Quality Control, Quality Assurance & Quality
Quality Management System (QMS)
Management System •
Set of coordinated activities to regulate a lab in order to continually improve
Quality
performance efficiency
Degree of congruence between expectations and realization.
Quality Management System in a Histopath
Customer – Customer – checks checks if the quality of
Lab
service fulfills the set requirements o
Courteous and skilled staff
o
Release of results on time as per requirement of the physician and patient
o
Short turnaround time
o
Correct diagnosis/findings
o
Non specimen mix-ups
Quality Control
Set of procedures or technical te chnical activities
•
Skilled histotechnologist/technician
•
Proper specimen collection
•
Proper processing of specimens
•
Efficient processing of results
•
High quality of reagents and equipment
•
Preventive maintenance of equipment
•
Continuous professional education of staff
•
Documentation and control
•
Proper coordination
•
Timely customer’s feedback
on fulfilling quality requirements
Good Laboratory Practices Quality Assurance
•
3 Phases of examination and Factors affecting them:
Aims to generate the confidence of the
o
patient to the final report
Pre-examination (Preanalytical)
Receipt of the request/specimen – request/specimen –
o
Examination (Analytical)
release of the report
o
Post-examination (Post-
Includes availability of reagents,
analytical)
preventive maintenance & monitoring of equipment & evaluation of the quality of service
Quality Assurance (QA) •
Pre-analytical Phase Factors •
Collection of the right specimen
•
The proper fixation of the specimen
•
The correct identification of the
In short, it is a means of: o f: •
•
•
•
•
•
getting the RIGHT test at the RIGHT time on the RIGHT specimen from the RIGHT patient with the RIGHT diagnosis and at the RIGHT price !!!!!
specimen •
The timely transportation of the specimen
Examination (Analytical) Factors •
Grossing of tissues
•
Processing
•
Procedure reliability using technical manuals
•
Reagent integrity and efficiency
Mounting/labeling
•
Cutting of paraffin sections
Microscopic exam
•
Staining
Release of reports
•
Slide labeling
•
Equipment reliability
•
Adequate calibration
•
Chemical name/names of all ingredients
•
Proficiency of personnel and co ntinuous
•
Manufacturer’s name& address/Person
Basic Information on Chemical Labeling
updating of their knowledge •
Good internal quality control
Post-examination (Post-analytical Factors •
•
Date purchased or made
•
Expiration date
•
Hazard warnings & safety procedures
Render histopathologic diagnosis (hard copy or electronic) free of clerical errors
•
making the reagent
Ensure that the report reaches the
Storage of Hazardous Chemicals •
appropriate clinician/surgeon. •
Filing of paraffin blocks must be in a
height •
cool area and rodent free •
Slides are stored for 10 years while
•
•
Dangerous reagents – reagents – plastic plastic or plastic coated glass bottles
•
reports may be longer in a safe and humidity free areas
Dangerous liquids – liquids – below below countertop
Flammables are never stored in a refrigerator or freezer, only in certified
•
Use of this chemicals are used only o nly in
Any possible remarks on the diagnosis
small quantities as needed and used up
obtained should also be included
completely
Frequent dialogues between the
•
pathologist and the surgeons/clinicians
Flow Chart for Surgical Biopsy Process
Do not store any leftover flammable liquid
Documentation
Receive/Record Specimen
Check request/Specimen
o
Surgical Pathology Report
Gross exam
o
Cytopathology Report
Tissue Processing
o
Autopsy Report
Cutting/staining
Mounting/Labeling
o
Patient’s Copy
Microscopic Exam
o
Doctor’s Copy
Release of reports
o
Pathology Department’s Copy
Flow Chart for Cytology
Receive/record specimen
Check request
Gross exam
Smear preparation
Staining
•
•
Histopath Reports
Number of Copies Prepared: (3)
Routine Turnover (Release) of Results •
Surgical Path & Cytopath Results – Results – 24 24 hrs
•
Frozen Section – Section – 5 5 to 15 minutes
•
Autopsy Report – Report – 1 1 week
•
Reagent integrity and efficiency
Mounting/labeling
•
Cutting of paraffin sections
Microscopic exam
•
Staining
Release of reports
•
Slide labeling
•
Equipment reliability
•
Adequate calibration
•
Chemical name/names of all ingredients
•
Proficiency of personnel and co ntinuous
•
Manufacturer’s name& address/Person
Basic Information on Chemical Labeling
updating of their knowledge •
Good internal quality control
Post-examination (Post-analytical Factors •
•
Date purchased or made
•
Expiration date
•
Hazard warnings & safety procedures
Render histopathologic diagnosis (hard copy or electronic) free of clerical errors
•
making the reagent
Ensure that the report reaches the
Storage of Hazardous Chemicals •
appropriate clinician/surgeon. •
Filing of paraffin blocks must be in a
height •
cool area and rodent free •
Slides are stored for 10 years while
•
•
Dangerous reagents – reagents – plastic plastic or plastic coated glass bottles
•
reports may be longer in a safe and humidity free areas
Dangerous liquids – liquids – below below countertop
Flammables are never stored in a refrigerator or freezer, only in certified
•
Use of this chemicals are used only o nly in
Any possible remarks on the diagnosis
small quantities as needed and used up
obtained should also be included
completely
Frequent dialogues between the
•
pathologist and the surgeons/clinicians
Flow Chart for Surgical Biopsy Process
Do not store any leftover flammable liquid
Documentation
Receive/Record Specimen
Check request/Specimen
o
Surgical Pathology Report
Gross exam
o
Cytopathology Report
Tissue Processing
o
Autopsy Report
Cutting/staining
Mounting/Labeling
o
Patient’s Copy
Microscopic Exam
o
Doctor’s Copy
Release of reports
o
Pathology Department’s Copy
Flow Chart for Cytology
Receive/record specimen
Check request
Gross exam
Smear preparation
Staining
•
•
Histopath Reports
Number of Copies Prepared: (3)
Routine Turnover (Release) of Results •
Surgical Path & Cytopath Results – Results – 24 24 hrs
•
Frozen Section – Section – 5 5 to 15 minutes
•
Autopsy Report – Report – 1 1 week
Signatories •
Four categories of tissues:
Request Forms (All Forms)
Patient’s doctor, Attending physician •
Epithelial tissues – tissues – derived derived from ALL THE THREE (3) GERM LAYERS
Result Forms (All Forms)
Connective tissues – tissues – from from mesoderm
Pathologist
Muscular tissues – tissues – from from mesoderm
Nervous tissues – tissues – from from ectoderm
Specimen Handling •
•
“Numbering” – system – system of assigning numbers to specimens received
A. Covering epithelia
chronologically as a form of labeling.
B. Glandular epithelia
Tools for Labeling o
Pencil
o
Diamond pencil
o
Gum Label
Retention Period •
Surgical Pathology specimen – specimen – 2 2 to 4 weeks after the issuance of a final report
•
Covering epithelia
Blood vessels are absent
Exposed to physical injury and infection
Classifications of Covering Epithelia: According to cellular arrangement:
– one-cell thick Simple – one-cell
– appear to be more Pseudostratified – appear
Tissue block, histopath slides & FNA
than one-cell thick but actually cells rest
slides – slides – 10 10 years
on common basement membrane
•
Cytology slides – slides – 5 5 years
•
Surgical pathology/cytology & other non-forensic reports – reports – 10 10 years
•
EPITHELIAL TISSUES:
Forensic autopsy reports - indefinitely
– many layers of cells Stratified – many
According to cell shapes:
cells (like “paving Squamous – flattened cells stones”)
Basic Histology •
Histology – Histology – study study of normal tissues o
Cuboidal – cube-like (isodiametric cells)
Columnar – cells that are taller than they are wide
A fertilized egg divides forms smaller cells
o
shape when the epithelium is stretched.
