed on the negative control strips only the band for serum control should be visible. Positive control: Almost all the virus specific bands at positions gp 160. gp 120, p66, p55/51, p31, p24, p17 and HIV-2 specific band should be visible along with the serum control band. The results should be interpreted as positive, indeterminate, negative or invalid based on the interpretation table in the instructions manual of the kit. INTERPRETATION TABLE INTERPRETATION

PATTERN

POSITIVE HIV-1 POSITIVE

a) 2 ENV + (either of 2 ENV gp 160, gp41, gp120)

1 GAG (p 24) +/1 POL (p31,p51,p66)

HIV-1 POSITIVE With HIV-2 INDICATED

b) 2 ENV + (either of 2 ENV; gp 160, gp41, gp120)

1 GAG (p24) +/1 POL (p31, p51, p66) + HIV-2 BANDz

HIV-1 NEGATIVE HIV-2 Indicated

Only control band + HIV-2 BAND

INDETERMINATE Viral specific bands present but pattern does not meet the criteria for POSITIVE

a) 1 ENV + GAG + POL (either of 1 ENV; (p17, p24, (p31,p51 gp 160, gp 41, gp120) p 55) p66) b) GAG + POL (p17, p24, p55) (p31, p51, p66) c) Only GAG (p17, p24, p55) d) Only POL (p31, p51, p66)

INDETERMINATE with HIV-2 Indicate

Viral specific bands present but pattern does not meet the criteria for POSITIVE + HIV-2 BAND

NEGATIVE

Only control band or control band with p51/55** band

INVALID

No control band

LIMITATIONS • The test has to be repeated when the test sample shows indeterminate results with the rapid procedure, with overnight incubation • Dark color background or greenish tinge on the bands p24, p55/51 and p66 will occur due to overexposure of strips to substrate buffer • In the indeterminate cases the test has to be retested using fresh specimen after 2–6 months. HIV-1 and HIV-2 viruses share many morphological and biological characteristics. It is likely that due to this, their antibodies have a cross-reactivity of 30–70%.


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SAFETY PRECAUTIONS • Handle all samples as potentially infectious • Handle all reagents with care and avoid contact with eye, mouth and skin • Discard used reagents and sample as per disposal procedure.

FLUORESCENT ANTINUCLEAR ANTIBODY TEST (FANA) PURPOSE Indirect immunofluorescence staining for detection of antinuclear antibodies present in human serum. PRINCIPLE The antibodies in the patient’s serum combines with the tissue antigen to form a complex. This complex binds with the fluorescent anti-human globulin conjugate and thus fluoresce in ultra-violet illumination. PERFORMANCE SPECIFICATIONS Always perform the test with freshly coated slides. Slides should be thoroughly washed after each step to get rid of non-specific fluorescence which might interfere with reading of the result. Reading of the result should be done by experienced personnel. PRIMARY SAMPLE • Use only serum as specimen for the test. • Collect 2 mL of venous blood in a plain red topped vacutainer tube or 0.1 N HCl washed tubes. • Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500–3000 rpm for 5–10 minutes. • Do not use lysed or contaminated serum. • Process the specimen on the same day. REAGENTS/ CONSUMABLES • • • •

Phosphate buffered saline at pH 7.2 Acetone Fluorescein conjugated anti-human immunoglobulin (polyvalent) Mounting medium - 50% glycerol in 0.05 M sodium barbitate pH 8.6


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Preparation of Glass Slides On the glass slides five circles are marked using diamond marker and the slides are dropped in distilled water containing 0.025% sodium meta silicate and it is kept in room temperature for half an hour. Then the slides are cleared well with tissue paper, then with methanol dipped tissue paper and again with clean tissue paper. Finally slides are wrapped with aluminium foil and it is kept in hot air oven for sterilization at 160°C for 1 hour. Coating of Slides • Inoculate into the wells of the specially made slides with HEp - 2 cells on the previous day which is incubated in 10% Carbon dioxide atmosphere overnight. • Next day observe the growth of the cells. • Then wash the slides in three changes of phosphate buffered saline (pH – 7.2) and fix the slides in cold acetone for 10 minutes. STEP BY STEP PROCEDURE • • • • • • •

•

Inactivate the patient’s serum before performing the test. Dilute the serum in saline from 1 in 4 up to 1 in 32 (four-fold dilution). Include positive control Include negative sera control at the same dilution as test. Mark the slide with diamond marker. Then add 10 μL of the 1 in 8, 1 in 16 and 1 in 32 diluted samples to three wells of the slide coated with cell lines. Keep in a moist chamber for 30 minutes. Wash thrice with PBS in slide chamber. Then add 5 μL of 1 in 20 diluted (diluted in PBS pH –7.2) rabbit antihuman Fluorescent Isothiocyanate (FITC) conjugate polyvalent serum to each of the wells. Keep for 30 minutes in moist chamber. Wash thrice in PBS and mount with glycerol mounting fluid and observe under 20X of fluorescent microscope with blue filter.

RESULTS Staining character

Antigenic determinant

Antibodies to

Rim and homogeneous

dS DNA DNA - histone complex Classes of histones

dS DNA DN protein LE - Cell ab

Variable large speckles

H3

Histone Contd.

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Contd. Speckled

Small nucleolar RNP RNA protein Complex protein

Smith Ag Nuclear RNP NSp I, NSpII

Nucleolar & Cytoplasm

Ribosomal fraction

Cytoplasmic RNP

Variable speckled in some cells

33 KD a protein

PCNA

Descrete speckled

Protein

Centromere

SAFETY PRECAUTIONS • Handle all samples as potentially infectious • Handle all reagents with care and avoid contact with eye, mouth and skin • Do not perform mouth pipetteing • Discard used reagents and sample as per disposal procedure.

UNIT 5

Ophthalmic Histopathology

Jyotirmay Biswas S Krishnakumar

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SPECIMENS ENTRY The different kinds of specimens received at the histopathology laboratory for investigations are: 1. Removed eyeballs. 2. Portion of the eye removed during various surgical procedures, e.g: a. Corneal button – after keratoplasty b. Lens – after cataract surgery c. Trabeculectomy specimen – after anti-glaucoma surgery d. Vitrectomy, membranectomy – after vitrectomy procedures 1. Biopsy portion of the tissue to establish the diagnosis which can be obtained from: a. Lid b. Conjunctiva c. Cornea d. Iris e. Orbital tissue f. Choroid or retino-choroidal tissue g. Any other ocular tissue 2. Aspiration from the anterior chamber and vitreous chamber or any cystic lesion of orbit. 3. Scraping from superficial structures: e.g. Conjunctiva or cornea. 4. Impression smear from conjunctiva using millipore filter paper. THINGS TO BE NOTED WHILE RECEIVING OCULAR PATHOLOGY SPECIMENS 1. Proper labelling of the specimen, i.e. it should contain the following information: a. Name of the patient b. Age of the patient c. The type of material d. The eye (right or left) e. Date and time of surgery f. Doctor’s name 2. Adequate clinical summary with clinical diagnosis. 3. Proper fixation in proper preservative. PRESERVATION OF THE SPECIMEN • Tissue should be preserved immediately in the proper fixative as drying of the tissue causes artifacts.


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• For routine surgical specimens 10% neutral buffered formalin is used, 70% acohol for IOLs, and 2.5% glutaraldehyde for specimens meant for electron microscopic study. • Approximate volumes of the fixative required for different kinds of specimens are: a. Cornea – 5 to 10 mL b. Eyeball – 150 to 200 mL c. Exenterated specimen – 500 mL d. Other biopsy material – according to the size of the specimen FIXATION TIME • Small specimens (Corneal button, retinal membranes, biopsy measuring<5 mm – 12 hrs • Globes, exenterated or large specimens measuring > 20 mm – 48 hrs • Smears of fluid specimens: 95% alcohol. Fixation should be done immediately. MAILING THE SPECIMEN In case specimen is needed to be sent for second opinion, one should avoid sending in a large volume at fixative. The fixed tissue can be transferred to smaller volume (about twice the volume of the specimen) or use formalin soaked gauze. HOW IS FROZEN SECTION ORDERED? Frozen section is usually done in the case of adnexal tumors. Histopathology lab has to be informed at least 6 hours prior to surgery to keep the cryostat machine ready. Reports of such study is ready by 20 to 30 minutes of time. SPECIMEN ACCESSION NUMBER PROVIDING PROCESS Tissues arriving at the laboratory are identified by means of an accession number. The accession number is marked both on the requisition form as well as on the container in which specimen is received. Once the tissue has been taken for processing, this accession number accompanies them through all stages.

FIXATION OF SPECIMENS “Fixation is the preparation of a histologic or pathologic specimen in a physical and also partly in a chemical state for the purpose of maintaining the existing form and structure of all of its constituents.”- It is one of the most important requirements in preparing good tissue sections.


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Fig. 5.1: Photograph showing various fixatives used in the laboratory

Purpose The purposes of fixation are as follows: 1. To prevent post-mortem changes, such as putrefaction and autolysis, by inhibiting autolytic enzymes and killing the organisms that cause decomposition. 2. To preserve the various tissues constituents as nearly as possible to their original form. 3. To harden the naturally soft tissue, permitting easier and safer manipulation during subsequent processing. 4. To render the various tissue constituents receptive to subsequent staining. The essentials for good fixation are: 1. Fresh tissue: Surgical specimens should be immersed in the fixing solution as soon as possible. Specimens obtained post-mortem would have undergone less autolysis if it is placed under refrigeration as soon as possible. 2. Proper penetration of the tissue by the fixative: Inadequate penetration produces poor fixation and subsequently bad staining. The mass of tissue excised may be so large that the fixative will not penetrate the tissue within a reasonably short time. In such situation, specimen is fixed for longer time, i.e., 48 to 72 hours depending on the size of the specimen. CHOICE OF FIXATIVE SOLUTION Choice of fixative is made with several factors in mind, e.g. structures and entities to be demonstrated and the effects of short-term and long-term storage. Some fixatives are restrictive, others are multi-purpose. Most commonly used fixatives are listed below:


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1. 10% Neutral Buffered Formalin Preparation 37 to 40% Formaldehyde ................................................. 100 mL Disodium hydrogen phosphate ...................................... 6.5 gm Sodium dihydrogen phosphate ...................................... 4.029 gm Distilled water ................................................................... 900 mL Except in special circumstances it is used routinely for fixation of all biopsy specimens and enucleated eyes. Advantages 1. Sections prepared from tissue fixed in formalin can be stained with almost any kind of special stains. 2. Formalin fixed tissue lends itself well to preparation of frozen sections and staining for fat. 3. Formalin does not cause excessive hardness or brittleness of the tissue. 2. Glutaraldehyde • Glutaraldehyde penetrates more slowly than formaldehyde and is useful in electron microscopy and in enzyme histochemistry. • There are many variations in the preparation of this fixative, including the percentage of glutaraldehyde, other additives, and buffers. Small blocks of tissues 1–2 mm in size, fix well at cold temperature 1°–4°C, and fixed tissue specimens can be stored in buffered solution for many months. • The slow penetration, cold temperature, and the need for a storage medium prevent the use of this fixative in routine diagnostic histotechnology. Electron microscopists, however, are able to use it with continued success. 3. Alcohol (Propanol/ Methanol/ Ethanol) It is used for cytology specimens. Refer cytology study.

