Quality Control, Quality Assurance & Quality
Management System
Quality
Degree of congruence between
expectations and realization.
Customer checks if the quality of
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 activities
on fulfilling quality requirements
Quality Assurance
Aims to generate the confidence of the
patient to the final report
Receipt of the request/specimen
release of the report
Includes availability of reagents,
preventive maintenance & monitoring
of equipment & evaluation of the
quality of service
Quality Assurance (QA)
In short, it is a means of:
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 !!!!!
Quality Management System (QMS)
Set of coordinated activities to regulate
a lab in order to continually improve
performance efficiency
Quality Management System in a Histopath
Lab
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 customers feedback
Good Laboratory Practices
3 Phases of examination and Factors
affecting them:
o Pre-examination (Pre-
analytical)
o Examination (Analytical)
o Post-examination (Post-
analytical)
Pre-analytical Phase Factors
Collection of the right specimen
The proper fixation of the specimen
The correct identification of the
specimen
The timely transportation of the
specimen
Examination (Analytical) Factors
Grossing of tissues
Processing
Procedure reliability using technical
manuals
 Reagent integrity and efficiency
Cutting of paraffin sections
Staining
Slide labeling
Equipment reliability
Adequate calibration
Proficiency of personnel and continuous
updating of their knowledge
Good internal quality control
Post-examination (Post-analytical Factors
Render histopathologic diagnosis (hard
copy or electronic) free of clerical errors
Ensure that the report reaches the
appropriate clinician/surgeon.
Filing of paraffin blocks must be in a
cool area and rodent free
Slides are stored for 10 years while
reports may be longer in a safe and
humidity free areas
Any possible remarks on the diagnosis
obtained should also be included
Frequent dialogues between the
pathologist and the surgeons/clinicians
Flow Chart for Surgical Biopsy Process
Receive/Record Specimen
Check request/Specimen
Gross exam
Tissue Processing
Cutting/staining
Mounting/Labeling
Microscopic Exam
Release of reports
Flow Chart for Cytology
Receive/record specimen
Check request
Gross exam
Smear preparation
Staining
Mounting/labeling
Microscopic exam
Release of reports
Basic Information on Chemical Labeling
Chemical name/names of all ingredients
Manufacturers name& address/Person
making the reagent
Date purchased or made
Expiration date
Hazard warnings & safety procedures
Storage of Hazardous Chemicals
Dangerous liquids below countertop
height
Dangerous reagents plastic or plastic
coated glass bottles
Flammables are never stored in a
refrigerator or freezer, only in certified
Use of this chemicals are used only in
small quantities as needed and used up
completely
Do not store any leftover flammable
liquid
Documentation
Histopath Reports
o Surgical Pathology Report
o Cytopathology Report
o Autopsy Report
Number of Copies Prepared: (3)
o Patients Copy
o Doctors Copy
o Pathology Departments Copy
Routine Turnover (Release) of Results
Surgical Path & Cytopath Results 24
hrs
Frozen Section 5 to 15 minutes
Autopsy Report 1 week
Signatories
Request Forms (All Forms)
Patients doctor, Attending physician
Result Forms (All Forms)
Pathologist
Specimen Handling
Numbering system of assigning EPITHELIAL TISSUES:
numbers to specimens received
chronologically as a form of labeling.
Tools for Labeling
o Pencil
o Diamond pencil
o Gum Label
Retention Period
Surgical Pathology specimen 2 to 4
weeks after the issuance of a final
report
Tissue block, histopath slides & FNA
slides 10 years
Cytology slides 5 years
Surgical pathology/cytology & other
non-forensic reports 10 years
Forensic autopsy reports - indefinitely
Basic Histology
Histology study of normal tissues
o A fertilized egg divides forms
smaller cells
o After morphogenetic
movements, these cells become
arranged in 3 germ layers:
1. ECTODERM
2. ENDODERM
3. MESODERM
Tissues group of cells of common origin and
common function
Four categories of tissues:
Epithelial tissues derived from ALL
THE THREE (3) GERM LAYERS
Connective tissues from mesoderm
Muscular tissues from mesoderm
Nervous tissues from ectoderm
A. Covering epithelia
B. Glandular epithelia
Covering epithelia
Blood vessels are absent
Exposed to physical injury and infection
Classifications of Covering Epithelia:
According to cellular arrangement:
Simple one-cell thick
Pseudostratified appear to be more
than one-cell thick but actually cells rest
on common basement membrane
Stratified many layers of cells
According to cell shapes:
Squamous flattened cells (like paving
stones)
Cuboidal cube-like (isodiametric cells)
Columnar cells that are taller than
they are wide
Transitional cells that change their
shape when the epithelium is stretched.