After morphogenetic movements, these cells become arranged in 3 germ layers:
1. ECTODERM 2. ENDODERM
Transitional – cells that change their
Combination of classifications I and II:
SIMPLE: o
Endothelium of blood vessels,
3. MESODERM
Tissues – group of cells of common origin and common function
Squamous – Squamous – Bowman’s capsule, capsule, Loop of Henley, Alveoli of lungs
o
Cuboidal – Cuboidal – in in walls of thyroid follicles and ducts of glands
o
Columnar – gallbladder
o
EXAMPLE: Mammary glands
(NONCILIATED), uterine tube
in milk secretion
(CILIATED)
STRATIFIED o
Holocrine o
Squamous – Epidermis of the
complete breakdown of the secretory cell
skin (keratinized), Vagina (Non-
o
EXAMPLE: Sebaceous glands
keratinized), Esophagus (Nonkera), Cervix (Non-kera) o
Cuboidal – sweat gland ducts
o
Columnar – Male urethra
o
Transitional – Urinary tract
OTHER CATEGORIES OF TISSUES
CONNECTIVE TISSUES •
Cells are usually widely separated by a large amount of intercellular substance
PSEUDOSTRATIFIED o
•
Columnar – much of female
Blood and blood-forming tissues, bone, and cartilage
reproductive tract (Nonciliated), trachea (Ciliated)
Glandular epithelia
I. General Connective Tissues
Loose Connective tissue
Dense Connective tissue
II. Special Connective Tissues Exocrine glands – glands with ducts
Tubular – Stomach, uterus
Acinar/Alveolar – pancreas, salivary glands
Tubulo-acinar - prostate
Methods of Secretions of Glands:
Merocrine o
No loss of cytoplasm
o
secretions accumulate below
Cartilage
Hematopoietic
Bone
Blood
Lymph
Loose Connective Tissues
Common examples include: o
jelly
the free surface of the cell
o
through which it is released o
EXAMPLES: Goblet cells, sweat
o
with cytoplasmic loss
o
secretions accumulate below the free surface but can only be
Mesenchyme – embryo and fetus
Apocrine o
Reticular – Bone marrow, lymph node
glands
Mucoid tissues – Wharton’s
o
Adipose - hypodermis
Dense Connective Tissues
Common examples include:
released by breaking away of
o
Dermis
the distal part of the epithelium
o
Capsules of organs
o
Tendons
o
Stroma of cornea
SPECIAL CONNECTIVE TISSUES
Cartilage
Clinical Chemistry
Immunology & Serology
Hematology
o
Bone
Blood Bank
o
Blood
Microbiology
o
Lymph
Clinical Microscopy
o
Hematopoietic
Cartilage: o
Hyaline - trachea
o
Fibrous – Intervertebral discs
o
Elastic – external ear, epiglottis
Bone: o
Cancellous/ Spongy – Epiphysis or ends of long bones
o
Clinical Pathology
Branches of Anatomical Pathology
Surgical or Morbid Pathology
Cytopathology
Autopsy Pathology
Forensic Pathology
Immunopathology
Molecular Pathology
Compact – Diaphysis or shaft
Hematopoietic: o
Myeloid – Bone marrow
o
Lymphoid - Spleen
Basic Concepts on Diseases Manifestations of disease
Signs o
MUSCLE TISSUES
disease.
Smooth (involuntary) – found in
o
ulcer, fracture, fever,
Striated (voluntary) – found in
hemorrhage, diarrhea, etc.
Symptoms o
Cardiac (striated but involuntary) -
o
NERVOUS TISSUES
Peripheral nervous system –
weakness, pruritus, fever, nausea, dizziness, pain,
Central Nervous system – brain and
numbness, etc.
spinal cord
subjective manifestations of a disease.
heart
Example :tumor, tenderness,
intestinal tracts and blood vessels skeletal muscles
objective manifestations of a
Idiopathic disease
peripheral nerves
o
Diseases that are not well understood
Special receptors – eye, ear, nose
o
The etiology or pathogenesis is not fully understood
General Pathology for Medical Technology Students Review
o
Impact to the population?
Periods of Disease Formation
Clinical Pathology
o
Etiology - Cause of the disease
Surgical or Anatomical Pathology
o
Pathogenesis - Sequence of mechanisms leading to disease
o
Prognosis - Probable outcome
There is an increase in the organelles and other components of
Cellular Injury “ To be viable, a living cell must
the cell.
maintain an organization capable
This will result to the compensation of function.
of producing energy.”
Hyperplasia Reversible Cell Injury
Functional Changes A. Cell and Tissue Accumulation
Hydropic change (Cloudy swelling)
Fatty change
Residual bodies
Hyaline change (Hyalinization)
B. Adaptive responses
Alternative metabolism.
Altered morphology and numbers.
Usually seen in labile and also in some stable cells.
Hyperplasia and hypertrophy usually goes together.
Metaplasia
Causes: o
Chronic irritation!!!
o
Chronic irritation!!!
o
Hypertrophy
o
Chronic irritation!!!
o
Hyperplasia
o
Metaplasia
Decrease in the size and function of Adaptive response reducing its need
“ It is the conversion of one adult cell type to another cell type.”
Irreversible Cell Injury Features:
Cell membrane disruption.
Nuclear change and deterioration.
for energy to the minimum but still
o
pyknosis
keeping the cell alive.
o
karyorrhexis
Effect: Conservation and limit use of
o
karyolysis
(energy) ATP to keep the cell alive.
Atrophy
the cell.
organ or tissue.
o
Atrophy
Increase in the number of cells in an
Lysosomal rupture and digestion.
Reversible Cellular Death
Hypertrophy
Increase in the size of cell
Apoptosis
Necrosis
accompanied by enhanced functional capacity.
Apoptosis
“programmed cellular death”
“cellular suicide”
Physiologic death of single or few cells
disease, more of idiopathic
Significance:
disease.
o
Replacement of old, worn
o
Genes controlling cell division and growth:
out and injured cells. Destruction of abnormally
o
Proto-oncogene
mutated cells that may lead
o
Tumor suppressor gene
to cancer or congenital
abnormalities. Necrosis
It is primarily a genetic
o
Mutant gene Oncogene
o
Classification of Neoplasms
Pathologic death of a large number
Benign tumors (Ok lang)
of cells.
Malignant Tumors (Deadly)
Results from the effects of diseases.
Leads to tissue or organ destruction.
Nomenclature
Types of Necrosis
Benign o
Coagulation necrosis
Liquefaction necrosis
Caseous necrosis
Non-caseous necrosis
Gangrenous necrosis
o
o
Dry gangrene
o
o
Wet gangrene
– oma microscopic & macroscopic
o
structure cell of origin
o
Malignant (Cancer) – carcinoma, - sarcoma microscopic & macroscopic structure o
cell of origin
Neoplasia Neoplasm (Tumor)
A neoplasm or tumor is a
Metastasis
is
the
single
most
proliferation of cells which persists
important feature of a malignant
after the stimulus which initiated it
tumor.
has been withdrawn.
Significant Information
o
Hematogenous route
Significant cause of death
o
Lymphatic spread
because of organ destruction
o
Local spread
Significance: o
o
Metastatic spread via:
early detection is necessary (difficult to attain)
Epidemiology of Neoplasms
Cancer can strike at any age.
Three leading cancers in men are lung, colon and rectal cancer.
Tumor Markers
of which are proteins, which are helpful
For women: breast, lung, colon &
in diagnosis and monitoring of
rectal ca.
Tumor cells produce substances, many
treatment.
Advancing age increases the risk of
o
cancer.
(A) Product enters blood stream (and/ or urine) where it can be
Current research indicates that
measured.
almost 90% of all cancers are related
o
(B) Histological diagnosis is
to lifestyle and environmental
improved by identifying the specific
factors.
product using immuno-staining in the cytoplasm of the tumor cells.
Environmental & Non-environmental Causes of Cancer
Chemical carcinogens o
Nitrosamines
o
Aflatoxin B1
o
Asbestos, silicon
(Used as Immunohistostains as well as in Serologic determinations.)
Goals and uses: o
Used as a screening test for cancer.
o
Used as a diagnostic tool for cancer .
o
To monitoring the effects of treatment for:
Physical Carcinogens o
UV rays
o
Radioactive/ionising waves
o
Hepatitis B
o
HPV
o
AIDS
Inherited ( Familial) Cancer Syndrome o
Neurofibromatosis
o
Retinoblastoma
Histologic and Cytologic Methods o
biopsy, autopsy , endoscopy, pap’s test
Molecular diagnosis
Flow Cytometry
Immunocytochemistry & Tumor Markers
-
Possible recurrence
-
Response to chemotherapy and radiotherapy.
Viral Oncogenes
Laboratory Diagnosis of Cancer
Polycyclic aromatic hydrocarbons
o
Immunocytochemistry & Tumor Markers
Tumor Markers
the biologic behavior of a
Staging and Grading
malignant neoplasm will be.
Staging and grading schema have been devised for malignant neoplasms, because the stage and/or grade may determine the treatment and the prognosis. In general, the higher the stage, the larger a neoplasm is and the farther it has likely spread.
Staging (TNM classification) o
A “T” score – size and/or extent of invasion
o
A “N” score – extent of lymph node envolvement
o
A “M” score – whether a distant metastasis is seen
Screening Test for Malignancy
There is often a stage at which they are clinically pre-malignant
It is important to detect cancers early, because with appropriate treatment, the neoplasm can be removed before it metastasizes and kills the patient
Screening Modalities for Malignancies (Cancer)
Cervical smear
Mammography
Endoscopic biopsy
Skin surveillance for pigmented tumors
Self examination (breast, testes)
Tumor markers o
Grading o
based upon the microscopic appearance of a neoplasm with H & E.
o
In general, a higher grade means there is a lesser degree of differentiation and the worse
blood, urine, tissue
Autopsy Autopsy (Necropsy)
This is the branch of Anatomic Pathology that deals with the
complete medical examination of
that are not close relatives to the
the body after death.
patient.