GROSSING TECHNIQUE Presentation of whole or representative tissue for further processing. The submitted material will be embedded and sectioned according to the physician’s instructions, and the final sections should be the representative of the pathologic changes noted or suspected from gross pathologic examination. I. GROSS DESCRIPTION OF GLOBE 1. Identify as right or left 2. Mark superior pole with a blue pencil

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3. Measure the globe in mm as follows: a. Anteroposterior b. Horizontal c. Vertical 4. Measure the cornea in mm as a. Horizontal b. Vertical 5. Pupil .......... × ........... mm 6. Measure the optic nerve segment in mm 1. Length attached to the globe 2. Diameter

Fig. 5.2: Photograph of the posterior part of the eyeball showing optic nerve and posterior ciliary veins running horizontally. Note inferior oblique muscle on the temporal side

7. Describe the external features of interest, if any, indicate the meridian, anteroposterior location (usually in relation to optic nerve, equator, or limbus) and size. Significant external markings include scars, corneal leucomas, staphylomas, areas of unusual pigmentation, distinct, abnormal shadows on transillumination, and others.

Fig. 5.3: Schematic diagram of eyeball for grossing


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Fig. 5.4: Transillumination of a globe showing ciliary staphyloma

Fig. 5.5: Sectioning technique of the globe

Fig. 5.6: Cut section of a case of malignant melanoma the choroid showing a of mushroom appearance

Fig. 5.7: Cut section of the globe of a case of retinoblastoma showing chalky filling the whole globe white mass

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8. Transillumination: Before an eye is opened it is to be transilluminated. The normal eye transmits light well except at the ciliary body/iris and at any optic nerve of significant length. Abnormal shadows (mark the defects externally) should be described. 9. Opening the eye: Tissue submitted for processing typically consists of a cross-section of the optic nerve and a pupil optic nerve section. The eye can be opened in any meridians. The meridian chosen should add the most possible additional information of the pupil optic nerve section. If, for instance, there is a limbal scar from previous cataract surgery and a peripheral iris coloboma at 12 o’ clock, the eye is opened vertically. If there is no history of prior injury or other accurately localized pathology, and if there is an abnormal shadow on transillumination which is localized, the eye is opened in the meridian of the shadow. If neither the external findings/history nor the findings on transillumination suggest a meridian for opening the eye, then open the eye horizontally to include the macular region the pupil optic nerve section. 10. Describe the abnormalities systemically proceeding posteriorly as follows: 1. Cornea 2. Anterior chamber 3. Angle 4. Lens 5. Vitreous cavity 6. Retina 7. Choroid 8. Sclera 9. Optic nerve head 11. Gross photography is done if required. Under the dissecting microscope with the attached camera, if needed, photographs can also be taken. 12. Submit sections for processing. One half of the calotte is processed and the other calottes are saved in 10% buffered formalin. 13. Indicate the type of stain adopted. 14. In case of retinoblastoma suspect, always make a section of optic nerve (after measurements) before opening the globe and the surgical end of the optic nerve could be marked with either help of hematoxylin or blue pencil. In case of Melanomas look for evidence of extrascleral extension by taking separate section of vortex veins before the globe is sectioned. 15. Make a rough diagram of the cut section of the eyeball on the grossing form. II. GROSSING OF THE BIOPSY SPECIMENS 1. Mark the surgical surface with hemotoxylin if tumor is suspected. 2. Bisect along the longitudinal axis and cut surface down in block. 3. If the specimen is very minute in size, put a drop of hemotoxylin or eosin on it before processing so as to spot it. 4. Small fragments of tissue are processed after wrapping in butter paper.


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III. CORNEAL BUTTON 1. Measure diameter of cornea in mm 2. Describe points of interest, if any 3. Bisect into equal halves. Process one half and save the other half in fixative. 4. After impregnation, embed the corneal button on the long edge.

DECALCIFICATION Cutting of thin sections by ordinary method is impossible when the tissue has become partly calcified. Such tissues must be treated to remove the calcium and phosphate salts, which are deposited in them. Methods of decalcification involve the use of acids, in which the bone salts are dissolved. All such acid solutions are injurious to the organic ground substances of the tissues, which must therefore be protected by adequate fixation before decalcification. Fixation Fix all tissue samples in 10% neutral buffered formalin. All fixed specimens from biopsies are washed in slowly running tap water for a minimum of 30 minutes. Larger specimens are washed up to a maximum of one hour. Avoid rinsing in rapidly running tap water. To avoid loosing small biopsy specimen carefully decan the fixative. The volume of decalcification used should be at least one oz/gm of tissue and should be changed once or twice a day until decalcification is completed. DECALCIFYING FLUID A. Formic acid—Sodium citrate mixtures 1. Stock citrate solution Sodium citrate ................................................ 100 gm Water ............................................................... 500 mL 2. Stock formic acid Concentrated formic acid ............................. 250 mL Distilled water ................................................ 250 mL Well-fixed calcified tissues are placed in a mixture of equal parts of stock citrate and formic acid solutions. Change daily until decalcification is complete. Then wash in running water for 4 to 8 hours. Then dehydrate, clear and embed. ADVANTAGES OF FORMIC ACID DECALCIFICATION • This has proved to be one of the most popular methods, since formic acid is gentler on tissues than nitric acid.

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• Tissues are not ruined if they remain in formic acid beyond the completion of decalcification. • It is safer to handle • Addition of citrate accelerates decalcification by chelating the calcium as it is liberated from the bone. B. Von Ebner’s hydrochloric acid—Sodium chloride mixture Conc. hydrochloric acid (Specific gravity 1.19) ............ 15 mL Sodium chloride ................................................................ 175 gm Distilled water ................................................................... 1000 mL Procedure: Add 1 ml of conc. Hydrochloric acid to each 200 ml of the above mixture, place the fixed calcified tissues in the solution. Change daily until decalcification is complete. END-POINT OF DECALCIFICATION End-point of decalcification can be checked by the following tests: Physical Test The physical test includes bending the specimen or inserting a pin, scalpel directly into the tissue. The disadvantage of inserting a pin or scalpel is the introduction of tears and pin hole artifacts slightly bending the specimen is safer and less disruptive but will not conclusively determine if all calcium salts have been removed. After checking for rigidity, wash thoroughly prior to processing. Chemical Test 1. Stock 5% Ammonium hydroxide Ammonium hydroxide 28% ............................................ 5 mL Distilled water ................................................................... 95 mL 2. Stock 5% Ammonium oxalate Ammonium oxalate .......................................................... 5 gm Distilled wate .................................................................... 100 mL Working ammonium hydroxide/ammonium oxalate solution Add equal parts of 5% ammonium hydroxide and 5% ammonium oxalate solution Procedure: To 5 mL of decalcification solution withdrawn from underneath the specimen, add working 10 mL of ammonium hydroxide/ammonium oxalate solution, mix well and allow to stand for 15–30 minutes. When no precipitate is observed, it indicates the completion of decalcification. Wash the tissue thoroughly in running water, prior to processing.


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TISSUE PROCESSING The term tissue processing refers to treatment of tissue necessary to impregnate them with a solid medium to facilitate the production of sections for microscopy. The aim of tissue processing (Fig. 5.8) is to embed the tissues in a solid medium firm enough to support the tissues and give it sufficient rigidity to enable thin sections to be cut, and yet soft enough to enable the knife to cut the sections with little damage to the knife or tissue.

Fig. 5.8: Automatic tissue processor

Introduction The more satisfactory embedding material for routine histology is paraffin wax. It is essential that the embedding medium thoroughly permeates the tissue in fluid form and that it solidifies with little damage to the tissue. When the tissue is received it is usually partly or completely fixed in a suitable fixative, nearly always an aqueous fixative. Before the tissue can be embedded in paraffin wax, the tissue must be subjected to the following: 1. Completion of fixation. 2. Gentle but completes dehydration to remove aqueous fixative and any tissue water. 3. Clearing with a substance which is totally miscible with both the dehydrating agent, which precedes it and the embedding agent which follows it. 4. Embedding: These four processes depend on complete impregnation of the tissue by the fluid being used. (i) Dehydration First stage in the processing of fixed tissue involves the removal of aqueous and some of the lipid tissue fluids by a variety of compounds, many of which are alcohols of varying types. Several are hydrophilic and attract

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water from the tissues, whereas others affect dehydration by repeated dilution of the aqueous tissue fluids. Numerous dehydrants are readily available and are used in a series of increasing strength, beginning by immersing the tissue in, for example, 70% alcohol, progressing through 95% and finally several changes of absolute alcohol. Common dehydrating fluids used are isopropyl alcohol, ethyl alcohol, methanol. Whatever agent is used its amount in each stage should not be less than ten times the volume of tissue to be dehydrated. Dehydration is also essential before stained sections are mounted. (ii) Clearing or Dealcoholization The use of a clearing agent becomes necessary as the dehydrating agent i.e., alcohol is not miscible with the impregnating medium (paraffin wax). The essential requirements of a clearing agent are that it is miscible with both dehydrating agent and embedding agent. The word ‘Clearing agent’ is used because in addition to removing alcohol many of these substances have the property of making the tissue transparent. This is possible because the refractive index of the clearing agent is approximately equal to that of the tissues. Clearing agents suitable for routine use are: Xylene, Chloroform. (iii) Paraffin Wax Impregnation This process involves the impregnation of the tissues with a medium that will fill all natural cavities, spaces and interstices of tissues even the spaces within the constituting cells and that will set to sufficiently a firm consistency to allow the cutting of thin sections without undue distortion and without alteration of the spatial relationships of the tissue and cellular elements. Vacuum Impregnation This technique is the transferring of cleared tissues to a heated sealed container of molten paraffin wax and applying suction to the container. Vacuum impregnation or embedding under reduced pressure is of use, for certain purposes: a. Lung or other tissues that contain much air. b. Dense pieces of tissues, e.g. skin and embryo which tend to become excessively hard in routine processing. c. To rapidly eliminate the clearing agents. MANUAL PROCESSING SCHEDULE When tissues are to be processed regularly by manual means, an arrangement of containers allows fewer possibilities of error and greater speed.


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Table 5.1: Processing schedule for smaller biopsies measuring less than 5 mm thickness Container 1. 2. 3. 4. 5. 6. 7. 8. 9.

Fluid

Time

100% alcohol 100% alcohol 100% alcohol Xylene Xylene Paraffin Paraffin Paraffin under vacuum Embed

30 min 30 min 30 min 30 min 30 min 30 min 1 hr 30 min

Table 5.2: Manual processing schedule for eyeball Container 1. 2. 3. 4. 5. 6. 7. 8. 9.