Combination of classifications I and II:
SIMPLE:
o Squamous Bowmans capsule,
Endothelium of blood vessels,
Loop of Henley, Alveoli of lungs
o Cuboidal in walls of thyroid
follicles and ducts of glands
 o Columnar gallbladder
(NONCILIATED), uterine tube
(CILIATED)
STRATIFIED
o Squamous Epidermis of the
skin (keratinized), Vagina (Non-
keratinized), Esophagus (Non-
kera), Cervix (Non-kera)
o Cuboidal sweat gland ducts
o Columnar Male urethra
o Transitional Urinary tract
PSEUDOSTRATIFIED
o Columnar much of female
reproductive tract (Non-
ciliated), trachea (Ciliated)
Glandular epithelia
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
the free surface of the cell
through which it is released
o EXAMPLES: Goblet cells, sweat
glands
Apocrine
o with cytoplasmic loss
o secretions accumulate below
the free surface but can only be
released by breaking away of
the distal part of the epithelium
o EXAMPLE: Mammary glands
in milk secretion
Holocrine
o complete breakdown of the
secretory cell
o EXAMPLE: Sebaceous glands
OTHER CATEGORIES OF TISSUES
CONNECTIVE TISSUES
Cells are usually widely separated by a
large amount of intercellular substance
Blood and blood-forming tissues, bone,
and cartilage
I. General Connective Tissues
Loose Connective tissue
Dense Connective tissue
II. Special Connective Tissues
Cartilage
Hematopoietic
Bone
Blood
Lymph
Loose Connective Tissues
Common examples include:
o Mucoid tissues Whartons
jelly
o Reticular Bone marrow,
lymph node
o Mesenchyme embryo and
fetus
o Adipose - hypodermis
Dense Connective Tissues
Common examples include:
o Dermis
o Capsules of organs
o Tendons
 o Stroma of cornea Clinical Pathology
Clinical Chemistry
SPECIAL CONNECTIVE TISSUES Immunology & Serology
Cartilage Hematology
o Bone Blood Bank
o Blood Microbiology
o Lymph Clinical Microscopy
o Hematopoietic
Cartilage: Branches of Anatomical Pathology
o Hyaline - trachea Surgical or Morbid Pathology
o Fibrous Intervertebral discs Cytopathology
o Elastic external ear, epiglottis Autopsy Pathology
Bone: Forensic Pathology
o Cancellous/ Spongy Epiphysis Immunopathology
or ends of long bones Molecular Pathology
o Compact Diaphysis or shaft
Hematopoietic: Basic Concepts on Diseases
o Myeloid Bone marrow Manifestations of disease
o Lymphoid - Spleen Signs
o objective manifestations of a
MUSCLE TISSUES disease.
Smooth (involuntary) found in o Example :tumor, tenderness,
intestinal tracts and blood vessels ulcer, fracture, fever,
Striated (voluntary) found in hemorrhage, diarrhea, etc.
skeletal muscles Symptoms
Cardiac (striated but involuntary) - o subjective manifestations of a
heart disease.
o weakness, pruritus, fever,
NERVOUS TISSUES
nausea, dizziness, pain,
Central Nervous system brain and
numbness, etc.
spinal cord
Peripheral nervous system 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 o Impact to the population?
Students Review
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
Cellular Injury
To be viable, a living cell must
maintain an organization capable
of producing energy.
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.
o Atrophy
o Hypertrophy
o Hyperplasia
o Metaplasia
Atrophy
Decrease in the size and function of Features:
the cell.
Adaptive response reducing its need
for energy to the minimum but still
keeping the cell alive.
Effect: Conservation and limit use of
(energy) ATP to keep the cell alive.
Reversible
Hypertrophy
Increase in the size of cell
accompanied by enhanced
functional capacity.
There is an increase in the
organelles and other components of
the cell.
This will result to the compensation
of function.
Hyperplasia
Increase in the number of cells in an
organ or tissue.
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 Chronic irritation!!!
It is the conversion of one adult
cell type to another cell type.
Irreversible Cell Injury
Cell membrane disruption.
Nuclear change and deterioration.
o pyknosis
o karyorrhexis
o karyolysis
Lysosomal rupture and digestion.
Cellular Death
Apoptosis
Necrosis
Apoptosis
programmed cellular death
cellular suicide
 Physiologic death of single or few o It is primarily a genetic
cells disease, more of idiopathic
Significance: disease.
o Replacement of old, worn Genes controlling cell division and
out and injured cells. growth:
o Destruction of abnormally o Proto-oncogene
mutated cells that may lead o Tumor suppressor gene
to cancer or congenital Mutant gene
abnormalities. o Oncogene

Necrosis 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
Benign
Types of Necrosis o oma
Coagulation necrosis o microscopic & macroscopic
Liquefaction necrosis structure
Caseous necrosis o cell of origin
Non-caseous necrosis Malignant (Cancer)
Gangrenous necrosis o carcinoma, - sarcoma
o Dry gangrene o microscopic & macroscopic
o Wet gangrene structure
o cell of origin
Neoplasia
Neoplasm (Tumor) Significant Information
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. Metastatic spread via:
Significance: o Hematogenous route
o Significant cause of death o Lymphatic spread
because of organ destruction o Local spread
o early detection is necessary
(difficult to attain) Epidemiology of Neoplasms
Cancer can strike at any age.
 Three leading cancers in men are Tumor Markers
lung, colon and rectal cancer. Tumor cells produce substances, many
For women: breast, lung, colon & of which are proteins, which are helpful
in diagnosis and monitoring of
rectal ca.
treatment.
Advancing age increases the risk of
o (A) Product enters blood stream
cancer. (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 Immunocytochemistry & Tumor Markers
(Used as Immunohistostains as well as
Chemical carcinogens
in Serologic determinations.)
o Polycyclic aromatic
Goals and uses:
hydrocarbons
o Used as a screening test for cancer.
o Nitrosamines o Used as a diagnostic tool for cancer.
o Aflatoxin B1 o To monitoring the effects of
o Asbestos, silicon treatment for:
Physical Carcinogens - Possible recurrence
o UV rays - Response to chemotherapy
o Radioactive/ionising waves and radiotherapy.
Viral Oncogenes
o Hepatitis B Tumor Markers
o HPV
o AIDS
Inherited ( Familial) Cancer Syndrome
o Neurofibromatosis
o Retinoblastoma

Laboratory Diagnosis of Cancer

Histologic and Cytologic Methods
o biopsy, autopsy , endoscopy,
paps test
Molecular diagnosis
Flow Cytometry
Immunocytochemistry & 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)

Grading
o based upon the microscopic
appearance of a neoplasm with
Autopsy (Necropsy)
H & E.
o In general, a higher grade
means there is a lesser degree
of differentiation and the worse
Cervical smear
Mammography
Endoscopic biopsy
Skin surveillance for pigmented tumors
Self examination (breast, testes)
Tumor markers
o blood, urine, tissue
Autopsy
This is the branch of Anatomic
Pathology that deals with the
 complete medical examination of
the body after death.
postmortem examination
It is a medical procedure practiced
since ancient times to analyze
organic alterations caused by
disease or trauma.