“postmortem examination”
It is a medical procedure practiced
autopsy should be explained fully
since ancient times to analyze
and clear enough to be understood
organic alterations caused by
by the relatives before seeking
disease or trauma.
consent.
Autopsia – “Seeing with one’s own
The procedures done during the
In medico legal cases, deaths with questionable circumstances, a
eyes.”
medico legal officer or an appropriate agency like the NBI or
Main Goal of Autopsy To identify the cause of death and to
improve medical science.
PNP is consulted.
Relatives of the patient are not supposed to be in the autopsy room
Other Values of Autopsy:
Diagnosis and treatment quality control.
during the procedure.
Source of accurate epidemiologic
Extent of the Autopsy Partial autopsy specific body component is
data
only involved.
Material for medical residents,
head only, thorax only
students and staff learning
Material for scientific research
Monitor changes in disease pattern.
General System or Procedure of Autopsy:
Gross examination
Autopsy proper and acquisition of specimen for microscopic
Complete (Full) autopsy whole body involvement
Incision for the autopsy:
The “Y” incision
Evisceration Techniques
examination.
En masse (Letulle)
Microscopic examination
En bloc (Ghon)
Releasing of reports
Individual organs (Virchow)
In situ (Rokitansky)
Pre-Autopsy Requirements
A consent for the autopsy should be obtained first from the nearest of kin.
Included in the consent form are at least three (3) witness signatures
En Masse (Letulle) Bulky single aggregate of organs
removed en masse and transferred to the dissecting
table for further dissection.
En Bloc Dissection (Ghon)
Organ Weights
Thoracic pluck (block)
Right lung: 300-400 gm
Coeliac block
Left lung: 250-350 gm
Intestinal block
Heart: 250-300 gm
Urogenital block
Liver: 1100-1600 gm
Neurological block (if needed)
Adrenals: 4 gm or so each
Individual Organ Removal (Virchow) Organs are removed one by one
sequentially
Thyroid: 10-50 gm Spleen: 60-300 gm Brain: 1150-1450 gm
Isolated and dissected immediately
Postmortem Changes
after removal In Situ Method (Rokitansky Technique)
Death – complete cessation of
Body is cut open in the usual fashion
metabolic and functional activities
Cavities and organs are thoroughly
of the organism or body as a whole.
inspected
Three (3) Primary Changes (Death)
Fluids are collected if needed
o
Circulatory Failure
Organs are dissected while still
o
Respiratory Failure
o
Nervous (CNS) Failure
inside the body Organ removal is optional
Secondary Changes After Death Special Considerations
Rigor Mortis
Atherosclerotic plaque (atheroma)
of coronary vessels – for
muscles.
decalcification
Severe rigidity or spasm of the
Interlocking of the actin and
Severe atheroma (75%) is required
myosin secondary to lactic acid
before it is said to be the cause of
buildup
death
Immediate - onset
The heaviest organ – liver, brain
1-6 hours - manifestation
Lung sinking in a pan of water
6-24 hours - maximum
indicates drowning or severe edema
Persists 12-36 hours (3-4 days)
Muscle tissue is stretched on a cardboard before fixing
Livor mortis (Lividity)
settling of the blood in the lower (dependent) portion of the body, causing a purplish red discoloration of the skin
heavy red blood cells sink through the serum by action of gravity.
starts immediately after death
congealed in the capillaries in 2 to 4
Adipocere
hours.
Maximum lividity occurs within 8-12
hours.
after death resulting to petechiae
Rate Method o
Tardeau’s Spots/Petechial Hemorrhages – rupture of capillaries
Methods for the Estimation of Time of Death Body changes, blood changes, plant or animal indicators
Concurrence Method o
environmental changes that
like hemorrhages.
may indicate a particular time
May indicate asphyxia if evident in
of death
the sclera. Algor Mortis
Postmortem Clot vs Premortem Clot Postmortem Clot
following death.
a steady decline until matching ambient temperature
1 to 7° Fahrenheit per hour until the body nears ambient temperature
(Thrombus)
reduction in body temperature
Glaister equation – Formula used for determining the approximate time period since death based on body temperature.
Settling/separation of RBC
“chicken fat” appearance
“currant jelly” appearance
Assumes the shape of the vessel
Rubbery in consistency
Premortem Clot (thrombus)
Granular and friable
Fibrin precipitation
Seldom assumes the shape of the vessel
Not easily detached from the bld vessel
Ocular Changes Description Corneal film Schleral discoloration “tachy noir” Corneal cloudiness Corneal opacity Exophthalmos
wall Eyes Open minutes Minutes to several hours
Eyes Closed Several hours ----------------
2 hours or less
12-24 hours
-----------------------w/ gas formation Endophthalmos w/ advance decomposition
3 postmortem day Same
POSTMORTEM TISSUE CHANGES
Decomposition o
Autolysis
o
Putrefaction
Mummification
Skeletonization
r
same
Do not have a rubbery consistency
Cytopathology/Cytology Manner of Cellular Exfoliation & Evaluation
Spontaneous shedding from organ surfaces. (Exfoliative Cytology)
Physical removal of cells from organ surfaces. (Imprint/Abraded Cytology)
General Applications of Cytopathology
Screening for the early detection of asymptomatic cancer.
Diagnosis of symptomatic cancer.
Surveillance of patients treated for cancer.
Detection of an infectious process and
difficulty in the interpretation of
its etiology.
cytology.
Determination of genetic sex.
For the assessment of female hormonal
o
Inadequate cell concentration or poor adhesion.
activity.
o
Thick smears cause overlapping of cells.
Cytologic Evaluation
o
Poor fixation.
o
Poor or over stained preparations.
Exfoliation (Exfoliative Cytology)
This refers to the examination of cells that are shed spontaneously into body fluids or secretions. o
Sputum
Vaginal smears
o
CSF
Endometreal and endocervical smears
o
Urine
Prostatic and breast secretions
o
Effusions in body cavities.
Gastric and bronchial secretions
o
Nipple discharge
Pleural and peritoneal fluids
o
Vitreous and aqueous humor
Sputum
Abrasive method (Imprint/Abraded Cytology)
Usual Specimens For Cytology (Cytopathology)
Smears of urine sediments
Encompasses methods by which cells
CSF
are dislodged by various tools from
Wound secretions
body surfaces.
FNAB derived
o o
Vaginal and cervical smears.
CT guided or deep organ aspiration
biopsy
Endoscopic brushing from the GIT, respiratory and urinary tracts.
o
o
Scraping of cutaneous, oral, vaginal or conjunctival lesions.
Preparation of Body Fluids for Microscopic
Washing or lavage during
Study
endoscopy or surgery.
Fine – Needle Aspiration Biopsy (FNAB)
It is a technique that uses cells obtained by aspiration under negative pressure
Smear preparation o
Crush Technique
o
Pull-Push Technique
Tissue (Cell) block
through a thin-guage needle.
The Cell Block Causes of Error in Cytology
Poor fixation of the smears or
Paraffin – embedded specimen from different fluids and aspirated materials
Inadequate sampling is one major cause of false negative diagnosis.
Can be prepared from all types of fluid specimen but particularly:
inadequate preservation of a fluid.
o
Effusions
Sub-optimal laboratory preparation and
o
endometrial aspirates
staining can cause considerable
o
brush samples
o
fine needle washings
C. Sputum and Bronchial Aspirates
Adjunct for establishing a more
fresh early morning collection
definitive cytopathologic diagnosis
5 consecutive day collection
(Malignancy and/or metastasis)
Prepare 5-8 slides and fix for 1 hour
Architectural evaluation
Categorization of tumors not possible in smears
D. Gastric Aspirate
Special stains & immunohistochemistry
Immunophynotyping, molecular studies
to aspirates.
(CISH, FISH, PCR)
Best obtained by brushing as compared Fix right away in 95% ethanol and cool in ice.
Archive material
E. Prostatic Secretions Cell Block Preparation
Three Specimens Obtained at Random
1) Centrifuge fluid to obtain cell button.
1. Voided urine before massage.
2) Decant supernatant fluid and re-
2. Smears from a prostatic massage.
suspend cells in normal saline.
3. Urine after massage.
3) Re-centrifuge at 2500 rpm for 5 min 4) Decant supernatant fluid. 5) Warm the tube at 45 C under hot water.