Fluid

Time

60% alcohol 80% alcohol 95% alcohol 100% alcohol Xylene Xylene Wax Wax Embed

Leave overnight 8 hours Overnight 24 hours 8 hours Leave overnight 2 hours 4 hours

AUTOMATIC TISSUE PROCESSING Automatic tissue processor enhances the processing of tissue specimens by using heat, vacuum, pressure and agitation. Processor also allows the three stages to be carried out and without the presence of personnel. Routine use of graded alcohols from a lower to higher concentration is standard. The sequential steps given in the processing schedules those follow are for small biopsies and routine specimens. SHORT SCHEDULE (BIOPSIES AND SMALL FRAGMENTS OF TISSUE MEASURING LESS THAN 5 MM THICKNESS) Total processing time—3 to 4 hours (approximately) • Hold if necessary in 80% alcohol • 95% alcohol 3 changes, 15 to 20 mins each • Absolute alcohol, 3 changes, 15 mins each • Equal parts of absolute alcohol and xylene—15 mins • Xylene, two changes—15 mins each • Paraffin, 3 changes—15 mins each • Paraffin under vacuum - 15 to 20 mins and then embed

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OVERNIGHT SCHEDULE (FOR ROUTINE SPECIMENS–LID, CONJUNCTIVA, ORBITAL BIOPSIES) Total processing time—14 to 16 hours (approximately) • 80% alcohol—1 hour • 95% alcohol, 3 changes—1 hour each • Absolute alcohol, 3 changes—1 hour each • Xylene, 3 changes—1 hour each • Paraffin, 3 changes—1 hour each • Paraffin, under vacuum—1 hour and then embed. OVERNIGHT PROCEDURE FOR EYEBALLS, EXENTERATED SPECIMENS AND LARGER BIOPSIES MEASURING MORE THAN 20 MM) Total processing time 17 hours (approximately) • 60% alcohol–4 hours • 95% alcohol 2 changes–1½ hours each • Absolute alcohol - 4 changes–45 mins each • Xylene - 3 changes–1 hour each • Paraffin - 2 changes–2 hours each • Embed Delay in operation of tissue processor: During the overnight processing (14–16 hrs schedule), the processing schedule can be delayed by 4–5 hrs with the help of the timer provided in the instrument, so that the tissues can be prevented from over exposure in hot paraffin which might cause shrinkage. In such conditions, the tissues would remain in 80% alcohol during the delayed period. In case the tissue processor needs to be operated during holidays, the processing schedule can be delayed by 24 hrs or more depending on number of holidays. During this period of delay, the first jar should be filled with 10% neutral buffered formalin so that the tissues remain in the fixative itself and avoid over exposure of alcohol.

EMBEDDING Tissues that have been completely dehydrated and cleared are impregnated with paraffin wax by immersion in a succession of molten wax baths. A physical advantage is provided in the handling of small specimens by surrounding the specimen with a mask or block of embedding material, thus allowing the specimens to be handled and fixed to the microtome block without damage to the actual tissue. PARAFFIN WAX EMBEDDING Paraffin embedding is the technique used in virtually all-general pathology laboratories and almost all-ophthalmic pathology laboratories (Fig. 5.9).


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Fig. 5.9: Embedding equipment

Advantages of Paraffin Embedding 1. Sections embedded in paraffin can be cut thinner. 2. A wide variety of special histochemical stains can be used 3. Serial sections can be readily prepared. Remaining uncut tissue residing in the paraffin block can be kept permanently. EMBEDDING EQUIPMENT There are several specialized materials and equipment that facilitate paraffin embedding. Embedding molds are used for casting/shaping liquid paraffin into blocks. These are: i. Stainless steel molds are perhaps the most widely used and are considered ideal for embedding purpose. It is manufactured in various sizes to accommodate different sizes of tissue specimens. They are re-usable but periodic cleaning is required. ii. L pieces consist of two L-shaped pieces of metal resting on a flat metal base. The L pieces can be moved to adjust the size of the mold so that, it will match the size of the tissues. PARAFFIN EMBEDDING PROCEDURE Fresh molten wax (melting point of 56°C to 60°C)is poured into the mold, which is to be used for embedding. The wax touching the mold will quickly form a thin solid layer. The tissues are then lifted from the final wax with a previously warmed forceps and placed in the bottom of the mold. The side of the tissue from which it is desired to take sections is placed face down and all other tissue must be carefully oriented, so that the plane of the sectioning will be correct. It is necessary to press down the tissue specimen in the mold for few seconds until it is held by the cooling wax. It is also necessary to flame the forceps periodically to prevent wax and tissue from adhering to its points. A label bearing the pathology number of the tissue is placed in the mold next to the tissue. When the wax becomes partly solid

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the mold should be placed in a basin of cold water (10° to 18° C) or in a refrigerator to cool. This method of cooling lessens the tendency of some waxes to crystallize when allowed to set at room temperature and give blocks of uniform, smooth and solid consistency. TRIMMING When blocks are hardened it is removed. Excess wax is cut leaving 2 to 3 mm of wax between edge and tissue, so that the block forms a four-sided prism. A small paper tag bearing the tissue number is affixed to the block with the help of hot knife or spatula. To attach the block to the wooden block holder, simultaneously heat both and press together while molten and seal the edges by means of a hot knife.

PARAFFIN SECTION CUTTING Equipment required 1. Microtome 2. Water bath, thermostatically controlled 3. Drying oven 4. Forceps and small squirrel hair brush 5. Coated slides 6. Slide rack 7. Tissue paper 8. Ice cubes. 1. Microtome (Fig. 5.10): It is an instrument designed for the accurate cutting of thin sections of tissues. Two types of microtomes used for light microscopy are rotary microtome—where the block moves, is the most widely used. Sliding microtome— where the knife moves, it is particularly useful when sectioning large blocks. 2. Water bath: With temperature of about 54°C to 58°C as required.

Fig. 5.10: Microtome


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3. Drying ovens: The fixing of sections to slides can be done in an oven at 54 to 58°C. 4. Forceps and brush: It is necessary for the manipulation of sections during cutting and for removal of folds and creases in sections after floating out. 5. Section adhesive: Certain adhesive mixtures are used for coating the slides so that the section remains adherent to the slide during subsequent staining. They are all protein solution and since proteins retain many stains, the amount and concentration of the adhesive used for the slides must be kept to a minimum. These adhesives mainly act by reducing the surface tension and thus produce closer capillary adhesion of the section to the slide. Coating of Slides 1. Chrome alum gelatin solution Gelatin ................................................................................ 3 gm Chromium potassium sulfate ......................................... 0.5 gm Distilled water ................................................................... 1000 mL Method: Heat the water to 60°C and completely dissolve the gelatin with the aid of a magnetic stirrer. Stir in the chromium potassium sulfate (the solution should turn pale blue) add few crystals of thymol as a preservative. Dip clean slides in the warm solution, blot the edges and then stand the slides on air dry. Once dried, store in dust-free container until ready for use. 2. 3-Aminopropyltriethoxy silane (APES) Method: 1. Place slides in a rack and immerse in acetone for 5 min. 2. Immerse in a solution of 2% silane in acetone v/v for 5 min. 3. Rinse in two consecutive baths of acetone for 5 min. each. Allow to dry in room temperature or 60° in oven for 45 minutes and store at room temperature indefinitely. PRACTICAL SECTION CUTTING Section cutting may be carried out with the operator standing or sitting. Setting of Microtome (Rotary) The floatation bath should be filled with distilled water at the appropriate temperature. The block is removed from the ice, wiped dry and clamped firmly in the clamp of the microtome. The object holder is moved until the surface of the wax just touches the knife edge, cutting then being commenced with regular even strokes (there must be no jerking motion).

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It is important to tighten knife clamp screws securely, similarly block clamp screws must also be firm. Trimming of tissue block: In order to trim away any surplus wax and to expose a suitable area of the tissue for sectioning, the section thickness is adjusted over 5 to 10 microns. A series of glass slides is numbered with a diamond marker pencil. When the block has been trimmed a new blade is used for cutting sections. The section thickness is now set to appropriate level, for routine purpose, 4-6 microns, eyeballs - 8 microns. Cutting Sections Paraffin blocks are cooled with ice cubes before cutting, such cooling can cause the production of flat sections and also make the wax block harder and firmer, thus facilitating the cutting of thin sections. It is important that the upper and lower surfaces of the block to be cut are parallel in order that straight ribbons of sections are obtained. The first section is raised carefully with the index finger or camel‘s hairbrush. As the knife strikes the block to start cutting the next section, locally generated heat and pressure weld the edge of section number 2 to the back edge of section number one. This continues with succeeding sections. When the ribbon is several cms long, handling is greatly eased, the first section being held with the finger or forceps and the last section being detached from the knife by means of a small brush. The ribbon of sections may be immediately floated onto the water bath. To obtain flat sections, it is necessary to spend time in cutting and gentle stretching of the ribbon, before floating on the water bath. Floating out sections (Fig. 5.11): The action in floating out must be smooth, with the trailing end of the ribbon making contact with the water first. The slight drag produced when the ribbon touches the water surface is sufficient to produce tension in the ribbon and remove some folds from the sections. When the ribbon has come to rest on the water, any remaining wrinkles

Fig. 5.11: Tissue floatation bath


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and folds are removed by tearing apart, using forceps. Complete flattening and expansion of sections is achieved after several minutes on the water surface, this may be hastened by transferring the sections on a slide to second water bath at higher temperature. Prolonged floating out of sections on the water bath may be avoided as tissues may expand beyond their original size and become distorted. Picking up sections: The ribbons of sections floating on water may be split into individual or groups of sections by the use of forceps. Picking up of a section on slide is achieved by immersing the slide, lightly smeared with adhesion, vertically in the water bath to three-fourth of its length and maneuvering the section into contact with the slide. On lifting the slide vertically from water, the section will flatten onto the slide. The sections are correctly positioned on the slide and blotted lightly with moistened blotting paper to remove excess water and to increase contact between section and the slide. Drying sections: The sections are dried in an oven at 54oC to 58 oC temperature, minimum of 45 minutes. The advantage of oven drying is that dust is less likely to settle on sections. After removal of sections from the oven, the cool sections are stored in dust free container.

FROZEN SECTION Purpose: To prepare the slides for histopathologic diagnosis within short duration to rule out malignancy. Various special stain techniques or rapid diagnosis are performed on fresh or fixed or frozen tissues. This is applicable for all tissues required except for aspirates or scrapped sample. Principle When the tissue is frozen, the water within the tissue turns to ice and in this stage, the tissue is firm with the ice acting as the embedding medium. Cryostat (Fig. 5.12) A cryostat consists of a rotary type microtome enclosed in mechanically refrigerated cabinet; virtually, it is a microtome in a deep freeze. It has a platform or chamber to freeze tissue rapidly and mostly have self-defrosting capability. In the cryostat’s controlled environment, the microtome knife, cabinet interior and instruments are all maintained at the same operating temperature, so that the sectioning operation is rarely affected by room temperature. The freezing platforms or chambers eliminate the need to use liquid nitrogen or other freezing agents for the vast majority of tissue types.