Autopsia Seeing with ones own
eyes.
Main Goal of Autopsy
To identify the cause of death and to
improve medical science.
Other Values of Autopsy:
Diagnosis and treatment quality
control.
Source of accurate epidemiologic
data
Material for medical residents,
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
examination.
Microscopic examination
Releasing of reports
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
that are not close relatives to the
patient.
The procedures done during the
autopsy should be explained fully
and clear enough to be understood
by the relatives before seeking
consent.
In medico legal cases, deaths with
questionable circumstances, a
medico legal officer or an
appropriate agency like the NBI or
PNP is consulted.
Relatives of the patient are not
supposed to be in the autopsy room
during the procedure.
Extent of the Autopsy
Partial autopsy
specific body component is
only involved.
head only, thorax only
Complete (Full) autopsy
whole body involvement
Incision for the autopsy:
The Y incision
Evisceration Techniques
En masse (Letulle)
En bloc (Ghon)
Individual organs (Virchow)
In situ (Rokitansky)
En Masse (Letulle)
Bulky single aggregate of organs
removed en masse
and transferred to the dissecting
table for further dissection.
En Bloc Dissection (Ghon)
Thoracic pluck (block)
Coeliac block
Intestinal block
Urogenital block
Neurological block (if needed)
Individual Organ Removal (Virchow)
Organs are removed one by one
sequentially
Isolated and dissected immediately
after removal
In Situ Method (Rokitansky Technique)
Body is cut open in the usual fashion
Cavities and organs are thoroughly
inspected
Fluids are collected if needed
Organs are dissected while still
inside the body
Organ removal is optional
Special Considerations
Atherosclerotic plaque (atheroma)
of coronary vessels for
decalcification
Severe atheroma (75%) is required
before it is said to be the cause of
death
The heaviest organ liver, brain
Lung sinking in a pan of water
indicates drowning or severe edema
Muscle tissue is stretched on a
cardboard before fixing
Organ Weights
Right lung: 300-400 gm
Left lung: 250-350 gm
Heart: 250-300 gm
Liver: 1100-1600 gm
Adrenals: 4 gm or so each
Thyroid: 10-50 gm
Spleen: 60-300 gm
Brain: 1150-1450 gm
Postmortem Changes
Death complete cessation of
metabolic and functional activities
of the organism or body as a whole.
Three (3) Primary Changes (Death)
o Circulatory Failure
o Respiratory Failure
o Nervous (CNS) Failure
Secondary Changes After Death
Rigor Mortis
Severe rigidity or spasm of the
muscles.
Interlocking of the actin and
myosin secondary to lactic acid
buildup
Immediate - onset
1-6 hours - manifestation
6-24 hours - maximum
Persists 12-36 hours (3-4 days)
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 Methods for the Estimation of Time of Death
hours. Rate Method
o Body changes, blood changes,
Tardeaus Spots/Petechial
plant or animal indicators
Hemorrhages rupture of capillaries
Concurrence Method
after death resulting to petechiae 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
reduction in body temperature (Thrombus)
following death. Postmortem Clot
a steady decline until matching Settling/separation of RBC
chicken fat appearance
ambient temperature
currant jelly appearance
1 to 7 Fahrenheit per hour until the
Assumes the shape of the vessel
body nears ambient temperature
Rubbery in consistency
Glaister equation Formula used for Premortem Clot (thrombus)
determining the approximate time Granular and friable
period since death based on body Fibrin precipitation
temperature. Seldom assumes the shape of the vessel
Not easily detached from the bld vessel
Ocular Changes wall
Description Eyes Open Eyes Closed
Do not have a rubbery consistency
Corneal film minutes Several
hours
Schleral Minutes to ---------------- Cytopathology/Cytology
discoloration several hours
tachy noir
Corneal 2 hours or less 12-24 hours Manner of Cellular Exfoliation & Evaluation
cloudiness
Corneal ----------------- rd
3 post- Spontaneous shedding from organ
opacity -------- mortem day
Exophthalmos w/ gas Same surfaces. (Exfoliative Cytology)
formation Physical removal of cells from organ
Endophthalmos w/ advance same
decomposition surfaces. (Imprint/Abraded Cytology)

POSTMORTEM TISSUE CHANGES

General Applications of Cytopathology
Decomposition
Screening for the early detection of
o Autolysis
asymptomatic cancer.
o Putrefaction
Diagnosis of symptomatic cancer.
Mummification
Surveillance of patients treated for
Skeletonization
cancer.
 Detection of an infectious process and difficulty in the interpretation of
its etiology. cytology.
Determination of genetic sex. o Inadequate cell concentration
For the assessment of female hormonal or poor adhesion.
activity. o Thick smears cause overlapping
of cells.
Cytologic Evaluation o Poor fixation.
o Poor or over stained
Exfoliation (Exfoliative Cytology) preparations.
This refers to the examination of cells
that are shed spontaneously into body
fluids or secretions. Usual Specimens For Cytology (Cytopathology)
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) Smears of urine sediments
Encompasses methods by which cells CSF
are dislodged by various tools from Wound secretions
body surfaces. FNAB derived
o Vaginal and cervical smears. CT guided or deep organ aspiration
o Endoscopic brushing from the GIT, biopsy
respiratory and urinary tracts.
o Scraping of cutaneous, oral, vaginal
or conjunctival lesions. Preparation of Body Fluids for Microscopic
o Washing or lavage during Study
endoscopy or surgery. Smear preparation
Fine Needle Aspiration Biopsy (FNAB) o Crush Technique
It is a technique that uses cells obtained o Pull-Push Technique
by aspiration under negative pressure Tissue (Cell) block
through a thin-guage needle.