F. Urinary Sediments
Males – voided urine
Female – a catheterized specimen is
6) Add 2% agar solution cooled at 50 C and
recommended
mix 7) Centrifuge at 1000 rpm for 2 mins 8) Remove the agar-cell prep and place it in 10% formalin solution for 1 hour to
Things to Consider in Specimen Preparation
Smears should be even and lump free.
Patients identification should be labeled
transform the agar into an irreversible
on the slide immediately after
gel.
smearing.
9) Process by routine histologic method.
Smears should immediately be placed in fixative while still moist.
Varied Specimens and their Preparation
A. Cervical and Vaginal Smears
Specimens transported should be fixed and air dried. (no longer absolute)
swab, aspirate, brush
Fixed in 95% ethyl alcohol
the collected material may be
Fix for 15 minutes before staining
refrigerated
Can spray with hair spray for transport. o
(ether alcohol and polyethylene
If smears cannot be made immediately,
Excessive blood is removed with 2-5 ml conc. Hac per 100 ml.
glycol)
Adhesives for Cytologic Smears B. Pleural/Peritoneal Fluids •
Fresh fluid is centrifuged
•
Can add 300 units of heparin
•
Most specimen contain a natural adhesive, albumin.
•
Specimens Requiring Adhesives:
o
urinary sediments
o
Bronchial lavage specimen
container forcefully to prevent
o
Specimen releasing proteolytic
dislodging the material from the slide.
enzymes
Avoid striking the bottom of the fixing
Identify the slides before preparing smears.
Characteristics of a Good Adhesive Agent
It must be permeable to both fixative
Have the individual fixative bottle open before preparing the smear.
and stain.
It must NOT retain the stain.
Adhesive Agents: 1. Pooled human serum or plasma
Sexual Determination Smears for Sexual Determination
2. Celloidin Ether Alcohol 3. Leuconostoc Culture
Fixation of Cytologic Smears Common Fixatives Used
Taken from scrapings of buccal mucosa and vagina.
Identification of the Barr Body.
100 cells are evaluated for Barr Bodies
20-90% …… positivity for female sex
< 4% ………. supports a male sex
Equal parts of 95% ethyl alcohol & ether.
Vaginal Cytology (“Pap’s Smear)
95% ethyl alcohol
Carnoy’s fluid
described its use in detecting malignant
Butanol-ethanol
cells of the uterine cervix.
Ethanol-acetic acid
Dr. George N. Papanicolaou, first
Today its usefulness is used as a primary screening method for the
Advantages of Ethyl Alcohol
detection of cervical malignancy, and
Alcohol is a protein coagulant and
especially its precursor lesions.
smears of predominantly protein material will remain better attached to
General Purposes
the slide during staining.
Alcohol gives good chromatin
(In addition as a screening test)
preservation and delineation.
Alcohol is easily dispensed to clinics and
infections.
can be stored without undue deterioration.
Detect the etiology of certain Determine ovarian function based on hormonal cytology
Used as one of the battery of tests used in infertility workup.
Precautions Observed During Fixation
Smears should be placed into the fixative immediately after preparation.
Place each smear in fixative by a single uninterrupted motion to avoid rippling of smeared material.
Monitor treatment for diagnosed cancer
Determine phenotypic sex.
Medico-legal examination of sexual assualt.
Sites for Sample Retrieval
Instruments to Obtain Samples
Lateral vaginal wall (just above the level
Speculum
of the cervical os, or from the posterior
Wooden spatula
fornix)
Modified spatula (Ayer’s spatula)
Ectocervix
Cervical brush
Endocervix
Cervical broom (Cervex-brush or
Papette)
Upper 3rd of Lateral Vaginal Wall
Cotton swab
Assessment of the hormonal status
Evaluation of possible inflammatory
Precautions Observed During Vaginal Smear
condition of the vagina
Preparation
Classify the microbiologic flora
Detection of malignant lesions of the vagina
Ectocervix
Detection of ectocervical tumors
Inflammatory conditions
hours.
Instruments should be perfectly dry.
No lubricant or powder should be used on the examiner’s glove.
Endocervix
Detection of endocervical tumors
May detect some intrauterine lesions
Inflammatory conditions
Ectocervix-Endocervix Junction
Smears should be spread thinly in a rotatory motion instead of pull apart method.
All materials should have been prepared before the procedure.
Instruments are soaked in soap solution
Most commonly sampled for cervical
for 20 minutes, rinsed with KOH and
cancer screening.
dried.
transformation (T or junctional) zone.
Transformation (T or junctional) Zone
No manipulation for at least 24-48
Represents an area where an abrupt
Epithelial Cells Evaluated in Vaginal Smears
Lateral Vaginal Wall (Squamous cells)
transition from the ectocervical
o
Mature superficial cells
epithelium abruptly changes to
o
Intermediate (Navicular, Boat) cells
endocervical type of epithelium.
It is a dynamic, ever changing
o
Parabasal cells.
environment adapting to chronic
o
Basal cells
inflammation and infection.
Endometrial cells (Columnar cells)
Metaplasia, dysplasia and carcinoma-in-
Endocervical cells (Columnar cells)
situ occurs
Ectocervical cells (Squamous cells)
Eventually progresses to invasive carcinoma.
This is the reason why most malignancy arises from this particular anatomic site.
Superficial and Intermediate Cells •
Nucleus o
5-6 u
o
Pyknotic
o
Centrally placed
•
•
Cytoplasm o
40-50 u
o
Polygonal, well defined
o
Pink (eosinophilic) or blue
o
endocervical cells o
Eccentrically placed
Cytoplasm
(cyanophilic)
o
Scanty, low columnar
Cellular Arrangement
o
Delicate, cyanophilic
o
Usually single cells
Cellular arrangement o
Intermediate cells
Chromatin is coarser than
Often tight, 3D clusters (from small clusters to large
Nucleus
fragments
o
10-18 u, round to oval
o
Finely granular, evenly
o
Single cells are very difficult to identify
distributed chromatin o
Centrally placed
Cytoplasm o
o
Endocervical Cells
20 – 50 u, polygonal, well
o
9-20 u, round to oval
defined
o
Finely granular, evenly
Blue (cyanophilic) or pink
distributed chromatin
(eosinophilic) o
May contain glycogen
o
Cellular Arrangement o
Nucleus
Cytoplasm o
Usually single cells
Columnar, delicate, fragile, may have cilia
o
Parabasal Cells
Eccentrically placed
Often contain single, large secretory vacuole (mucus)
Nucleus
o
Variable, round to oval, central
o
Finely granular, evenly
Cellular arrangements o
Sheets (honeycomb) or in palisading strips (picket fence)
distributed chromatin
Cytoplasm o
12-30 u, round, dense, well
Papanicolaou Staining Technique
defined
the nucleus.
o
Usually cyanophilic
The cytoplasm is rendered translucent.
o
May contain glycogen
The staining of squamous cell cytoplasm
Cellular arrangement o
Provides an excellent demonstration of
Single cells or sheets
range from: o
FRIED EGG APPEARANCE
basophilic (green or brown) staining of the least mature and deepest layer.
Endometrial Cells
Nucleus o
o
Eosinophilic or acidophilic (pink or orange) staining of the most
8-10 u, round, often “wrinkly”
mature and superficial.
It is a regressive, indirect and counter type of staining.
Ferning
pattern
Stains used: o
o
EA 36 (EA 50) EA 65 is for non-
High & persistent estrogen effect
gynecologic
Formation of salt crystals
One basis of early pregnancy
•
Eosin Y
•
Bismark Brown
•
Light-Green SF
OG 6
o
Cervical mucus shows a “palm leaf”
Leptothrix spp
Orange G crystals
Harris’ Hematoxylin
long, slender, gram negative, non spore forming anaerobic organisms
Non-pathogenic by themselves, but 75%-80% of cases have associated
Factors that Diminish the Accuracy of a Pap’s
Smear
Trichomonas vaginalis
Other associated infective organisms
Contamination with blood or lubricants.
include Candida and Garderella
Mislabeled or unlabeled slides.
vaginalis
Inadequate clinical history.
Inadequate sampling of the transformation zone.
Hormonal Evaluation Hormonal Cytology
Slide material too thick or inadequate.
Performing pap’s in spite of obvious
to stimulation by steroid hormones,
infection.
mainly estrogens and progesterone and
Principle: vaginal epithelium responds
to a lesser degree, to androgens and
Artifacts Seen in Poorly Prepared Smears 1. Enlarged nuclei, ill defined weakly
adrenal steroids.
Hormonal evaluation should be
staining chromatin and indistinct cell
performed on samples from the lateral
outline. (Drying Effect/Atrophy)
vaginal wall.