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Fig. 5.12: Cryostat

Cryostat Knife/Disposable Blades In a disposable knife system the blade must be cleaned before it is mounted in the knife holder. Most disposable blades are coated with silicone or oil, which can be removed with xylene followed by absolute alcohol. Knife angle is important but seems to be a little less critical than in the paraffin technique. Temperature Each type of tissue has a optimum cutting temperature, but it is impossible especially during sectioning for rapid diagnosis, to adjust the cryostat temperature for every tissue. Machines are set at (–19°C to – 21°C) and performed adequately for most tissues. The cabinet, microtome, microtome knife and any tools (forceps, paintbrushes, etc.) must all be at the same temperature. It is a good idea to keep a spare sharp microtome knife stored in the cabinet so that if the regular knife becomes dull or nicked one can instantly change knifes without regard for cool-down time. Sections Adhesion Adhesives are infrequently necessary when performing an H and E stain on fresh tissue. The proteins in the tissue and the tissue fluids coagulate in the first alcohol or fixative and help the section adhered to the slide. The sections of fixed tissue have a greater tendency to float off the slide, and


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many special staining techniques will loosen both fresh and fixed tissues. Thorough washing of fixed tissues before sectioning can also help prevent section loss. If the sections just will not stay, several adhesives may be used, provided that the adhesives not interfere with the stain to be performed. The common adhesive used is chrome alum gelatin solution (as in case of paraffin sections). Cutting Technique • A small amount of commercially available liquid embedding medium is spread evenly on the appropriate object holder. The tissue is placed on the surface of the object holder and completely surrounded with embedding medium. The embedding medium should provide adequate support for sectioning and have at least 2 mm margins. Care must be taken to orient the tissue properly. • The object holder is placed on the freezing platform in the cryostat to start the freezing process. If necessary, more embedding medium is added as the tissue freezes. The freezing process can be accelerated if the object holder is at - 20°C, the same temperature as the cryostat chamber. • When the tissue is completely frozen, the object holder is securely mounted to the microtome head. • Preliminary facing of the tissue : – Adjust the knife holder so that the tissue just clears the knife – Release the automatic advance pawl from the toothed ratchet wheel – Manually advance the ratchet wheel until the tissue extends over the knife-edge approximately 0.1–0.5 mm. – Release the lock on the drive wheel – Rotate the drive wheel counter clock-wise 180° to cut the rough tissue section. Immediately return the drive wheel to the upright position. – Repeat the above steps until the desired cutting plan is reached. Lock the drive wheel. • Engage the automatic advance pawl and the toothed ratchet wheel. Carefully clean the knife edge with dry gauze. • Unlock the drive wheel. Slowly and smoothly rotate the wheel clockwise. As the section begins to form on the knife edge, a cold paint brush may be used to gently guide the section down the face of the knife to help keep the section flat. Some machines have plastic anti-roll devices attached to guide the sections over the knife. • Mount the section on the slide by bringing the surface of room temperature slide very close to the section. The section will seem to jump on the slide and the mounting medium will melt immediately. • Slide is ready to be placed in fixative or stained. • Remember to lock the drive wheel and clean the tissue debris from the cabinet mechanism and knife.

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STAINING The following rapid hematoxylin and eosin procedure is used: • Alcoholic formalin (90 mL of 80% alcohol + 10 mL of formalin) .......................................... 15 seconds. • 70% alcohol ...................................................... 10 dips • Rinse well in distilled water • Harri’s hematoxylin ....................................... 45 seconds–1 minute • Rinse well in tap water • Ammonia water .............................................. till the sections turn blue • Rinse in tap water • Counter stain in eosin/phloxine solution ... 30 seconds • Dehydrate with 5 quick dips in each of two changes of 95% alcohol. • Complete dehydration and clearing in two changes of absolute alcohol and two changes of xylene, 10 dips in each solution. • Mount the slides in DPX.

STAINING TECHNIQUES If unstained sections of tissue are examined under the microscope with transmitted light, little details other than nuclear and cellular boundaries can be identified. Staining the sections with one or more dyes permits the evaluation of the physical characteristics and relationships of the tissues and their constituent cells. This is facilitated if two contrasting stains are used such as hemotoxylin (which stains the nuclear detail) and eosin (which stains the cytoplasmic details of the cell and extracellular tissues). The most commonly used routine staining method in histopathology is hematoxylin-eosin. Routine Staining Stains for Nuclei The most important of these is hematoxylin. This is a basic dye that causes staining of the acidic nucleoproteins. The resulting color is a bluish purplishblack. Substances that stain with this basic dye are described as basophilic. Counter Stain Sections that have been stained with hematoxylin alone are unsatisfactory for general examination unless a counterstain is used to demonstrate cytoplasmic details, and details of cellular tissues. Eosin is, by far, the most commonly used of this background or contrast stains. It gives a bright red color to erythrocytes and muscles. It imparts a pale pink color to the cytoplasm and to proteins in edema fluid. It is an acidic dye. Substances that stain with eosin are described as acidophilic.


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1. Harri’s Hematoxylin This stains the nuclei only. Enhancement of the blue color is accomplished by washing the slides in running tap water. Preparation Hematoxylin ........................................................ 5 gm 100% ethyl alcohol ..............................................50 mL Potassium or ammonium alum ........................ 100 gm Distilled water ..................................................... 1000 mL Mercuric oxide ....................................................2.5 gm Use a 2000 ml flask for the alum and water and a small flask for alcohol and hematoxylin, completely dissolve the alum in the distilled water with the aid of heat and a magnetic stirrer. Vigorously shake to dissolve the hematoxylin in the alcohol at room temperature. Remove the alum and distilled water from the heat. Slowly combine the two solutions. Return the combine solution to heat. Bring to a boil as rapidly as possible, approximately 1 minute or less. Remove from heat and slowly add the mercuric oxide. Return the solution to the heat until it becomes dark purple, remove it from the heat and plunge it into a basin or sink of cold water to cool. The solution is ready for use. Add 20 ml of glacial acetic acid to intensify the nuclear stain. Always filter before each use. 2. Eosin stock solution Eosin Y, water soluble ........................................1 gm Distilled water ..................................................... 100 mL 3. Phloxine stock solution Phloxine B ............................................................ 1 gm Distilled water ..................................................... 100 mL 4. Eosin-Phloxine working solution Eosin stock solution ............................................ 100 mL Phloxine stock solution ...................................... 10 mL 95% Alcohol ......................................................... 780 mL Glacial acetic acid ............................................... 4 mL The solution is used for approximately one week. ** Routine staining of virtually all specimens are done using hematoxylin and eosin stain Staining Procedure a. Deparaffinize the slides • Xylene I ........................................................... 10 mins • Xylene II ......................................................... 10 mins • Xylene III ........................................................ 10 mins • 100% Alcohol I ..............................................4 mins


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b. c. d. e. f. g. h. i. j.

• 100% Alcohol II ............................................. 4 mins • 95% Alcohol I ................................................ 4 mins • 95% Alcohol II ............................................... 4 mins • 80% Alcohol ................................................... 4 mins • 70% alcohol ....................................................4 mins • 60% alcohol ....................................................4 mins • Water .............................................................. 4 mins Stain in Harri’s hematoxylin ............................. 8 mins Wash in tap water ............................................... 3 mins Differentiate in 1% acid alcohol (99 mL of 70% alcohol + 1 mL of HCl) 1 to 2 dips Wash in briefly in tap water Place in weak ammonia water (0.4% of ammonia in D. water) or saturated lithium carbonate solution until the sections are bright blue Wash thoroughly in running water Counter stain in eosin-phloxine solution ........ 1-2 minutes Dehydrate and clear through two changes each of 95% alcohol, absolute alcohol and xylene ......2 minutes each Dry and mount the slides in DPX mountant

RESULTS Nuclei ...................................................................Blue Cytoplasm ............................................................ Pink to red Most other tissue structures .............................. Pink to red

Fig. 5.13: Hematoxylin and eosin stain showing Flexner Wintersteiner Rosettes in a case of retinoblastoma. Note: Nuclei are stained blue and cytoplasm is stained pink in color

SPECIAL STAINS * Paraffin sections with thickness of 6 to 10 microns are required


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Periodic Acid and Schiff Stain (PAS) Stains glycogen, basement membrane, e.g. descemet’s membrane, Bruch’s membrane, lens capsule, vascular basement membrane and fungi. Principle: The reaction is based on oxidation of certain tissue elements to aldehydes by periodic acid. Schiff reagent is prepared by treating basic fuchsin (pararosaniline) with sulphurous acid. Reduction causes the loss of quinoid structure and masking of chromosphores. A colorless compound referred to as eucofuchsin is formed. Following the Schiff reaction, washing in running water causes the loss of the bound sulphurous acid group attached at the central carbon atom, the restoration of quinoid structure in the dye bound by the aldehyde, and the visualization of the typical schiff color. Preparation 1. 0.5% Periodic acid solution: Periodic acid ........................................................ 0.5 gm Distilled water ..................................................... 100 mL 2. 1N Hydrochloric acid solution Hydrochloric acid, specific gravity (1.19) .......83.5 mL Distilled water ..................................................... 916.5 mL 3. Coleman’s Schiff reagent Basic fuchsin ........................................................ 1 gm Distilled water heat to 60°C .............................. 200 mL and bring just to boil point. Cool and then add Potassium metabisulfite .....................................2 gm 1 N hydrochloric acid ......................................... 10 mL Let bleach for 24 hours then add activated carbon .......................................... 1 gm Shake for one minute, then filter through coarse filter paper. Repeat filtration until the solution is colorless. Store in refrigerator Staining Procedure 1: a. Deparaffinize and hydrate to water b. Oxidize in periodic acid solution ..................... 5 minutes c. Rinse in distilled water d. Schiff reagent .......................................................15 minutes e. Wash in lukewarm tap water ............................10 minutes f. Harris hematoxylin ............................................. 6 minutes g. Wash in tap water ............................................... 15 minutes h. Dehydrate and clear through 2 changes of 95% alcohol, 100% alcohol and xylene ............ 2 minutes each i. Dry and mount the slides using resinous medium


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Staining Procedure 2: a. Deparaffinize and hydrate to water b. Oxidize in periodic acid solution ..................... 4 mins c. Running tap water ..............................................1 minute d. Distilled water ..................................................... 10 dips e. Schiff reagent .......................................................4 minutes f. Running tap water ..............................................5 minutes g. Harris hematoxylin ............................................. 1 minute h. Running tap water ..............................................1 minute i. Ammonia water .................................................. 5 dips j. Running tap water ..............................................1 minute k. 95% Alcohol ......................................................... 10 dips (2 changes) l. 100% Alcohol .......................................................10 dips (2 changes) m. Xylene ...................................................................10 dips (2 changes) n. Dry and mount using resinous medium RESULTS Glycogen, mucin, and some basement membranes.....red to purple Fungi ..................................................................... red to purple Nuclei ...................................................................blue

Fig. 5.14: PAS stain showing goblet cells in a case of conjunctival papilloma (x 100)

Fig. 5.15: PAS stain of the section of corneal button showing Descemet’s membrane staining pink