The Cell Block
Causes of Error in Cytology Paraffin embedded specimen from
Inadequate sampling is one major cause different fluids and aspirated materials
of false negative diagnosis. Can be prepared from all types of fluid
Poor fixation of the smears or 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
Adjunct for establishing a more
definitive cytopathologic diagnosis
(Malignancy and/or metastasis)
Architectural evaluation
Categorization of tumors not possible in
smears
Special stains & immunohistochemistry
Immunophynotyping, molecular studies
(CISH, FISH, PCR)
Archive material
Cell Block Preparation
1) Centrifuge fluid to obtain cell button.
2) Decant supernatant fluid and re-
suspend cells in normal saline.
3) Re-centrifuge at 2500 rpm for 5 min
4) Decant supernatant fluid.
5) Warm the tube at 45 C under hot
water.
6) Add 2% agar solution cooled at 50 C and
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
transform the agar into an irreversible
gel.
9) Process by routine histologic method.
Varied Specimens and their Preparation
A. Cervical and Vaginal Smears
swab, aspirate, brush
Fixed in 95% ethyl alcohol
Fix for 15 minutes before staining
Can spray with hair spray for transport.
o (ether alcohol and polyethylene
glycol)
B. Pleural/Peritoneal Fluids
Fresh fluid is centrifuged
Can add 300 units of heparin
C. Sputum and Bronchial Aspirates
fresh early morning collection
5 consecutive day collection
Prepare 5-8 slides and fix for 1 hour
D. Gastric Aspirate
Best obtained by brushing as compared
to aspirates.
Fix right away in 95% ethanol and cool
in ice.
E. Prostatic Secretions
Three Specimens Obtained at Random
1. Voided urine before massage.
2. Smears from a prostatic massage.
3. Urine after massage.
F. Urinary Sediments
Males voided urine
Female a catheterized specimen is
recommended
Things to Consider in Specimen Preparation
Smears should be even and lump free.
Patients identification should be labeled
on the slide immediately after
smearing.
Smears should immediately be placed in
fixative while still moist.
Specimens transported should be fixed
and air dried. (no longer absolute)
If smears cannot be made immediately,
the collected material may be
refrigerated
Excessive blood is removed with 2-5 ml
conc. Hac per 100 ml.
Adhesives for Cytologic Smears
Most specimen contain a natural
adhesive, albumin.
Specimens Requiring Adhesives:
 o urinary sediments
o Bronchial lavage specimen
o Specimen releasing proteolytic
enzymes
Characteristics of a Good Adhesive Agent
It must be permeable to both fixative
and stain.
It must NOT retain the stain.
Adhesive Agents:
1. Pooled human serum or plasma
2. Celloidin Ether Alcohol
3. Leuconostoc Culture
Fixation of Cytologic Smears
Common Fixatives Used
Equal parts of 95% ethyl alcohol &
ether.
95% ethyl alcohol
Carnoys fluid
Butanol-ethanol
Ethanol-acetic acid
Advantages of Ethyl Alcohol
Alcohol is a protein coagulant and
smears of predominantly protein
material will remain better attached to
the slide during staining.
Alcohol gives good chromatin
preservation and delineation.
Alcohol is easily dispensed to clinics and
can be stored without undue
deterioration.
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.
Avoid striking the bottom of the fixing
container forcefully to prevent
dislodging the material from the slide.
Identify the slides before preparing
smears.
Have the individual fixative bottle open
before preparing the smear.
Sexual Determination
Smears for Sexual Determination
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
Vaginal Cytology (Paps Smear)
Dr. George N. Papanicolaou, first
described its use in detecting malignant
cells of the uterine cervix.
Today its usefulness is used as a
primary screening method for the
detection of cervical malignancy, and
especially its precursor lesions.
General Purposes
(In addition as a screening test)
Detect the etiology of certain
infections.
Determine ovarian function based on
hormonal cytology
Used as one of the battery of tests used
in infertility workup.
Monitor treatment for diagnosed
cancer
Determine phenotypic sex.
Medico-legal examination of sexual
assualt.
Sites for Sample Retrieval
Lateral vaginal wall (just above the level
of the cervical os, or from the posterior
fornix)
Ectocervix
Endocervix
Upper 3rd of Lateral Vaginal Wall
Assessment of the hormonal status
Evaluation of possible inflammatory
condition of the vagina
Classify the microbiologic flora
Detection of malignant lesions of the
vagina
Ectocervix
Detection of ectocervical tumors
Inflammatory conditions
Endocervix
Detection of endocervical tumors
May detect some intrauterine lesions
Inflammatory conditions
Ectocervix-Endocervix Junction
Most commonly sampled for cervical
cancer screening.
transformation (T or junctional) zone.
Transformation (T or junctional) Zone
Represents an area where an abrupt
transition from the ectocervical
epithelium abruptly changes to
endocervical type of epithelium.
It is a dynamic, ever changing
environment adapting to chronic
inflammation and infection.
Metaplasia, dysplasia and carcinoma-in-
situ occurs
Eventually progresses to invasive
carcinoma.
This is the reason why most malignancy
arises from this particular anatomic site.
Instruments to Obtain Samples
Speculum
Wooden spatula
Modified spatula (Ayers spatula)
Cervical brush
Cervical broom (Cervex-brush or
Papette)
Cotton swab
Precautions Observed During Vaginal Smear
Preparation
No manipulation for at least 24-48
hours.
Instruments should be perfectly dry.
No lubricant or powder should be used
on the examiners glove.
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
for 20 minutes, rinsed with KOH and
dried.