2. Presence of contaminants (talcum crystals)
Other Cells and Structures Seen (Normal and Pathologic)
Bacteria
Virus
Parasites
Spermatozoa
Fungi
Blood
WBC
Cancer cells
Accompanying data:
Patient name
Patient age
LMP
History of hormone or IUD use
History of medicine intake, operations, radiation, infection or disease.
Evaluation
Count 100 cells and classify the types of squamous cells seen.
Reporting: o
Hormonal Evaluation:____/_____/____
First blank = parabasal cells
Second blank = intermediate cells
Third blank = superficial cells
Classes not transferrable to histology terms
No classes for non-cancerous entities
Summary of Maturation Index Basic Features of the Bethesda System
Birth ------------------ 0/95/5
Childhood ------------ 90/10/0
Pre-ovulatory -------- 0/30/70
Pre-menstrual -------- 0/70/30
General categorization of abnormalities
Pregnancy ----------- 0/95/5 (navicular
Interpretation
cells)
Result
Lactation ------------- 90/10/0
Pre-menopausal & menopausal ---
Includes specific statements on adequacy of specimen.
Abnormal Cellular Findings (Bethesda 2001)
0/100/0
Atypical glandular cells (AGC)
Post-menopausal ------ 100/0/0
Atypical squamous cells of undetermined significance (ASCUS)
Cancer Diagnosis
o
It is a matter of finding the physical
undetermined significance
presence of malignant (cancer) cells.
(ASC-US) o
(Pap’s Smear)
Papanicolaou Numerical Classification System (1928) Class I – absence of atypical or abnormal cells
Class II – atypical cytologic picture but no evidence of malignancy
Class III – cytologic picture suggestive but not conclusive of malignancy. Dysplasia
Class IV – Cytologic picture conclusive of malignancy
Pap Classes are out because:
Do not reflect current understanding of Pathology
High grade squamous intraepithelial lesion (HGSIL or HSIL)
2. Bethesda System (1988) 3. Revised Bethesda System 2001
Low grade squamous intraepithelial lesion (LGSIL or LSIL)
1. Papanicolaou Numerical Classification System (1928)
Atypical squamous cells cannot exclude HSIL (ASC-H)
Reporting Systems for Vaginal Cytology
Atypical squamous cells of
Squamous cell carcinoma
Basic histopathologic techniques
process, cut and stain well
Histopathology
It is a basic component of a t ertiary
Human tissue & body fluids are
Tissue Exposure to Fixative
processed
Duration of Fixation
Tissue slides are produced for
o
Optimal thickness of 3-5 mm:
microscopic examination
o
Minimum – 6 hrs
Read and assigned a diagnosis by an
o
Maximum – 48 hrs
anatomical pathologist
o
To allow immunohistochemistry and/or in situ hybridization
Proper specimen handling does not start only upon reaching the lab
No amount of troubleshooting will solve poor fixation!!!!
hospital laboratory
Inadequately fixed tissues will not
o
5mm intervals
Pre-analytical Factors o
Warm Ischemia o
o
o
Properly Filled-up Surgical Pathology Request
initial anoxic insult a tissue undergoes during surgery
Accessioning Procedure
depends on the particular
o
Unlabeled specimens
circumstances of the
o
Incomplete specimens
procedure, skill & speed of the
o
Numbering – assigning of an
surgeon and other factors
accession number for proper
surrounding the procedure
and convenient identification
beyond the control of the lab
o
Questions of identification between 2 samples may require
Cold Ischemia o
Hollow organs are opened to allow full fixative penetration
Pre-analytical Factors
Large organs are sectioned at
expensive DNA identification
lack of oxygen once the sample is removed from the body
o
before the stoppage of
FIXATION
continuous metabolic processes by fixation o
o
further changes.
The earlier a tissue is fixed,
Important – preservation of the tissue
Properly preserved tissues are
at the exact moment when it is
more resistant to processing
removed from the body
artifacts Fixative to Tissue Ratio o
Alteration of tissues by stabilizing protein to become more resistant to
better preservation
Fixation o
Killing, penetration and hardening of tissues
both the operating room and laboratory can be responsible
10:1
Taking a “snapshot” of the disease
Functions of Fixing Agents
TYPES OF FIXATIVE
To set organs or parts of organs so that
According to COMPOSITION
the microanatomical arrangement of
According to ACTION
tissue elements will not be altered by subsequent processing.
To set intracellular inclusion bodies so that the histologic and cytologic
According to COMPOSITION A. Simple fixatives 1. Aldehydes
conditions of cells maybe studied.
a.
Formaldehyde
To arrest autolysis & putrefaction
b. Glutaraldehyde
To bring out differences in the
2. Metallic fixatives
refractive index of tissues.
a.
To enhance staining of tissues.
b. Chromate fixatives
To harden tissues for cutting very thin
c.
sections.
Mercuric chloride Lead fixatives
3. Heat B. Compound fixatives
Fixation – consideration
Masking of antigen by fixation leads to the failure of antibodies used in
A. Microanatomical Fixatives
immunohistochemistry to determine
B. Cytological Fixatives
the antigenic site.
Result- false negative
Solution – reversed by using antigen retrieval techniques
According to ACTION
1. Nuclear Fixative -
Contains glacial acetic acid, 4.6 pH or less
-
Flemming’s fluid,
Lipids and carbohydrates are very prone
Carnoy’s fluid, Bouin’s
to be lost during processing
fluid, Newcomer’s fluid & Heidenhain’s Susa
Solution – use alternative fixation methods
2. Cytoplasmic fixative -
Desirable Characteristics of Fixative: 1. Cheap 2. Stable & safe to handle
acetic acid, > 4.6 pH 3. Histochemical Fixatives -
Chemical components (protein,
3. Kills the cell instantly
carbohydrates,
4. Inhibit bacterial growth and damage
enzymes, hormones,
5. Minimum tissue shrinkage
etc.)
6. Rapid and even tissue penetration 7. Hard enough to allow cutting of thin
Never contains glacial
-
Lipid – cryostat or frozen section,
sections
chemical
8. Does not prevent staining reactions -
Carbohydrates (glycogen) – alcoholic fixatives
-
Protein – neutral buffered formol saline
Factors affecting fixation
or formaldehyde vapor
Temperature o
Increases fixation but increases rate of autolysis
Other classifications
o
Temperatures up to 45º C has
Additive or non-additive
little effect on tissue
Coagulant or Non-coagulant
morphology o
satisfactory to most
Additive fixatives
Bonds and adds itself to the tissues
altering protein structures
Specimen Size o
o
glutaraldehyde, osmium tetroxide and
Volume Ratio o
Non-additive fixatives Do not chemically bind with tissues
Removes water from the tissue protein groups
o
Ideal ratio of fixative volume: 15-20 is to 1
o
o
Prone to excessive shrinking and
Current recommendations: 10:1
Time Cold ischemia time •
hardening of tissues
Critical factor – involvement of operating room staff
zinc sulfide or chloride
3 mm sections are required for formalin fixed tissue processing
Formaldehyde, mercuric chloride, chromium trioxide, picric acid,
Formalin penetrates to a rate of 3-4 mm/hr
Implicated in the failure of immunohistochemical staining
Room temperature is
perform fixation
Acetones and alcohols •
time
Creates a network out of the protein o
structures that increases solution
Fixation time •
penetration to the interior of the tissue
10% neutral buffered
ethyl alcohol, methyl alcohol and
Non-coagulant fixatives
formalin
Creates a gel making penetration of tissues difficult
Overfixation: o
HER-2 false negative
o
Adversely affect staining qualities
Tissues are cut thinly for better penetration Formaldehyde, glutaradehyde, acrolein,
o
Over hardening of tissues
Properly fixed (ideal time) o
potasium dichromate, acetic acid, osmium tetroxide
Minimum of 6 hrs and a maximum of 48 hrs in
Zinc salts, mercuric chloride, picric acid, acetone
60 min is the maximum time before fixation
Coagulant fixatives
20 – 30 minutes ideal to
almost immune to processing artifacts
Underfixation o
Cannot be processed well
o
Subject to harmful effects of
solutions o
Gendre’s Fixative o
Irreversible artifacts and
Formalin + ethyl alcohol + glacial acetic acid
distortions
o
post fixation for immune staining
Aldehyde Fixatives
o
Formaldehyde (Formalin) o
o
Glutaraldehyde (2.5% & 4%)
routine fixative for paraffin