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ALCIAN BLUE STAINING (pH 2.5) FOR ACID MUCOPOLYSACCHARIDES Principle: Alcian blue is a copper phthalocyanine basic dye that is water soluble and is colored blue because of its copper content. When used in a 3% acetic acid solution (pH 2.5) Alcian blue stains both sulfated and carboxylated acid mucopolysaccharides and sulfated and carboxylated sialomucins (glycoproteins) Reagents Required 1. 3% acetic acid Glacial acetic acid ............................................... 3 mL Distilled water ..................................................... 97 mL 2. Alcian blue solultion (pH 2.5) Alcian blue 8G X .................................................1 gm 3% acetic acid ...................................................... 100 mL 3. Nuclear fast red (Kernechtrot) solution 0.1 gm nuclear fast red in 100 mL of 5% aluminium sulfate solution. Heat to boiling slowly, cool, filter and add a grain of thymol as a preservative. Staining Procedure a. Place in 3% acetic acid solution for 3 minutes b. Stain in Alcian blue solution for 30 minutes c. Wash in running water for 10 mintues d. Rinse in distilled water e. Counterstain in filtered nuclear fast red solution for 5 minutes f. Wash in running water for 1 minute

Fig. 5.16: Alcian blue staining in a case of macular dystrophy of cornea showing blue staining of acid mucopolysaccharide (x200)

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g. Dehydrate and clear through 95% ethyl alcohol, absolute alcohol and xylene, 2 changes each, 2 minutes each h. Mount with resinous media. Result Weakly acidic sulfated mucosubstances Hyaluronic acid and sialomucins ........................... dark blue Nuclei ......................................................................... red to pink Cytoplasm .................................................................. pale pink CONGO RED (BENHOLD’S) METHOD FOR AMYLOID Principle: Congo red is a benzidine derivative that can react with cellulose and it is a linear molecule and allows azo and amine group of the dye to form hydrogen bonds with hydroxy radicals of the amyloid. * Paraffin sections with thickness of 8 to 12 microns required for this stain Reagents Required 1. 1% Congo Red solution Congo red ............................................................ 1 gm Distilled water ..................................................... 100 mL 2. 1% Sodium Hydroxide solution Sodium hydroxide .............................................. 1 gms Distilled water ..................................................... 100 mL 3. Alkaline-Alcohol solution 1% Sodium Hydroxide solution ....................... 1 mL 50% Ethyl Alcohol solution ............................... 99 mL Mayer’s/Harri’s Hematoxylin Solution Staining procedure: a. Stain in filtered Congo red solution for 1 hr b. Rinse brief in distilled water c. Differentiate rapidly in alkaline-alcohol solution d. Wash in running tap water for 5 mins e. Counterstain in Mayer’s/Harri’s hematoxylin for 5 minutes f. Wash in running tap water for 15 minutes g. Dry and mount using resinous media Result: Amyloid ........... Pink to red and “apple green” birefringence with polarized light Nuclei ............... Blue


Fig. 5.17: Section of corneal button showing red rose color of the amyloid in a case of lattice dystrophy of the corneal stroma (x100)

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Fig. 5.18: The same slide with polarizer showing apple green birefringence (x100)

GOMORI’S ONE STEP TRICHROME STAIN FOR MUSCLE FIBERS Principle: A plasma stain (Chromotrope 2R )and a connective tissue fiber stain (light green or aniline blue) are combined in a solution of phosphotungstic acid to which glacial acetic acid has been added. Phosphotungstic acid favors the red staining of muscle and cytoplasm. The tungstate ion is specifically taken by the collagen and the connective tissue fiber stain is subsequently bound to this complex coloring the collagen green or blue. Reagents Required 1. Bouin’s fixative solution: To 750 ml saturated aqueous picric acid solution add 250 ml formalin (37–40%) and 50 ml glacial acetic acid (saturated picric acid: 2 gm in 100 ml distilled water) 2. Weigert’s iron hematoxylin: Stock solution A: Hematoxylin ........................................................ 1 gm 95% ethyl alcohol ................................................ 100 mL


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Stock solution B: 29% Ferric chloride ............................................. 4 mL Distilled water ..................................................... 95 mL Concentrated HCl ............................................... 1 mL Working solution: Equal parts of stock solutions A and B, as 100 ml A and 100 ml B (It can be stored for about 2 weeks) 3. Gomori’s trichrome stain: Chromotope 2R ................................................... 0.6 gm Light green (or) Aniline blue ............................0.3 gm Glacial acetic acid ............................................... 1 mL Phosphotungstic acid ......................................... 0.8 gm Distilled water ..................................................... 100 mL Staining Procedure a. Place in Bouin’s fixative in oven at 56°C for 1 hr or at room temperatureovernight b. Wash well in running water till sections are clear c. Stain in Weigerts iron hematoxylin working solution for 10 minutes d. Rinse in tap water e. Stain in Gomori’s trichrome stain for 15–20 minutes f. Rinse in 1% acetic acid g. Rinse in distilled water; dry and mount in resinous medium Result Muscle fibers .......................................................red Collagen ............................................................... green or blue Nuclei ...................................................................black

Fig. 5.19: Trichrome staining of the eyelid showing muscle fibers staining red, collagen fibers staining blue


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Fig. 5.20: Section of corneal button showing red staining of the hyaline material in a case of granular dystrophy of the corneal stroma

VERHOEFF’S ELASTIC STAIN Principle The ferric chloride and iodine serves as mordants and they also have an oxidizing function that assists in converting hematoxylin to hematin. Differentiation is accomplished by using excess mordant or ferric chloride, to break the tissue mordant—dye complex. The dye will be attracted to larger amount of mordant in the differentiating solution and will be removed from the tissue. The elastic tissue has strongest affinity for the iron hematoxylin complex and will retain the dye longer than other tissue elements. This allows other tissue elements to be decolorized and the elastic fibers remain stained. Sodium thiosulfate is used to remove excess iodine, Van Gieson solution is used as counterstain. Reagents 1. 10% Alcoholic hematoxylin: Hematoxylin ........................................................ 10 gm Ethanol ................................................................. 100 mL 2. 10% Ferric chloride: Ferric chloride ..................................................... 10 gm Distilled water ..................................................... 100 mL 3. Verhoeff’s Iodine solution: Iodine .................................................................... 2 gm Potassium iodide ................................................ 4 gm Distilled water ..................................................... 100 mL Mix the crystals of iodine and potassium iodide in a flask, shake vigorously, then add DW 20 ml at a time.

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4. Verhoeff’s elastic stain working solution: Mix equal parts of above solutions and ethanol 5. 2% Ferric chloride (Differentiation solution): 10% Ferric chloride ............................................. 20 mL Distilled water ..................................................... 80 mL 6. Van Gieson solution: 1% Acid Fuchsin .................................................5 mL Saturated Picric acid ...........................................95 mL 7. 5% Sodium Thiosulfate solution Staining Procedure a. b. c. d. e. f. g. h.

Stain the deparaffinized slide in Verhoeff’s elastic solution…..15 minutes Wash in lukewarm running water………20 minutes Place in D water Differentiate in 2 % ferric chloride, check microscopically, elastic fibers are black and sharply fine and the background is grey Place in 5% sodium thiosulphate solution for 1 minute Wash in tap water for 5 minutes Counterstain with Van Gieson solution for exactly 1 minute Dehydrate through 2 changes of 95% alcohol, 100% alcohol and xylene— 2 minutes each. Dry and mount.

Results Elastic fibers ......................................................... Black Nuclei ...................................................................Black Other tissue structures ....................................... Yellow

Fig. 5.21: Temporal artery biopsy showing rupture of elastic lamina and granulomatous inflammation in a case of giant cell arthritis


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GRAM STAIN FOR BACTERIA Principle Crystal violet is applied first and then followed by an iodine mordant forming a dye lake. At this point both Gram positive and Gram- negative organisms are stained. Both types of bacteria have a cell wall composed of peptidoglycan, and the wall of Gram-positive bacteria are thicker than Gram-negative organisms, and the Gram negative bacteria also contain a layer of lipopolysaccharide external to the cell wall. The large crystal violetiodine molecular complex cannot be easily washed out of the intact peptidoglycan layer of the Gram positive cells, however, it is easily removed from Gram negative bacteria because the acetone disrupts the outer lipopolysaccharide layer and the remaining thin peptidoglycan cell wall cannot retain the complex. Undamaged Gram positive cell walls will retain the crystal violetiodine complex. After decolorization, a counter stain is applied to color the Gram negative bacteria (Fig. 5.22). Reagents Required 1. Crystal violet: Crystal violet .......................................................1 gm Distilled water ..................................................... 100 mL 2. Grams iodine: Iodine .................................................................... 1 gm Potassium iodide ................................................ 2 gm Distilled water ..................................................... 300 mL 3. 1% Basic fuchsin: Strong carbol fuchsin ......................................... 10 mL Distilled water ..................................................... 90 mL 4. Gallego’s solution: Distilled water ..................................................... 100 mL 37–40% Formaldehyde ....................................... 2 mL Acetic acid (glacial) ............................................. 1 mL 5. Picro acetone: 1 gm picric acid in 100 ml acetone 6. Acetoxylene: Equal parts of acetone and xylene Staining Procedure a. 1% crystal violet .................................................. 1 minute b. Wash in water c. Grams iodine .......................................................1 minute

412 d. e. f. g. h. i. j. k. l. m. n.

Manual of Medical Laboratory Techniques Wash in water Acetone ............................................... 1 dip (Decolorization) Wash in water 1% Basic fuchsin ................................ 2 minute Wash in water Gallego’s solution ............................. 2 minutes (for differentiation) Wash in water Acetone ............................................... 30 seconds Picroacetone ....................................... 2 minutes Acetoxylene ....................................... 2 dips Dry and mount

Results Gram positive bacteria ..................... blue Gram negative bacteria .................... red Back ground ...................................... yellow

Fig. 5.22: Gram stain showing Gram-positive bacteria staining blue

ACID FAST BACILLI STAIN (FIG. 5.23) Reagents required: 1. Carbol fuchsin (strong) 2. 20% Sulfuric acid Concentrated sulfuric acid .............. 20 mL Distilled water ................................... 80 mL 3. Stock methylene blue solution Methylene blue solution .................. 1.4 gm 95% ethyl alcohol .............................. 100 mL 4. Working solution: Stock methylene blue ....................... 10 mL Distilled water ................................... 90 mL


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Staining Procedure a. Stain sections with commercially available Carbol Fuchsin and heat it carefully till fumes come, allow it to stain for 5 minutes b. Wash thoroughly with water c. Decolorize with 20% sulfuric acid till section becomes colorless d. Wash in water e. Counter stain with working methylene blue solution for 2 to 3 minutes f. Wash in water. Result Acid fast bacilli ....................................................bright red Erythrocytes ......................................................... yellow orange Other tissue elements ......................................... blue

Fig. 5.23: AFB staining showing multiple acid fast bacilli staining bright red in blue background (x400)

GROCOTT’S METHENAMINE SILVER NITRATE (GMS) METHOD FOR FUNGI (FIG. 5.24) Principle Polysaccharides in the fungal wall are oxidized to aldehydes by the chromic acid. Chromic acid is a strong oxidant, further oxidizing many of the newly released aldehyde groups to breakdown products that will not react. Only substances that possess large quantities of polysaccharides such as the fungal walls, glycogen and mucins will remain reactive with the methenamine-silver, reducing it to visible metallic silver. Methenamine gives the solution the alkaline properties necessary for proper reaction and the sodium borate acts as the buffer. Gold chloride is a toning solution and the thiosulfate removes any unreduced silver.