Epithelial Cells Evaluated in Vaginal Smears
Lateral Vaginal Wall (Squamous cells)
o Mature superficial cells
o Intermediate (Navicular, Boat)
cells
o Parabasal cells.
o Basal cells
Endometrial cells (Columnar cells)
Endocervical cells (Columnar cells)
Ectocervical cells (Squamous cells)
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
(cyanophilic)
Cellular Arrangement
o Usually single cells
Intermediate cells
Nucleus
o 10-18 u, round to oval
o Finely granular, evenly
distributed chromatin
o Centrally placed
Cytoplasm
o 20 50 u, polygonal, well
defined
o Blue (cyanophilic) or pink
(eosinophilic)
o May contain glycogen
Cellular Arrangement
o Usually single cells
Parabasal Cells
Nucleus
o Variable, round to oval, central
o Finely granular, evenly
distributed chromatin
Cytoplasm
o 12-30 u, round, dense, well
defined
o Usually cyanophilic
o May contain glycogen
Cellular arrangement
o Single cells or sheets
FRIED EGG APPEARANCE
Endometrial Cells
Nucleus
o 8-10 u, round, often wrinkly
o Chromatin is coarser than
endocervical cells
o Eccentrically placed
Cytoplasm
o Scanty, low columnar
o Delicate, cyanophilic
Cellular arrangement
o Often tight, 3D clusters (from
small clusters to large
fragments
o Single cells are very difficult to
identify
Endocervical Cells
Nucleus
o 9-20 u, round to oval
o Finely granular, evenly
distributed chromatin
o Eccentrically placed
Cytoplasm
o Columnar, delicate, fragile, may
have cilia
o Often contain single, large
secretory vacuole (mucus)
Cellular arrangements
o Sheets (honeycomb) or in
palisading strips (picket fence)
Papanicolaou Staining Technique
Provides an excellent demonstration of
the nucleus.
The cytoplasm is rendered translucent.
The staining of squamous cell cytoplasm
range from:
o basophilic (green or brown)
staining of the least mature and
deepest layer.
o Eosinophilic or acidophilic (pink
or orange) staining of the most
mature and superficial.
 It is a regressive, indirect and counter Ferning
type of staining. Cervical mucus shows a palm leaf
Stains used: pattern
o EA 36 (EA 50) EA 65 is for non- High & persistent estrogen effect
gynecologic Formation of salt crystals
Eosin Y One basis of early pregnancy
Bismark Brown
Light-Green SF Leptothrix spp
o OG 6 long, slender, gram negative, non spore
Orange G crystals forming anaerobic organisms
o Harris Hematoxylin Non-pathogenic by themselves, but
75%-80% of cases have associated
Factors that Diminish the Accuracy of a Paps Trichomonas vaginalis
Smear 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 Hormonal Evaluation
transformation zone. Hormonal Cytology
Slide material too thick or inadequate. Principle: vaginal epithelium responds
Performing paps in spite of obvious to stimulation by steroid hormones,
infection. mainly estrogens and progesterone and
to a lesser degree, to androgens and
Artifacts Seen in Poorly Prepared Smears adrenal steroids.
1. Enlarged nuclei, ill defined weakly 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 Accompanying data:
crystals) Patient name
Patient age
Other Cells and Structures Seen (Normal and LMP
Pathologic)
History of hormone or IUD use
Bacteria
History of medicine intake, operations,
Virus radiation, infection or disease.
Parasites Evaluation
Spermatozoa Count 100 cells and classify the types of
Fungi squamous cells seen.
Blood Reporting:
WBC o Hormonal
Cancer cells Evaluation:____/_____/____
First blank = parabasal cells
 Second blank = intermediate cells
Third blank = superficial cells
Summary of Maturation Index
Birth ------------------ 0/95/5
Childhood ------------ 90/10/0
Pre-ovulatory -------- 0/30/70
Pre-menstrual -------- 0/70/30
Pregnancy ----------- 0/95/5 (navicular
cells)
Lactation ------------- 90/10/0
Pre-menopausal & menopausal ---
0/100/0
Post-menopausal ------ 100/0/0
Cancer Diagnosis
It is a matter of finding the physical
presence of malignant (cancer) cells.
Reporting Systems for Vaginal Cytology
(Paps Smear)
1. Papanicolaou Numerical Classification
System (1928)
2. Bethesda System (1988)
3. Revised Bethesda System 2001
Classes not transferrable to histology
terms
No classes for non-cancerous entities
Basic Features of the Bethesda System
Includes specific statements on
adequacy of specimen.
General categorization of abnormalities
Interpretation
Result
Abnormal Cellular Findings (Bethesda 2001)
Atypical glandular cells (AGC)
Atypical squamous cells of
undetermined significance (ASCUS)
o Atypical squamous cells of
undetermined significance
(ASC-US)
o Atypical squamous cells -
cannot exclude HSIL (ASC-H)
Low grade squamous intraepithelial
lesion (LGSIL or LSIL)
High grade squamous intraepithelial
lesion (HGSIL or HSIL)
Squamous cell carcinoma

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
 Basic histopathologic techniques
Histopathology
It is a basic component of a tertiary
hospital laboratory
Human tissue & body fluids are
processed
Tissue slides are produced for
microscopic examination
Read and assigned a diagnosis by an
anatomical pathologist
Proper specimen handling does not
start only upon reaching the lab
Pre-analytical Factors
Pre-analytical Factors
Warm Ischemia
o initial anoxic insult a tissue
undergoes during surgery
o depends on the particular
circumstances of the
procedure, skill & speed of the
surgeon and other factors
surrounding the procedure
o beyond the control of the lab
Cold Ischemia
o lack of oxygen once the sample
is removed from the body
o before the stoppage of
continuous metabolic processes
by fixation
o both the operating room and
laboratory can be responsible
Fixation
o The earlier a tissue is fixed,
better preservation
o Properly preserved tissues are
more resistant to processing
artifacts
Fixative to Tissue Ratio
o 10:1
Inadequately fixed tissues will not
process, cut and stain well
No amount of troubleshooting will solve
poor fixation!!!!