o
electron microscopy
embedded sections
o
Slower penetration, less tissue
usually buffered to pH 7 with a
shrinkage
phosphate buffer o
Advantages:
o
Cheap, available, easy
o
Compatible with most
o
4-6 hrs fixation
stains
o
45 mins fixation for biopsy
Do not over harden
specimen o
Penetrate tissues well o
Irritating fumes
slight reduction in staining
Causes dermatitis
ability of tissues
Shrinkage of tissues
Produces black 10% ideal, 15% for
Metallic Fixatives
Mercuric Chloride o
10% Formol-Saline o
Microanatomical fixative
o
Saturated formaldehyde +
o
o
Extremely toxic, expected not to be available commercially
o
De- zenkerizationis needed
examination
(alcoholic iodine solution) prior
Slow fixative
to staining
10% Neutral Buffered Formalin o
ideal fixative of choice
o
prevents precipitation of acid formalin pigments
o
Must always be freshly prepared
sodium chloride CNS and histochemical
Most commonly used metallic fixative
brain tissue
o
Long term tissue storage with
o
Better performance than formalin
Disadvantages:
precipitates on staining
Smallest aldehyde, 40% aqueous solution
tissues
fixation is limited best at 2 hrs
GLYOXAL
to prepare, stable
o
also for electron microscopy
o
Routine fixative of choice for preservation of cell detail in tissue photography
o
Recommended for renal tissue,
10% ideal most surgical path
fibrin, connective tissue and
specimen, 15% for brain tissue
muscle o
Brown pigment formation beyond 24 hrs, treated with
saturated picric acid or sodium
o
Preservation of myelin sheath
hydroxide
Lead Fixatives
Types
Zenker’s fluid (mercuric chloride +
glacial acetic acid) o
Used as 4% aqueous solution lead acetate
recommended for fixing small
Mucin & mucopolysaccharide fixation
pieces of liver, spleen,
Forms insoluble lead carbonate with
connective tissue fibers &
CO2, dissolved by acetic acid
nuclei
Zenker-formol (Helly’s solution) o
o
Picric Acid Fixatives
microanatomical fixative for
Excellent for glycogen demonstration
pituitary gland, bone marrow,
Imparts a yellow color (stain)
spleen & liver
Avoided in DNA or DNA staining
Highly explosive when dry
recommended for lymph node biopsies
Heidenhain’s Susa Solution o
recommended mainly for skin
Types:
tumor biopsies]
o
B-5 Fixative o
recommended for bone
Chromate Fixative
Highly toxic and carcinogenic
o
better than Bouin’s fixative
o
excellent fixative for glycogen
Glacial Acetic Acid o
Used as 1-2% aqueous solution
o
Usually a component of a
o
Used as a 3% aqueous solution
o
Cytologic fixative for
Used as component/additive of compound fixatives
o
Potassium Dichromate o
Penetrates very rapidly – soft tissues
compound fixative
Brasil’s Alcoholic Picroformol Fixatives
Chromic acid o
recommended for fixing embryos
marrow biopsies
Bouin’s Solution
Useful in the study of nucleic acids
o
Destroys mitochondria & golgi bodies
mitochondria o
Lipid preservation
Regaud’s (Moller’s) Fluid o
mitochondria, mitotic figures, golgi bodies, colloid Orth’s fluid o
Recommended for demonstrating chromatin,
Alcohol Fixatives
Study of early degenerative processes and necrosis
Can be used both as fixative and dehydrating agent
Excellent for glycogen preservation
Used routinely in cytologic studies
Contraindicated for lipid studies
Considerable tissue shrinkage
Tends to harden tissues excessively causing distortion
Trichloroacetic acid
Types
Methyl Alcohol 100% o
Excellent for fixing dry & wet smears, blood smears & bone
Incorporated with other fixatives to counter shrinkage
Weak decalcifying agent
marrow smears o
Highly toxic
Ethyl alcohol o
o
Used in histochemical studies
Routine fixative for cytologic
Rabies studies
smears (Paps smear)
Solvent for metallic salts in Freeze
Used for preserving tissues for enzyme studies
Acetone
Substitution techniques
Carnoy’s fluid o
HEAT FIXATION
Preserves Nissl granules &
1. Direct heat
cytoplasmic granules
2. Microwave fixation/stabilization
Alcoholic Formalin (Gendre’s) fixative
Newcomer’s fluid o
distortion.
Considered to be the most rapid fixative
o
Considerable tissue shrinkage and
Recommended for fixing
Zinc sulfate
mucopolysaccharides & nuclear
chloride
proteins
Osmium Tetroxide (OSMIC ACID)
pale yellow powder
Fixes myelin & peripheral nerves well
Fixative for ultrathin microtomy
Kept in a dark-colored, chemically clean
Proposed as a replacement for mercuric
o
Less toxic moderate health risks
o
Preserves tissue antigenicity
Cons o
Precipitation of zinc
Microwave fixation/stabilization
bottle to prevent evaporation &
controlled heat fixation, protein denaturation
reduction by sunlight
optimum temperature is 45-55 C
Choice for fixing lipids/fat tissue
allows routine light microscopic
Good fixative for nasal mucosa and
techniques, special stains,
conjunctiva
histochemistry and
Extremely volatile, hazardous and
immunocytochemical techniques
expensive
Types:
good secondary fixative post osmium tetroxide fixed
Flemming’s Solution
Flemming’s Solution w/o Acetic Acid o
Cytoplasmic stain
accelerates staining (immunocytochemistry)
only penetrates tissue to a thickness of 10-15 mm
Chief values:
-
fixed right through the block in a very short time
-
non-chemical technique
TISSUE PROCESSING AIM of Tissue processing
firm enough to support the tissue and
Secondary Fixation
give it sufficient rigidity to enable thin
To improve the demonstration of
sections to be cut yet soft enough not
particular substances.
To ensure further and complete hardening and preservation of tissues.
Secondary Fixation Technique
to damage the knife or tissue.
To make special staining techniques possible.
10% formalin o o
mercuric chloride easier tissue cutting and
Post-fixation treatment (Washing Out)
3% potassium dichromate) o
acts as a mordant
o
good for the demonstration of the mitochondria.
FACTORS AFFECTING FIXATION OF TISSUES RETARDED BY: Size & thickness of the tissue specimen Mucus Fat
solutions.
Agitation Warm temperature
excess formalin
o
excess osmic acid
excess picric acid from Bouin’s solution
Iodine
removal of mercuric deposits
General steps of tissue processing 1. Dehydration 2. Clearing 3. Infiltration 4. Embedding
DECALCIFICATION
Cold temperature Size & thickness of the tissue
o
50-70% alcohol
Blood
ENHANCED BY:
excess chromates from tissues fixed in Helly’s, Zenker’s and Flemming
subsequent staining is more
Post-chromatization (formalin – 2.5 –
removal of fixation artifacts
Tap water
brilliant and intense
remove excess fixative to improve staining,
flattens better o
To embed the tissue in a solid medium
-
Should be done after fixation
-
A good decalcifying agent should: 1. Remove calcium salts completely 2. Minimal destruction of cells & tissues 3. Minimal adverse effect to staining
Types of Decalcifying Agent
decalcify bone more rapidly (but more
1. Acids 2. Chelating agents
harmful to the tissue).
3. Ion exchange resins 4. Electrical ionization (electrophoresis)
More concentrated acid solutions
Recommended ratio of decal. fluid to tissue volume 20 to 1.
.
Heat hastens decalcification (but may damage tissues).
1. ACID DECALCIFYING AGENTS
1. Nitric acid A. Formol-nitric acid
Van Gieson’s stain for collagen fibers.
B. Perenyi’s Fluid C. Phloroglucin-Nitric Acid
3. Formic Acid – moderate acting
At 55 C = tissue will undergo complete digestion within 24-48 hours.
2. Hydrochloric Acid – slower A. Von Ebner’s Fluid
At 37 C = impaired nuclear staining of
Optimum temperature o
= RM TEMP (18-30 C)
The ideal time required: 24-48 hours.
Dense bone tissues require up to 14
4. Trichloroacetic Acid – weak
days or longer
5. Sulfurous Acid – weak 6. Chromic Acid (Flemming’s Fluid)
Decalcifying agents
2. CHELATING AGENTS -
EDTA (Versene)
-
Combines with calcium forming an
Nitric acid o
MOST COMMON
o
examples: Perenyi’s fluid – acts as BOTH tissue softener and
insoluble complex -
Very slow, 1-3 weeks
3. ION EXCHANGE RESIN
decalcifying agent.
Phloroglucin-Nitric Acid o
4. ELECTROPHORESIS (ELECTRICAL IONIZATION) MEASURING EXTENT OF DECALCIFICATION 1. Physical or Mechanical Test
AGENT!
5% formic acid is considered to be the BEST GENERAL DECALCIFYING AGENT
Formic acid is recommended for small pieces of bones and teeth.