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Reagents Required 1. 5% chromic acid Chromium trioxide ............................................. 5 gm Distilled water ..................................................... 100 mL 2. 5% silver nitrate solution Silver nitrate ........................................................ 5 gm Distilled water ..................................................... 100 mL 3. 3% methenamine solution Hexamethylene tetramine ................................. 3 gm Distilled water ..................................................... 100 mL 4. Methenamine—Silver nitrate stock solution 5% silver nitrate .................................................. 5 mL 3% methenamine .................................................100 mL 5. 5% borax solution Sodium borate ..................................................... 5 gm Distilled water ..................................................... 100 mL 6. Methenamine—Silver nitrate working solution Methenamine—Silver nitrate stock .................. 25 mL Distilled water ..................................................... 25 mL 5% borax ............................................................... 2 mL * Prepare fresh prior to use, do not use if cloudy. Prewarm the solutions seperately in the oven at least 40 minutes before use. 7. 1% Sodium bisulfite: 1 gm in 100 ml distilled water 8. 0.1% Gold chloride solution: 10 ml of 1% gold chloride stock solution and 90 ml of distilled water 9. 2% Sodium thiosulfate (Hypo) Sodium thiosulfate ..............................................2 gm Distilled water ..................................................... 100 mL 10. 0.1% light green solution 0.1 gm light green in 0.1% acetic acid Staining Procedure a. Oxidize in freshly prepared 5% chromic acid for 1 hour, discard the solution after use b. Rinse in tap water c. 1% sodium bisulfite for 1 minute, discard the solution d. Wash in tap water for 10 minutes


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e. Rinse 4 times in distilled water f. Place in working methenamine silver solution in oven at 58° to 60° for 45 minutes to 60 minutes or until sections turn a yellow-brown. Using paraffin coated or Teflon forceps, remove a control slide, rinse in the warmed distilled water and check microscopically for adequate silver impregnation. Fungi should be a dark brown. Rinse in distilled water and return to the silver solution if reaction is too pale. g. Rinse in 6 changes of distilled water h. Tone in 0.1% gold chloride for 2–5 minutes. Solution may be refiltered and reused for about 100 slides i. Rinse in distilled water j. Treat with 2% sodium thiosulfate for 2–5 minutes, discard solution k. Wash thoroughly in tap water for 5 minutes l. Counterstain in light green solution for 30–45 seconds m. Dry and mount in resinous medium. Result Fungi ................................................................ sharply delineated in black Pneumocystis carinii ..................................... black Mucin ............................................................... taupe to dark grey Inner parts of mycelia and hyphae ............. old rose Back ground ................................................... pale green

Fig. 5.24: GMS stain showing fungi as black in a case of fungal keratitis

OIL O RED METHOD FOR LIPIDS (FIG. 5.25) Principle Staining with oil soluble dyes is based on the greater solubility of the dye in the lipoid substances than in usual hydroalcoholic dye solvents.


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FOR FROZEN SECTION Reagents Required 1. Oil O Red Stain (Stock solution) Oil O Red .............................................................0.5 g 99% Iso-propanol ................................................ 100 mL Working Solution Oil O Red stock ................................................... 6 mL Distilled water ..................................................... 4 mL 2. Mayer’s/Harri’s Hematoxylin Procedure Prepare the working solution of Oil O Red and allow to stand for 5 minutes and then filter. The filtrate can be used for several hours. a. Stain thin frozen sections for 10–15 minutes b. Wash in water. c. Stain nuclei briefly (10–30 seconds) in Mayer’s/Harri’s Hematoxylin d. Blue in water. e. Drain slides and mount the section with glycerol/water soluble mount Result Lipid ..................................................................... Red Nuclei ...................................................................Blue

Fig. 5.25: Oil O’ red stain showing positivity of lipids in a case of sebaceous gland carcinoma (Frozen Section)


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For Permanent Section Reagents Required: 1. 100% propylene glycol 2. 0.5 % Oil O Red solution Oil O Red .............................................................0.5 g Propylene glycol .................................................100 mL Add a small amount to propylene glycol to the Oil O Red and mix well. Crush larger pieces. Gradually add the remainder of the propylene glycol stirring periodically. Heat gently until the solution reaches 96°C. Do not allow to go over 100° C. Stir while heating. Pass through coarse filter paper while still warm. Allow to stand overnight at room temperature. Filter through medium glass filter with aid of vacuum. If the solution becomes turbid refilter. 3. 85% propylene glycol solution 100 % propylene glycol ...................................... 85 mL Distilled water ..................................................... 15 mL 4. Mayer’s/Harri’s hematoxylin solution Staining Procedure a. b. c. d. e. f. g. h.

Rinse the slides containing the permanent sections in distilled water Place in absolute propylene glycol for 3–5 minutes Stain in Oil O Red solution for 48–72 hrs Differentiate in 85 % propylene glycol solution for 1–2 minutes. Rinse in 2 changes of distilled water Stain in hematoxylin solution for 5 minutes Rinse thoroughly in distilled water Mount in warmed glycerin jelly solution.

Result Lipids .................................................................... red color Nuclei ...................................................................blue BLEACHING OF MELANIN PIGMENTS Principle Melanin pigments are bleached by using oxidizing agents such as potassium permanganate followed by oxalic acid to clear the sections of color. Reagents Required 1. 0.25% Potassium permanganate Potassium permanganate ..................................0.25 gm Distilled water ..................................................... 100 mL

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2. 5% Oxalic acid Oxalic acid ........................................................... 5 gm Distilled water ..................................................... 100 mL Bleaching Technique a. 0.25% potassium permanganate ....................... 5 minutes (if bleaching is not completed, time can be extended) b. Wash in water ..................................................... 5 minutes c. 5% oxalic acid ...................................................... 3 minutes d. Wash in water ..................................................... 2 minutes Continue with routine staining PERL’S IRON STAIN FOR HEMOSIDERIN PIGMENTS Principle The sections are treated with an acidic solution of potassium ferrocyanide and any ferric iron present reacts to form an insoluble bright blue pigment called Prussian blue. Reagents Required 1. 20% Hydrochloric acid HCl ........................................................................ 20 mL Distilled water ..................................................... 80 mL 2. 10% Potassium ferrocyanide stock solution Potassium ferrocyanide .....................................10 gm Distilled water ..................................................... 100 mL 3. HCl-Potassium ferrocyanide working solution Equal parts of 20% HCl and 10% potassium ferrocyanide solution 4. Nuclear fast red solution (Refer Alcian blue stain) Staining Procedure a. Place slides in freshly mixed HCl-potassium ferrocyanide working solution for 30 minutes b. Rinse slides in distilled water c. Counter stain with nuclear fast red solution for 5 minutes d. Wash thoroughly in running tap water for 2 minutes e. Mount slides with resinous medium. Result Hemosiderin and some oxides and salts of iron .. blue Nuclei and cytoplasm ..............................................pink to red


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ALIZARIN RED FOR CALCIUM Principle Alizarin red reacts with cations (calcium), forms alizarin red-calcium complex in a chelation process (Fig. 5.26). Reagents 1. Alizarin red solution: Alizarin red S .......................................................2 gm Distilled water ..................................................... 100 mL Adjust the pH to 4.0 to 4.3 by adding drop by drop of ammonium hydroxide 2. Acetone 3. Aceto-xylene solution 4. Xylene Staining Procedure a. Place the deparaffinzed slide in alizarin red solution——30 seconds –5 minutes examine microscopically, when orange red appears, shake off the excess stain b. Dehydrate and clear through acetone, aceto-xylene and xylene c. Dry and mount in resinous medium. Results Most calcium salts .................................... Birefringent red precipitate Calcium oxalate ......................................... No reaction

Fig. 5.26: Alizarin red stain of a case of retinoblastoma showing calcium material staining red (x200)


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TOLUIDINE BLUE STAIN FOR MAST CELLS Principle Mast cells will stain metachromatically with toluidine blue that will stain in different color from the dye solution from rest of the tissue. The color shift called metachromasia. Reagents 1. Toluidine blue solution: Toluidine blue ..................................................... 0.1 gm Distilled water ..................................................... 100 mL Staining Procedure a. Stain the deparaffinized sections in toluidine blue solution……10 minutes b. Rinse in distilled water c. Dehydrate and clear through 95% alcohol,100% alcohol and xylene d. Dry and mount in DPX. Results Mast cells .............................................................. Deep violet Background ......................................................... Blue GIEMSA STAIN Principle Giemsa stain used to differentiate nuclear and/or cytoplasmic morphology of RBC’s, WBC’s, parasites and also demonstration of some microorganisms. The pH of the staining solution is critical and ideally should be adjusted. More pH level gives more selective chromatin staining and less cytoplasmic basophilia; less acidic pH levels give denser nuclei and increased cytoplasmic basophilia. Reagents Required 1. Giemsa stock solution: Giemsa powder ................................................... 1 gm Methanol .............................................................. 65 mL (51.0 gm) Glycerine .............................................................. 40 mL (51.0 gm) * Measure volume of Methanol and weigh the volume.Weigh an equal volume of glycerine. Use a measured volume of methanol to rinse the glycerine. Combine glycerine and methanol by shaking it slightly. Mix it well with help of sterile glass beads or magnetic stirrer. Add giemsas powder, tighten the cap and shake


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well about once an hour for 3 days. When ready, filter through coarse filter paper for using as working solution 2. Buffer solution: Disodium monohydrogen phosphate anhydrous .. 6.0 gm Potassium dihydrogen phosphate ............................ 5.0 gm * Mix the buffer salts and weigh 1 gm unit and place in a well stoppered vial. One unit is dissolved in 1 liter of distilled water to give the buffer approximately pH.7.0 3. Giemsa working solution: Giemsa stock solution ........................................3 mL Buffer .................................................................... 97 mL * Never introduce pipette directly into stock solution. To avoid contamination pour stock solution into another container and pipette required amount and discard the unused solution. 4. Glacial acetic acid—water solution Glacial acetic acid ............................................... 1 mL Distilled water ..................................................... 499 mL Staining Procedure a. Deparaffinize the slides. If necessary treat the slides with alcoholic iodine to remove mercuric pigments. b. Place the slides in buffer solution ..................... 30 minutes c. Working giemsa solution ..................................overnight d. Quickly rinse in buffer solution e. Glacial acetic acid water solution ..................... 1 minute f. Absolute alcohol- 2 changes .............................. 15 seconds each g. Xylene 2 changes .................................................2 minutes each h. Dry and mount in DPX. Results Malarial parasite .................................................Blue Malarial pigments ............................................... Black Nuclei of tissues .................................................. Blue