Tissue Exposure to Fixative
Duration of Fixation
o Optimal thickness of 3-5 mm:
o Minimum 6 hrs
o Maximum 48 hrs
o To allow immunohistochemistry
and/or in situ hybridization
o Large organs are sectioned at
5mm intervals
o Hollow organs are opened to
allow full fixative penetration
Properly Filled-up Surgical Pathology
Request
Accessioning Procedure
o Unlabeled specimens
o Incomplete specimens
o Numbering assigning of an
accession number for proper
and convenient identification
o Questions of identification
between 2 samples may require
expensive DNA identification
FIXATION
Killing, penetration and hardening of
tissues
Alteration of tissues by stabilizing
protein to become more resistant to
further changes.
Important preservation of the tissue
at the exact moment when it is
removed from the body
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. According to COMPOSITION
To set intracellular inclusion bodies so A. Simple fixatives
that the histologic and cytologic 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. Mercuric chloride
To enhance staining of tissues. b. Chromate fixatives
To harden tissues for cutting very thin c. Lead fixatives
sections. 3. Heat
B. Compound fixatives
Fixation consideration
Masking of antigen by fixation leads to According to ACTION
the failure of antibodies used in A. Microanatomical Fixatives
immunohistochemistry to determine B. Cytological Fixatives
the antigenic site. 1. Nuclear Fixative
Result- false negative - Contains glacial acetic
Solution reversed by using antigen acid, 4.6 pH or less
retrieval techniques - Flemmings fluid,
Lipids and carbohydrates are very prone Carnoys fluid, Bouins
to be lost during processing fluid, Newcomers fluid
Solution use alternative fixation & Heidenhains Susa
methods 2. Cytoplasmic fixative
- Never contains glacial
Desirable Characteristics of Fixative: acetic acid, > 4.6 pH
1. Cheap 3. Histochemical Fixatives
2. Stable & safe to handle - Chemical components
3. Kills the cell instantly (protein,
4. Inhibit bacterial growth and damage carbohydrates,
5. Minimum tissue shrinkage enzymes, hormones,
6. Rapid and even tissue penetration etc.)
7. Hard enough to allow cutting of thin - Lipid cryostat or
sections frozen section,
8. Does not prevent staining reactions chemical
- Carbohydrates
(glycogen) alcoholic
fixatives
 - Protein neutral Factors affecting fixation
buffered formol saline Temperature
or formaldehyde vapor o Increases fixation but increases
rate of autolysis
Other classifications o Temperatures up to 45 C has
Additive or non-additive little effect on tissue
Coagulant or Non-coagulant morphology
o Room temperature is
Additive fixatives satisfactory to most
Bonds and adds itself to the tissues Specimen Size
altering protein structures o Formalin penetrates to a rate of
Implicated in the failure of 3-4 mm/hr
immunohistochemical staining o 3 mm sections are required for
Formaldehyde, mercuric chloride, formalin fixed tissue processing
chromium trioxide, picric acid, Volume Ratio
glutaraldehyde, osmium tetroxide and o Critical factor involvement of
zinc sulfide or chloride operating room staff
Non-additive fixatives o Ideal ratio of fixative volume:
Do not chemically bind with tissues 15-20 is to 1
Removes water from the tissue protein o Current recommendations: 10:1
groups Time
Prone to excessive shrinking and o Cold ischemia time
hardening of tissues 20 30 minutes ideal to
Acetones and alcohols perform fixation
60 min is the maximum
Coagulant fixatives time before fixation
time
Creates a network out of the protein
o Fixation time
structures that increases solution
Minimum of 6 hrs and a
penetration to the interior of the tissue
maximum of 48 hrs in
Zinc salts, mercuric chloride, picric acid,
10% neutral buffered
ethyl alcohol, methyl alcohol and
formalin
acetone
Overfixation:
Non-coagulant fixatives
o HER-2 false negative
Creates a gel making penetration of
o Adversely affect staining
tissues difficult
qualities
Tissues are cut thinly for better
o Over hardening of tissues
penetration
Properly fixed (ideal time)
Formaldehyde, glutaradehyde, acrolein,
o almost immune to processing
potasium dichromate, acetic acid,
artifacts
osmium tetroxide
Underfixation
o Cannot be processed well
 o Subject to harmful effects of
solutions
o Irreversible artifacts and
distortions
Aldehyde Fixatives
Formaldehyde (Formalin)
o routine fixative for paraffin
embedded sections
o usually buffered to pH 7 with a
phosphate buffer
o Advantages:
Cheap, available, easy
to prepare, stable
Compatible with most
stains
Do not over harden
tissues
Penetrate tissues well
o Disadvantages:
Irritating fumes
Causes dermatitis
Shrinkage of tissues
Produces black
precipitates on staining
10% ideal, 15% for
brain tissue
10% Formol-Saline
o Microanatomical fixative
o Saturated formaldehyde +
sodium chloride
o CNS and histochemical
examination
o Slow fixative
10% Neutral Buffered Formalin
o ideal fixative of choice
o prevents precipitation of acid
formalin pigments
o 10% ideal most surgical path
specimen, 15% for brain tissue
Gendres Fixative
o Formalin + ethyl alcohol +
glacial acetic acid
o post fixation for immune
staining
o also for electron microscopy
Glutaraldehyde (2.5% & 4%)
o electron microscopy
o Slower penetration, less tissue
shrinkage
o fixation is limited best at 2 hrs
GLYOXAL
o Smallest aldehyde, 40%
aqueous solution
o 4-6 hrs fixation
o 45 mins fixation for biopsy
specimen
o Better performance than
formalin
o Long term tissue storage with