Test)
Hydrochloric acid o
DECALCIFICATION
Formic acid – BOTH fixative and decalcifying agent
2. X-ray or Radiological Method 3. Chemical Method (Calcium Oxalate
MOST RAPID DECALCIFYING
Von Ebner’s fluid – recommended for teeth and
A procedure whereby calcium or lime
small pieces of bones.
salts are removed from tissue FOLLOWING FIXATION
Should be done after fixation and before impregnation
Post-Decalcification •
After decalcification is complete, acid can be removed from tissues or
neutralized chemically by immersing
GLYCOL-ETHERS
the decalcified bone in either:
Primarily solvents
o
o
saturated lithium carbonate
Do not act as secondary fixative
sol’n.
Ethoxyethanol
5-10% aqueous sodium
Cellosolve - rapid dehydrant
bicarbonate
Polyethlene glycols – dehydrating, embedding agent
DEHYDRATION Ideal Dehydrating Solution 1. Rapid but minimal tissue shrinkage.
Other dehydrating agents
2. Slow evaporation 3. Dehydrates fatty tissue 4. Minimal tissue hardening 5. Stain friendly 6. Non-toxic
7. Not a fire hazard
Dehydrating agents ALCOHOLS
Acetone o
Rapid dehydrant
o
Coagulant secondary fixative
o
Best dehydrant for fatty tissues
o
Controlled substance
Tetrahydrofuran o
Less toxic than dioxane
o
Rapid acting
o
Clears as well as it dehydrates
Excessive vs incomplete dehydration
Clear, colorless, flammable and hydrophilic
Acts as a secondary coagulant fixatives
Dioxane – being phased out
Ethanol
o
o
Most commonly used
o
99.85%
o
Inexpensive, readily available
Excessive Incomplete o
o
Prevents entry of clearing agents
Methanol o
Most common processing problem
and low toxicity
hard, brittle or shrunken
o
Good substitute but rarely used
Soft and non-receptive to wax infiltration
Isopropanol or Isopropyl alcohol (IPA) o o o
versatile ethanol substitute fully miscible with water, most organic solvents and parrafin
o
Ideal Clearing Agent
causes less tissue shrinkage and hardening
o
CLEARING
efficient lipid solvent
presently used as a xylene substitute in rapid automated processing
Miscible with alcohol, paraffin, mounting media.
Reduced tissue shrinkage
Makes tissue transparent (optically clear)
CLEARING (De-alcoholization)
Xylene
Types:
Paraffin Wax Impregnation
o
few hours, 5 mm tissue size
Celloidin (Collodion) Impregnation
o
most rapid
Gelatin impregnation
o
makes tissue transparent
o
highly inflammable
Cheap
o
not suitable for lymph node and
easily handled
section production provides few
nervous tissue
1. Paraffin wax Impregnation
Toluene
Benzene
Chloroform
o
difficulties
not inflammable
Cedarwood oil
wide range of melting points mixture of hydrocarbons in the cracking of mineral oil
Aniline oil
melting point range is between 40 – 70 C
Clove oil
54 – 58 C
Carbon tetrachloride
2 – 5 C above melting point maintained
Dioxane
Ethylene glycol monoethyl ether
beeswax
(Cellosolve)
ceresin
rubber
diethylene glycol distearate
resin
o
under investigation
Terpenes, Limonene & Terpineol
Prolonged vs incomplete clearing
Prolonged clearing time o
Additives:
IMPREGNATION
Brittle
Paraffin o
Incomplete clearing o
uneven H&E staining
o
Poor nuclear chromatin
the man who introduced paraffin wax embedding: Butschlii
o
patterns
simplest, most common and the BEST infiltrating/embedding medium.
Impregnation(Infiltration)
Also known as INFILTRATION
Process of replacing the clearing agent
o
tissues ( the dehydrants and clearing agents used in the process dissolve and remove fat
with the infiltrating medium.
The medium used to infiltrate the tissue is usually the same medium used for embedding.
from the tissues).
2. Celloidin (Collodion) Embedding
thin (2%), medium (4%), thick (8%) solutions
Volume of the impregnating medium should be at least 25 times the volume
is NOT recommended for fatty
large tissues cut with base sledge or sliding microtome
of the tissue
good for CNS and eyeballs
no heat involved
50% decrease in impregnation time
difficult to cut
Lungs, muscle, spleen, decalcified bone,
Two Methods o
skin, CNS tissue
Wet Celloidin Method (bones, teeth, large brain sections, large
EMBEDDING/CASTING/
section) o
BLOCKING
Dry Celloidin Method ( whole eyeball) chloroform +
cedarwood oil
LVN (low viscosity nitrocellulose)
can be used in higher concentrations
penetrates well
explosive when dry
Embedding Tissue in Paraffin Wax
wax dispenser
cold plate
heated storage area for molds
Embedding molds 1. Leukhart’s or Dimmock embedding
3. Gelatin impregnation
mold
frozen section
2. Compound embedding unit
histochemical and enzymatic studies
3. Plastic embedding rings and base mold 4. Disposable Embedding molds
Substitiutes for Paraffin wax
a. peel-away
1. Paraplast (56-57 C)
-
b. plastic ice trays
highly purified paraffin and
c.
paper boats
synthetic plastic polymers
-
embeddol Bioloid (embedding eyes) Tissue mat ( paraffin + rubber
Orientation of Tissues
starts during gross time
Most tissues are cut from the largest
2. Ester wax (46 – 48 C)
-
harder than paraffin
area.
soluble in 95% alcohol sliding or sledge microtome
3. Water soluble waxes (polyethylene
section
surface and oriented so the surface is
38-42 C to 45 – 56 C Carbowax Does not require dehydration
cut first.
speed up impregnation and remove any residual air bubbles
500 mmHg max
Muscle biopsies are sectioned in both transverse and longitudinal planes.
and clearing
Vacuum impregnation
Skin and other epithelial biopsies are cut in a plane at right angles to the
glycols)
-
Tubular structures are cut in cross
When a particular tissue feature is present on one aspect only.
*melt wax to temperature 5 – 10 C above the melting point of wax
Other embedding methods
Celloidin or nitrocellulose method
Double – embedding method (Peterfi’s
Resin Embedding Media Areas Where Paraffin is Unsuitable
technique)
Unsuitable for ultrastructural studies. o
thin sections (30-80 nm)
Resin embedding o
paraffin wax is unable to withstand the high energy
Tissue Softeners
does not offer support for ultra
electron beam
For unduly hard tissues that may damage the microtome knives.
It doesn’t permit sufficiently thin
4% aq. phenol.
sections to be cut for high resolution
Molliflex - tissues immersed in Molliflex
microscopy.
may appear swollen and soapy
o
specially important for renal and lymph node biopsies.
2% HCl
1% HCl in 70% alcohol
It doesn’t provide support for extremely hard tissue for cutting.
Tissue Processing Methods
o
Resin Media
Manual Can be accelerated by: -
Microwaves
-
Ultrasonics
o
o
allows sections as thin as 30 – 40 nm
fixes, dehydrates, clears and o
infiltrates Two Main Types Tissue –Transfer (Dip & Dunk) Type o
widely used for electron microscopy
Automated Processing o
Epoxy resins
Carousel type (Elliott Bench-
3 Types •
Bisphenol A (Araldites)
•
Hlycerol (Epons)
•
Cyclohexene dioxide (Spurrs)
Type) Fluid-transfer (enclosed pump fluid)
Type o
Autotechnicon, Hypercenter
Acrylic resins o
earliest resin used
o
unstable to high energy electron beam
Advantages of automatic machines
o
recommended for light
1. Vacuum and heat at any stage
microscopy (High resolution
2. More rapid schedules
microscopy)
3. Fluid spillage containment
•
sections
4. Less toxic fumes emitted
Factors Influencing the Rate of Processing
o
provides support for cutting undecalcified bone
1. Agitation - 30% reduction 2. Heat – 45 C ideal
can support 1-2 um
o
Types
3. Viscosity
•
Butyl methacrylates
4. Vacuum
•
Methyl methacrylates
•
Glycol methacrylates
5. Ultrathin microtome 6. Vibrotome
Microtomy
for unfixed, unfrozen sections
enzyme studies
Microtome Cutting •
Microtome Knives
tissue is advanced for a predetermined distance then slide the object to the cutting tool, usually a steel knife.