IMMUNOHISTOCHEMISTRY Examination of tissue sections with the conventional H and E stain and histochemical characterization with special stains may not be sufficient to arrive at precise diagnosis. Immunohistochemical methods will help in some of these instances by providing additional information. For example, poorly differentiated or undifferentiated tumors can be identified for the origin of

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tumor cell type by their specific cytoplasmic or surface antigens, utilizing monoclonal or polyclonal antibodies directed to these antigens. oxidaseantiperoxidase (PAP) method or the avidin-biotin complex (ABC) method, both of which have higher sensitivity than the Immunoperoxidase method. FIXATION Fresh 10% neutral buffered formalin (pH 7–7.5) is most likely the optimum for immunohistochemistry. Tissue should be exposed to the fixative just long enough to achieve good preservation and morphology. Over-fixation will cause the formation of excess aldehyde linkages that can block or mask antigen binding sites and prevent the primary antibody from linking to the antigen. Tissues that have been exposed to formalin can be digested with proteolytic enzymes such as trypsin or pepsin; can also be treated with citrate buffer (pH 6.0). PROCESSING • Routine paraffin procedures are adequate for immunohistochemistry. • Do not allow processing temperature to exceed 60°C, since excess heat will destroy antigen and cellular morphology. • It is important to use clean paraffin compounds and to be certain that all traces of paraffin and plastic additives are removed from the tissue during the deparaffinizing and hydration phase. Residual embedding media can cause nonspecific staining, incomplete staining or suppress staining entirely. SECTIONING Paraffin sections are cut at 5 microns and the selected sections are picked up with pre-coated slides and dried horizontally. Optionally slides may be dried at 60°C for 30 mins but great care must be taken to insure that the slides are completely dry and not overheated. CONCENTRATIONS Valid staining of the target antigen can only be achieved if the antibody solution is used at ideal concentration (as indicated by the manufacturer). Correct antibody dilution is effected by every solution and procedure performed on the specimen: fixation, processing, buffer selection, staining temperature, humidity and staining, just to mention a few. Key to uniform, valid and significant result is consistency during all phases of tissue handling and staining.


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Methods Avidin Biotin Complex (ABC) Technique This technique uses three reagents a primary antibody, a secondary antibody. That is chemicals bound to vitamin biotin and a complex of the glycoprotein avidin that is bound to biotin and peroxidase. Avidin has the ability to bind nonimmunologically for molecules of biotin. Labeled Avidin-Biotin Technique Purpose The localization of tissue antigens. Principle This method utilizes three reagents: Primary antibody, biotinylated secondary antibody, and avidin conjugated with a marker enzyme. The primary antibody is specific for the antigen. The secondary or “link” antibody is capable of binding to the primary antibody and then binding the avidin conjugated with the marker enzyme, usually horseradish peroxidase or alkaline phosphatase. Avidin, either an egg-white protein or streptavidin, has the ability to nonimmunologically bind four molecules of the vitamin biotin. The chromogen is applied to develop an observable color. GENERAL LABORATORY TECHNIQUES The solutions used in these techniques can be very expensive. To minimize solution wastage slides can be stained horizontally on elevated rods in a humidity chamber. Use of humidity chamber is important as the slides must not be allowed to dry during any step of the procedure. Staining protocol is to be followed from the kit insert as provided by the manufacturer.

Fig. 5.27: Microphotograph showing positive staining with brown color with pan B cell marker (x200)

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Fig. 5.28: Microphotograph showing positive staining with brown color of T cells with pan T cell marker (x200)

CYTOLOGY STUDY Knowing the composition of cells and how cells work is fundamental to all of the biological sciences. Appreciating the similarities and differences between cell type is particularly important to the fields of diagnosis. These fundamental similarities and differences provide a unifying theme, allowing the principles learned from studying one cell type to be extrapolated and generalized to other cell type. Primary Sample This is applicable to the following specimens: 1.1 Aspirates—vitreous, aqueous, subretinal, lens, cystic and other fluids 1.2 Cytology of aspiration fluid/Biopsy material of intraocular tumor 1.3 Impression cytology of cornea/conjunctiva using millipore paper 1.4 Impression cytology from fresh orbit /lid biopsies 1.5 Scrapping material from conjunctiva or cornea 1.6 Intraocular lens. Method 1. Aspirates and FNAC material: All the aspirated fluids are cytocentrifuged by cytospin at 1000 rpm for 5 minutes or direct smear can be made from the material. The slide containing the material is dried and fixed in 95% alcohol for 5–30 minutes and is stained by H and E stain or any other stains required. Cell block preparation from the aspirates: Add equal volumes of 95% alcohol directly into the tube, containing the aspirate and let it stand for 2-3 hrs. This will coagulate into a soft mass which will sediment in the bottom. The soft mass can be removed by inverting the tube and tapping or by using a applicator stick. Fix the sediment in 10% formalin and submit for routine processing so that sections can be made.


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2. Impression cytology of cornea/conjunctiva using millipore paper: This technique is useful in evaluating patients with dry eyes, whose samples are examined for the presence or absence of goblet cells and changes of other epithelial cells. During this process, bits of cellulose acetate filter paper/millipore paper (pore size of 0.2 μ) is applied to the cornea/ conjunctival surface, gently pressed for about 30 seconds and removed. Superficial epithelial cells that adhered to the paper is then stained after it is fixed in 95% alcohol either by H and E, PAS or by modified technique as required. 3. Impression cytology from fresh orbit/lid specimens: The freshly sent sample can be used as a whole or cut into bits if it is large. The specimen is held using a forceps and is touched on surface of clean glass slide or a smear is made. The smear is air dried, fixed in 95% alcohol and stained with H and E stain or other stain. 4. Scrapping material from conjunctiva or cornea: The scraped material obtained in a clean glass slides and the area is marked with a glass marking pencil. On drying, the slides are fixed in 95% alcohol and stained. STAINING METHODS 1. Hematoxylin-Eosin Stain: a. Hematoxylin .................................................. 7 mins b. Wash in water c. Acid alcohol ................................................... 1 dip d. e. f. g. h.

Wash in water Ammonia water ............................................ 2 minutes Wash in water Eosin ............................................................... 30 seconds Dry and mount

2. Modified technique: a. 0.5% Periodic acid ......................................... 2 minutes b. Rinse in tap water c. Schiff reagent freshly diluted in distilled water in ratio (1:3) ......................................... 2 minutes d. DW .................................................................. 10 dips e. Tap water .......................................................2 minutes f. 0.5% Sodium metabisulfite .......................... 2 minutes g. Tap water .......................................................10 dips h. Hematoxylin .................................................. 2 minutes


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Fig. 5.29: Imprint smear of the conjunctiva with modified PAS staining technique showing sheaths of epithelial cells with Goblet cells in between

i. j. k. l. m. n. o. p. q. r. s.

Scott’s tap water substitute .........................10 dips D.W ................................................................. 2 minutes Tap water .......................................................10 dips Dehydration in 95% alcohol ........................ 10 dips Modified orange G solution ........................ 2 minutes 95% alcohol ....................................................3 minutes Eosin Y solution ............................................ 2 minutes 95% alcohol ....................................................5 minutes 100% alcohol .................................................. 2 minutes Xylene .............................................................5 minutes Dry and mount

STORING PROCESS Packing and storing of specimens 1. Half of the specimen which has not been processed is sealed in polythene cover with sufficient amount of fixative (formalin). 2. The sealed packet should carry the name of the patient, the accession details. This enables easy identification of the specimen. 3. The sealed packets are arranged serially in plastic boxes and these boxes are arranged year wise in racks. The room where the specimens are stored should be properly ventilated.


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Sealing and storing of blocks 1. Once the desired sections have been cut, remove the block from the block holder and seal the exposed surface with molten paraffin. 2. This insures that the tissues will not dry out and become hard and brittle and will facilitate re-sectioning of the blocks weeks, months and years later. 3. The blocks are then packed separately and used again whenever need arises. 4. The blocks are arranged in polythene packets serially arranged and each packet carries 10 numbers. 5. These packets are stored in cardboard boxes which are arranged in yearwise in racks.

Index Page numbers followed by f refer to figure and those followed by t refer to table A Abnormal RBC morphology 185f Absolute eosinophil count 190 Acanthamoeba culture 335 Acanthocytosis 185 Acetic acid 247, 250 suspension 237 Acetoacetic acid 241, 249 Acetone 249 Acetoxylene 411 Acid fast bacilli culture 320 stain 412 phosphatase 65, 67 Activated partial thromboplastin time 208 Acute viral infections 264 Advantages of formic acid decalcification 387 paraffin embedding 393 Agarose concentration 147 gel electrophoresis 146 Alanine aminotransferase 60 Albumin 22 Alcian blue staining 405, 405f Alcoholic formalin 400 Alizarin red-calcium complex 419 Amikacin 332 Amino acids 91 Amoxycillin 332 Analysis of CSF 259 Angiotensin converting enzyme 68 Anisocytosis 185 Antibiotic sensitivity 318 Antigen control tubes 362 Apple green birefringence 407f Arginine 98 Ascorbic acid 246 in urine 240 Aspartate transaminase 58 Automatic tissue processor 389f Avant genetic analyser 153f Avidin biotin complex technique 423

B Bacillus subtilis 316 Bacteria contain peroxidase 242 Barium chloride 252 Basic fuchsin 411 Bence Jones protein 257 Benedict’s qualitative reagent 246 Benhold’s method for amyloid 406 Benzidine 237 powder 237, 256 test 256 Beta hydroxyl butyric acid 241 Bile aesculin hydrolysis 315 Bilirubin 9, 241, 244 Bleaching of melanin pigments 417 technique 418 Blood test 239 Boiling test 248 Borax solution 414 Brick red precipitate 246 Brucella agglutination test 360 Buffer solution 421 Buffered substrate 72, 76 Bunsen burner 246, 247, 258

C Calcium 44 oxalate crystals 265 Calibration procedure 342 Carbohydrates 99 Carbonate buffer 83 Carboxylated sialomucins 405 Catalase test 308 Cefazolin 332 Cefotaxime 332 Ceftazidime 332 Cell count-using Neubauer chamber 261 Cerebrospinal fluid proteins 27 Ceruloplasmin 25 Chlamydia trachomatis 286, 337 Chloramphenicol 332 Chlorpromazine 241 Choice of fixative solution 382 Cholesterol 12

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Chromatography 89 Chromogen 371 Chromosome abnormalities 127 Ciprofloxacin 332 Citrate utilization test 313 Classification of chromosomes 128 Clindamycin 332 Clot retraction 217 Clotting time 215 Coagulase test 309 Coating of slides 377, 395 Cocktail preparation for setting PCR 144t Collection of specimens 303, 306 stool sample 107 test sample 106 urine sample 106 Complete blood count 170 Concentration method 235 for detection of mycobacterium 274 Congo red method for amyloid 406 Conjunctival papilloma 404f scraping 294, 332 swab 332 Contact lens 332 Coombs’ test 206, 207 Cornea 425 Corneal button 332, 381, 387 scraping 299, 300, 332 stroma 407f Counter stain 400 C-reactive protein 343 Creatinine 7 Cryostat knife 398 Culture methods 291 Cutting technique 399 Cyanmethemoglobin method 197 Cystine 102 crystals 265 Cytogenetic methods 116