slight reduction in staining
ability of tissues
Metallic Fixatives
Mercuric Chloride
o Most commonly used metallic
fixative
o Must always be freshly
prepared
o Extremely toxic, expected not
to be available commercially
o De- zenkerizationis needed
(alcoholic iodine solution) prior
to staining
o Routine fixative of choice for
preservation of cell detail in
tissue photography
o Recommended for renal tissue,
fibrin, connective tissue and
muscle
o Brown pigment formation
beyond 24 hrs, treated with
 saturated picric acid or sodium
hydroxide
Types
Zenkers fluid (mercuric chloride +
glacial acetic acid)
o recommended for fixing small
pieces of liver, spleen,
connective tissue fibers &
nuclei
Zenker-formol (Hellys solution)
o microanatomical fixative for
pituitary gland, bone marrow,
spleen & liver
o recommended for lymph node
biopsies
Heidenhains Susa Solution
o recommended mainly for skin
tumor biopsies]
B-5 Fixative
o recommended for bone
marrow biopsies
Chromate Fixative
Highly toxic and carcinogenic
Chromic acid
o Used as 1-2% aqueous solution
o Usually a component of a
compound fixative
Potassium Dichromate
o Used as a 3% aqueous solution
o Cytologic fixative for
mitochondria
o Lipid preservation
Regauds (Mollers) Fluid
o Recommended for
demonstrating chromatin,
mitochondria, mitotic figures,
golgi bodies, colloid
Orths fluid
o Study of early degenerative
processes and necrosis
o Preservation of myelin sheath
Lead Fixatives
Used as 4% aqueous solution lead
acetate
Mucin & mucopolysaccharide fixation
Forms insoluble lead carbonate with
CO2, dissolved by acetic acid
Picric Acid Fixatives
Excellent for glycogen demonstration
Imparts a yellow color (stain)
Avoided in DNA or DNA staining
Highly explosive when dry
Types:
Bouins Solution
o recommended for fixing
embryos
Brasils Alcoholic Picroformol Fixatives
o better than Bouins fixative
o excellent fixative for glycogen
Glacial Acetic Acid
o Penetrates very rapidly soft
tissues
o Used as component/additive of
compound fixatives
o Useful in the study of nucleic
acids
o Destroys mitochondria & golgi
bodies
Alcohol Fixatives
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
Types
Methyl Alcohol 100%
o Excellent for fixing dry & wet
smears, blood smears & bone
marrow smears
o Highly toxic
Ethyl alcohol
o Routine fixative for cytologic
smears (Paps smear)
o Used for preserving tissues for
enzyme studies
Carnoys fluid
o Considered to be the most
rapid fixative
o Preserves Nissl granules &
cytoplasmic granules
Alcoholic Formalin (Gendres) fixative
Newcomers fluid
o Recommended for fixing
mucopolysaccharides & nuclear
proteins
Osmium Tetroxide (OSMIC ACID)
pale yellow powder
Fixes myelin & peripheral nerves well
Fixative for ultrathin microtomy
Kept in a dark-colored, chemically clean
bottle to prevent evaporation &
reduction by sunlight
Choice for fixing lipids/fat tissue
Good fixative for nasal mucosa and
conjunctiva
Extremely volatile, hazardous and
expensive
Types:
Flemmings Solution
Flemmings Solution w/o Acetic Acid
o Cytoplasmic stain
Trichloroacetic acid
Incorporated with other fixatives to
counter shrinkage
Weak decalcifying agent
Acetone
Used in histochemical studies
Rabies studies
Solvent for metallic salts in Freeze
Substitution techniques
Considerable tissue shrinkage and
distortion.
HEAT FIXATION
1. Direct heat
2. Microwave fixation/stabilization
Zinc sulfate
Proposed as a replacement for mercuric
chloride
o Less toxic moderate health risks
o Preserves tissue antigenicity
Cons
o Precipitation of zinc
Microwave fixation/stabilization
controlled heat fixation, protein
denaturation
optimum temperature is 45-55 C
allows routine light microscopic
techniques, special stains,
histochemistry and
immunocytochemical techniques
good secondary fixative post osmium
tetroxide fixed
accelerates staining
(immunocytochemistry)
only penetrates tissue to a thickness of
10-15 mm
Chief values:
 - fixed right through the block in TISSUE PROCESSING
a very short time
AIM of Tissue processing
- non-chemical technique
To embed the tissue in a solid medium
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 damage the knife or tissue.
To make special staining techniques
possible.
Post-fixation treatment (Washing Out)
To ensure further and complete
remove excess fixative to improve
hardening and preservation of tissues.
staining,
removal of fixation artifacts
Secondary Fixation Technique
10% formalin
Tap water
o mercuric chloride
excess chromates from tissues fixed in
o easier tissue cutting and
Hellys, Zenkers and Flemming
flattens better
solutions.
o subsequent staining is more
o excess formalin
brilliant and intense
o excess osmic acid
Post-chromatization (formalin 2.5
50-70% alcohol
3% potassium dichromate)
excess picric acid from Bouins solution
o acts as a mordant
Iodine
o good for the demonstration of
removal of mercuric deposits
the mitochondria.

General steps of tissue processing

FACTORS AFFECTING FIXATION OF TISSUES
1. Dehydration
RETARDED BY:
2. Clearing
Size & thickness of the tissue specimen
3. Infiltration
Mucus
4. Embedding
Fat
Blood
Cold temperature DECALCIFICATION
ENHANCED BY: - Should be done after fixation
Size & thickness of the tissue - A good decalcifying agent should:
Agitation 1. Remove calcium salts
Warm temperature completely
2. Minimal destruction of cells &
tissues
3. Minimal adverse effect to
staining

Types of Decalcifying Agent
1. Acids
2. Chelating agents
3. Ion exchange resins
4. Electrical ionization (electrophoresis)
. damage tissues).