Knife materials 1. High carbon steel 2. Tungsten carbide-tipped knives
Types of Microtome
3. Glass knives
Rocking microtome (Cambridge)
4. Diamond knives
Rotary microtome (Minot)
5. Stellite-tipped knives
Sliding microtome
Freezing microtome
Ultrathin microtom
o
Non-rusting
Vibrotome
o
For cryostat
o
Not recommended for paraffin
o
tungsten)
embedded tissues
1. Rocking microtome (Cambridge)
most popular from 19th century to
(alloy of cobalt, chromium and
6. Disposable blades
1950’s
o
very sharp and cheap
Paldwell Trefall
o
can produce flawless 3-4 um
large blocks of paraffin embedded
sections, clearance angle of 3 to
tissues
8 degrees
cut surface of tissue block is beveled
also used in cryostats
o
replaced the conventional steel knives
o
Classification: 1. A. Low-profile blades
2. Rotary microtome (Minot)
commonly used today, even in cryostats
heavier and stable
provides a flat face to the tissue block
retracting mechanism
3. Sliding microtome
Adams (1789)
Base sledge/Standard sliding
for celloidin embedded tissues
4. Freezing microtome
Queckett (1848)
Cryostat (cold microtome)
2. B. High-profile blades
Profiles of Steel Knives 1. Plane wedge 2. Planoconcave
-
sliding microtome celloidin embedded
3. Biconcave
-
very keen edge rigidity is sacrificed
4. Tool edge
-
used with the hardest tissue and embedding media
Cutting edge profiles
Floating Out & Fishing Out Sections
1. Bevel angle – 27 to 32 ° 2. Clearance angle – 0 to 15°
“The sections are floated out on a water bath set at 45-50 C, approximately 6-10
Sharpening Microtome knives
C lower than the MP of the wax used
1. Honing – heel to toe 2. Stropping – toe to heel
for embedding the tissue.”
For routine work, 76 x 25 mm. slides that are 1.0-1.2 mm. thick are usually
Honing Stones
preferred because they do not break
1. Belgium Yellow
easily.
2. Arkansas 3. Fine Carborundum
Types of Tissue Sections & Thickness 1. Paraffin Sections – 4 to 6 u, 3 to 4 u
MOUNTING Desirable Characteristics
2. Celloidin sections – 10 to 15 u 3. Frozen sections
glass(1.518)
Trimming: o
to expose the tissue
It should set hard
o
apply ice over the surface after
Types:
Cutting Sections: o
Softer tissue, slower stroke.
o
Gentle exhaling breath on the block surface.
o
Sections should stick – to – stick
o
Ribbons are separated for every 6 to 8 sections
Aqueous Media
o
Resinous Media
Generally 1.40 to 1.45
For water miscible prep if xylene or alcohol dissolves the stain.
Usually for enzyme histochemistry
Most use:
Floating out sections o
o
Aqueous Media
(ribboning)
It does not interfere with the stain and distort tissues.
trimming
Refractive index approaching
o
Water temp 6 to 10 C lower
gum arabic & gelatin as solidifiers
than the melting point of the
o
wax
glycerol to prevent drying, increase refractive index, preservative
Adhesives 1. Mayer’s Egg Albumin 2. Dried Albumin 3. Gelatin 4. Starch paste 5. Plasma
Aqueous Mounting Media
Water
Farrants’ medium (gum arabic)
Apathy’s mountant (gum arabic)
Kaiser’s glycerol jelly (gelatin)
Polyvinyl pyrrolidone
Resinous Mounting Media
B. Pure Physical stain
Canada balsam (1.54) Dyes used in histopathology
natural, Abus Balsamea
stains fade after sometime
1. Natural Dyes
turns dark yellow after sometime
2. Synthetic (Artificial) Dyes
DPX (1.52)
neutral colorless solution
good for small tissue mounts
XAM (1.52)
Natural Dyes
Hematoxylin
Cochineal dyes
pale yellow or colorless solution
o
Cochineal bug (Coccus cacti)
preserves stain
o
Carmine dyes
dries w/o tissue retraction
o
Powerful chromatin and nuclear stain
Clarite (1.54)
Ringing Sealing the margins of the coverslip :
Orcein dyes o
vegetable dye (lichens)
o
litmus
Saffron
prevent escape of mountant
usually for aqueous mountants
prevent evaporation
immobilize the coverslip
prevent sticking of slides during
Coal Tar Dyes (Hydrocarbon benzene)
storage.
Aniline dyes
o
Crocus
Synthetic (Artificial) Dyes
Kronig cement (Paraffin + resin) Durofix (cellulose)
Basic Principles (Synthetic Dyes)
Chromophores are substances capable of producing colors.
STAINING
Chromogens are substances containing chromophores.
4 Major Applications of Stains
Auxochrome alters the chromogens to
•
Routine staining procedures
form salts to allow retention of the dye
•
Special stains
to the tissue.
•
Histochemical stains
•
Immunohistochemical stains
Mechanisms of tissue STAINING •
Chemical Staining Dye-to-Tissue
o
Classification of Dyes
Mechanisms
Acid dyes (Anionic dyes) o
2. Hydrogen bonding 3. Van der Waals Forces 4. Covalent bonding
react with cationic or basic components in cells
A. Chemical stain 1. Ionic or Electrostatic bonding
(-NH2, -COOH, -S03H, -OH)
Basic dyes (Cationic dyes) o
react with anionic or acidic components in cells
General Methods of Staining
Direct Staining
Indirect staining o
Progressive staining
Regressive staining
Haematoxylin
alcohol solvent usually takes away the stain
nuclear dye in the standard H & E, primary stain
granules and amyloid o
myelin, elastic, collagen, muscle striations, mitochondria, etc.
staining of cartilage, connective tissue, mucin, mast cell
most widely used natural dye in histotechnology
differentiation (decolorization)
Metachromatic staining o
Haematoxylin & Eosin
mordant
o
Staining
Source
logwood, Haematoxylon campechianum (order Leguminosae, genus Eucaesalpinieae)
Metachromatic Dyes
reddish color of its heartwood and
Methyl violet or Crystal violet
Cresyl blue
Safranin
Bismarck brown
(Campeche and Yucatan regions of
Basic fuchsin
Mexico)
Methylene blue
Thionine
Toluidine blue
Azure A, B, C
Counterstaining
young leaves
indigenous to Central America
grown commercially in the West Indies
Extraction
Crude product is obtained from chipped heartwood by hot water or steam.
Purified by ether extraction.
Dried
Recrystallized from water or precipitated with urea.
Microanatomical staining
Cytoplasmic staining
Alterations
Negative staining
The Spanish recognized that the color of the dye could be altered. 1. iron alum – black
Metallic impregnation
2. potash alum – blue
Gold chloride & Silver nitrate
3. salts of tin – red
Vital staining
Intravital staining (lithium, carmine, India ink)
Properties of Haematoxylin
haematoxylin itself is not a stain.
Supravital staining (Neutral red, Janus green, Trypan blue, Nile blue, thionine, toluidine blue)
It is important to realize that
It is its oxidation product, HAEMATIN, which is the natural dye.
Haematin is anionic, having a poor
•
similar features with Ehrlich’s
affinity for tissue.
•
15 – 20 mins
Mordants are required to enable a
3. Mayer’s haematoxylin
binding to the tissue site, nucleus.
•
chemically ripened with sodium iodate
Haematoxylin
•
used as regressive and progressive stain
Oxidation methods:
•
for glycogen demonstration
1. Natural oxidation (ripening)
slow process, 3-4 mos
longer shelf life
Ehrlich’s and Delafield’s
•
conversion is almost instantaneously
shorter shelf life
•
Alum haematoxylin
•
Iron haematoxylin
•
Tungsten haematoxylin
•
Molybdenum haematoxylin
•
Lead haematoxylin
•
Haematoxylin w/o mordants
chemically ripened with mercuric oxide, sodium or potassium iodate
•
provides a clear nuclear staining
•
routinely used in exfoliative cytology staining
Classification of Haematoxylin •
5 -10 mins
4. Harris’s haematoxylin
2. Chemical oxidation
used as a nuclear counterstain
(Papanicolaou) •
good for the staining of sex chromosomes
•
5 – 20 mins
5. Cole’s haematoxylin •
chemically ripened with alcoholic iodine solution
•
A. Alum Haematoxylins
most are used for routine staining
aluminum potassium sulphate,
6. Carazzi’s haematoxylin •
stains the nucleus- red
addition of glycerin stabilizes and prevents evaporation naturally ripened, 2 mos
•
generally used for regressive good for mucins of cartilage, bone
•
NOT ideal for frozen sections
•
15 – 40 mins
2. Delafield’s haematoxylin •
naturally ripened
good for frozen section
•
1 min
•
chemically ripened with sodium iodate
staining (routine H & E staining) •
•
7. Gill’s haematoxylin
1. Ehrlich’s haematoxylin •
chemically ripened using potassium iodate
aluminum ammonium sulphate
5 mins
•
good for regressive staining
•
5 – 15 mins
B. Iron Haematoxylins
iron salts are both used as a mordant and oxidizing agent
ferric chloride, ferric ammonium sulphate