D Decalcifying fluid 387 Dehydration 389 Descemet’s membrane staining pink 404f Detection of Bence Jones protein 257 hepatitis C virus antibodies 367 occult blood in stool 236 Diagnosis of ketonuria 250 Diluted serum 72

Direct and indirect Coombs’ test 206 bilirubin 11 Coombs’ test 207 smear examination 234 Disposable blades 398 Disposal of materials 110 Distilled water 35, 67, 81 Donor corneoscleral rim 332 Down syndrome 133f Drops of glacial acetic acid 257 Drying ovens 395 Durham’s tube 311

E Ehrlich’s aldehyde reagent 255 reagent 255 Electrophoresis 85, 200 buffer 148 of serum lipoproteins 86 of serum proteins 85 Electrophoretic techniques 145 Electrophoretogram 151 ELISA test 368 End-point of decalcification 388 Enterobacter aerogenes 315 Enterobacteriaceae 317 Enterococcus faecalis 315, 316 Eosin stock solution 401 Eosinophils 181 Eosin-phloxine working solution 401 Epinephrine 83 Epithelial cells 426f Equilibrated phenol 140 Erythrocyte sedimentation rate 186 Erythromycin 332 Escherichia coli 311, 312 Ethanol solution 135 Ethidium bromide 148 Examination of malarial parasite 223 Examination of microfilaria 226 stained smears 225, 228 External ocular specimens 332 Eye karyotyping 127

F Femoral tap 106 vein 106 Fermentation of glucose 317 test 311

Index Ferric chloride 409, 410 Fibrinogen assay 218 Fixation 387, 422 time 381 Fluorescent antinuclear antibody test 376 Formaldehyde 383 Formamide wash solution 135 Formation of air bubbles 236 Formula for calculation of annealing temperature 145f Fouchet’s reagent 252 Fragile-X syndrome 127 Fructose 240

G Gain of chromosome 127 Galactose 240 Gallego’s solution 411 Gatifloxacin 332 Gene expression 162 General laboratory techniques 423 Genescan PCR conditions 158t Genomic DNA extraction from blood 138 Gentamicin 332 Giemsa banding 124 buffer 124, 280 stain 125, 279, 420 stock solution 125 working solution 125, 421 Glacial acetic acid 237, 256, 421 Glass test tubes 249, 253, 255, 256 tube 237 Glucose 6-phosphate-dehydrogenase 73 tolerance test 4 Glucuronic acid 246 Glutaraldehyde 381, 383 Glutathione 39 peroxidase 79 Glycine 98 Glycoproteins 405 Goblet cells 404f Gold chloride solution 414 Gomori’s trichrome stain 408 Gram’s iodine 411 negative bacilli 317 stain 268 for bacteria 411 staining procedure 270

431 Granular casts 265 dystrophy of corneal stroma 409f Granulocytic leukocytes 239 Grocott’s methenamine silver nitrate 413 Grossing of biopsy specimens 386 technique 383

H Harri’s hematoxylin 400 Harvesting of chromosomes 121 HCl-potassium ferrocyanide working solution 418 Hematology 170 Hematoxylin 402f Hemoglobin electrophoresis 199 estimation 197 High density lipoprotein 15 urinary protein 242 Homocysteine 30 Homocystinuria 104 Homogentisic acid 246 Howell Jolly bodies 185 Hyaline casts 265 Hydrogen peroxide 237 solution 257 Hydroxy butyric acid 249 Hypochromasia 185

I Inborn errors of metabolism 99 Indirect Coombs’ test 207 Indole test 314 Infected red cell 226 suture 332 International System for Human Cytogenetic Nomenclature 132 Iron and iron-binding capacity 48 buffer reagent 50 color reagent 50 Isochromosome 127 Ivy method 213

K Ketone bodies 245, 249, 250 in urine 249 KOH-calcofluor white stain 271

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Manual of Medical Laboratory Techniques L

Lactate dehydrogenase 70 Lactophenol cotton blue stain 283 Leishman buffer 260, 262 stain 260, 262 Leukocytes 242, 244, 245, 265 Levey Jennings chart for SPC period 111f Lid margin swab 297 Light microscope 260 Litmus paper 234 Low-density lipoprotein cholesterol 15 Lower level of gastrointestinal tract 234 Lymphocyte 181

M Malignant melanoma choroid 385f Mannitol fermentation 312 motility test 312 Mantoux test 263, 264 Manual processing schedule for eyeball 391t Manufacturer of urine strip 244 Maple syrup urine disease 101 Mature trophozoites 226 Mayer’s/Harri’s hematoxylin solution 406, 417 Measles 264 Metaphase chromosomes 121 Metaphosphoric acid 56 Methenamine 414 solution 414 Methicillin 320 Methionine 98 Microtome 394, 394f Molecular cytogenetics 132 Morganella morganii 313 Moxifloxacin 332 Multiple acid fast bacilli staining 413f Mumps 264 Myocilin gene exons 148f

N Neubauer counting chamber 259, 260 Neutral buffered formalin 383 Neutrophils 181 Nigrosin stain 281 Nitrate reduction test 314 Nitric acid 387 Norfloxacin 332 Normal urine 248, 250

Novobiocin 320 Nuclear fast red solution 418

O Ofloxacin 332 Ornithine 78 aminotransferase 77 Oxalic acid 418 Oxidase test 309

P Packed cell volume 196 P-aminosalicyclic acid 241 Paraffin embedding procedure 393 section cutting 394 wax embedding 392 impregnation 390 Parasites, erythrocytes and pus cells 233 Pasteur pipette 248 Penicillin 332 Periodic acid and Schiff stain 403 Peripheral blood lymphocyte culture 117 smear study 182 Perl’s iron stain for hemosiderin pigments 418 Phenylalanine 98 Phenylenediamine 309 Phenylketones 241 Phenylketonuria 105 Phloxine solution 400 stock solution 401 Phosphate solution 81 Phosphorus 46 Picking up sections 397 Picro acetone 411 Piperacillin 332 Plasma amino acids 98 stain 407 Polio 264 Polyacrylamide gel electrophoresis 148 Polychromasia 185 Polymerase chain reaction 142 Porphobilinogen 241 Positive citrate control 314 Potassium ferricyanide 66, 67 ferrocyanide stock solution 418 hydroxide 271

Index Preparation of agarose gel 146 Coombs’ control cells 207 fresh smear 227 glass slides 377 hemolysate 200 Kovac’s reagent 315 nitrate reagent 314 oxidase reagent 309 smear 179, 278, 280 substrate 371 thin smear 225 Prometaphase chromosomes 121 Propylene glycol solution 417 Prostatic cancer 67 Protein 240 Proteus mirabilis 314 Prothrombin time 211 Protocol for cycle sequencing reaction 153t DNA extraction 140 setting RT reaction 163t Providencia alcalifaciens 314 Pseudomonas aeruginosa 311, 314

Q Qualitative detection of bile pigments by Fouchet’s method 251 bile salts by Hay’s test 252 free 256 identification of protein in urine by heat and acetic acid method 247 reducing sugar in urine by Benedict’s test 245 test 342 Quantification of DNA 141 gene expression 162

R RBC pipette 174f Red cell agglutination 185 Reticulocyte count 192 Retinal membranes 381 Retinoblastoma 133f, 385f, 402f Rheumatoid arthritis test 341 RNA extraction 162 purification 163 Rothera’s mixture 249 Rouleaux formation 185

433 Routine paraffin procedures 422 staining 400

S Salmonella typhi 314 Sanger dideoxy sequencing 151 Sartorius filter apparatus 168 Schistocytosis 185 Schuffner’s dots 226 Screw-capped bottles 167 Sebaceous gland carcinoma 416f Section of corneal button 407, 409f Sectioning technique of globe 385f Semi-quantitative estimation 356 test 343, 345, 349, 352 Sensitivity of blood test 242 Separates cell lines or clones 127 chromosome modal number 127 Serum antistreptolysin ‘o’ titer 347 glutamic oxaloacetic 58 pyruvic transaminase 60 Setting of lymphocyte culture 117 Sex chromosomes 127 Sickle cell preparation 231 Silver nitrate solution 414 stock solution 414 working solution 414 Slide agglutination method 203 coagulase for free coagulase 310 Sodium azide 81 bisulfite 414 chloride mixture 388 hydroxide solution 406 nitroprusside in test tube 249 solution 250 thiosulfate 414 solution 410 Sorensen’s buffer 125 Spherocytosis 185 Squamous epithelial cell 265 Stained cellular components 260 Staining method 322, 425 of blood film 180 techniques 268, 400

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Staphylococcus aureus 309 Steatorrhea 234 Sterile test tubes 260 Stomatocytosis 185 Stool examination 233 Streptococcus pneumoniae 309 Sulfanilic acid 314 Sulfur powder 253 Sulfuric acid 35 Superoxide dismutase 82 Symbols and abbreviations used in karyotyping 127t

U Urea 6 Urease test 312 Uric acid 28, 246 crystals 265 Urinary tract infections 242 Urine 321, 332 deposit examination 265f dropper 248 of menstruating females 244 of pregnant women 240 Urobilinogen 239, 241, 244, 255

V

T Tear drops 185 Temporal artery biopsy 410f Test tubes 246, 248, 258 Testing for urobilinogen 255 Tetracycline 332 Thermometer 258 Thin with white flakes 234 Thiobarbituric acid reactive substances 37 Threonine 98 Throat swab 331 Tissue floatation bath 396f processing 389 Tobramycin 332 Total bilirubin 11 erythrocyte count 173 proteins 20, 238 Touch-down PCR 144 Transaminase 58 Transitional epithelial cell 265 Treponema pallidum hemagglutination 353 Triacylglycerols 18 Trichrome staining of eyelid 408f Triglycerides 18 Triple phosphate 265 sugar iron agar test 313 Trophozoites 226 Tube agglutination method 203 Tuberculin syringe 263 Turk’s fluid 260 Types of PCR 144 Tyrosine 98, 103 needles 265

Vacuum impregnation 390 pump 119 Valine 98 van Gieson solution 410 Vancomycin 320, 332 Verhoeff’s elastic stain 409 working solution 410 iodine solution 409 Visualizing hybridization 137 Vitamin A 51 C 54 E 53 von Ebner’s hydrochloric acid 388

W Washing procedure 136 Waxy casts 265 WBC casts 265 pipette 174f Wedge-type blood film preparation 182f Weigert’s iron hematoxylin 407 Western blotting technique 372 Widal test 357 Working conjugate 373 diluent buffer 373 solution 412 of buffer 280

X Xylene 391

Z Ziehl-Neelsen stain 273, 277

Sankara Nethralaya’s Manual of Medical Laboratory Techniques

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