1. ACID DECALCIFYING AGENTS
1. Nitric acid
A. Formol-nitric acid
B. Perenyis Fluid
C. Phloroglucin-Nitric Acid
2. Hydrochloric Acid slower
A. Von Ebners Fluid
3. Formic Acid moderate acting
4. Trichloroacetic Acid weak
5. Sulfurous Acid weak
6. Chromic Acid (Flemmings Fluid)
2. CHELATING AGENTS
- EDTA (Versene)
- Combines with calcium forming an
insoluble complex
- Very slow, 1-3 weeks
3. ION EXCHANGE RESIN
4. ELECTROPHORESIS (ELECTRICAL
IONIZATION)
MEASURING EXTENT OF DECALCIFICATION
1. Physical or Mechanical Test
2. X-ray or Radiological Method
3. Chemical Method (Calcium Oxalate
Test)
DECALCIFICATION
A procedure whereby calcium or lime
salts are removed from tissue
FOLLOWING FIXATION
Should be done after fixation and
before impregnation
More concentrated acid solutions
decalcify bone more rapidly (but more
harmful to the tissue).
Recommended ratio of decal. fluid to
tissue volume 20 to 1.
Heat hastens decalcification (but may
At 37 C = impaired nuclear staining of
Van Giesons stain for collagen fibers.
At 55 C = tissue will undergo complete
digestion within 24-48 hours.
Optimum temperature
o = RM TEMP (18-30 C)
The ideal time required: 24-48 hours.
Dense bone tissues require up to 14
days or longer
Decalcifying agents
Nitric acid
o MOST COMMON
o examples: Perenyis fluid acts
as BOTH tissue softener and
decalcifying agent.
Phloroglucin-Nitric Acid
o MOST RAPID DECALCIFYING
AGENT!
Formic acid BOTH fixative and
decalcifying agent
5% formic acid is considered to be the
BEST GENERAL DECALCIFYING AGENT
Formic acid is recommended for small
pieces of bones and teeth.
Hydrochloric acid
o Von Ebners fluid
recommended for teeth and
small pieces of bones.
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 saturated lithium carbonate Do not act as secondary fixative
soln. Ethoxyethanol
o 5-10% aqueous sodium Cellosolve - rapid dehydrant
bicarbonate Polyethlene glycols dehydrating,
embedding agent
Dioxane being phased out
DEHYDRATION
Ideal Dehydrating Solution Other dehydrating agents
1. Rapid but minimal tissue shrinkage. Acetone
2. Slow evaporation o Rapid dehydrant
3. Dehydrates fatty tissue o Coagulant secondary fixative
4. Minimal tissue hardening o Best dehydrant for fatty tissues
5. Stain friendly o Controlled substance
6. Non-toxic Tetrahydrofuran
7. Not a fire hazard o Less toxic than dioxane
o Rapid acting
Dehydrating agents o Clears as well as it dehydrates

ALCOHOLS Excessive vs incomplete dehydration

Clear, colorless, flammable and hydrophilic Excessive
Acts as a secondary coagulant fixatives o hard, brittle or shrunken
Ethanol
Incomplete
o Most commonly used o Most common processing
o 99.85% problem
o Inexpensive, readily available o Prevents entry of clearing
and low toxicity agents
Methanol o Soft and non-receptive to wax
o Good substitute but rarely used infiltration
Isopropanol or Isopropyl alcohol (IPA)
o versatile ethanol substitute
o efficient lipid solvent CLEARING
o fully miscible with water, most Ideal Clearing Agent
organic solvents and parrafin Miscible with alcohol, paraffin,
o causes less tissue shrinkage mounting media.
and hardening Reduced tissue shrinkage
o presently used as a xylene Makes tissue transparent (optically
substitute in rapid automated clear)
processing
CLEARING (De-alcoholization) Types:
Xylene Paraffin Wax Impregnation
o few hours, 5 mm tissue size Celloidin (Collodion) Impregnation
o most rapid Gelatin impregnation
o makes tissue transparent 1. Paraffin wax Impregnation
o highly inflammable Cheap
o not suitable for lymph node and easily handled
nervous tissue section production provides few
Toluene difficulties
Benzene wide range of melting points
Chloroform mixture of hydrocarbons in the cracking
o not inflammable of mineral oil
Cedarwood oil melting point range is between 40 70
Aniline oil C
Clove oil 54 58 C
Carbon tetrachloride 2 5 C above melting point maintained
Dioxane Additives:
Ethylene glycol monoethyl ether beeswax
(Cellosolve) ceresin
o under investigation rubber
Terpenes, Limonene & Terpineol diethylene glycol distearate
resin
Prolonged vs incomplete clearing IMPREGNATION
Prolonged clearing time Paraffin
o Brittle o the man who introduced
Incomplete clearing paraffin wax embedding:
o uneven H&E staining Butschlii
o Poor nuclear chromatin o simplest, most common and
patterns the BEST infiltrating/embedding
medium.
Impregnation(Infiltration) o is NOT recommended for fatty
Also known as INFILTRATION tissues ( the dehydrants and
clearing agents used in the
Process of replacing the clearing agent
process dissolve and remove fat
with the infiltrating medium.
from the tissues).
The medium used to infiltrate the tissue
2. Celloidin (Collodion) Embedding
is usually the same medium used for
thin (2%), medium (4%), thick (8%)
embedding.
solutions
Volume of the impregnating medium
large tissues cut with base sledge or
should be at least 25 times the volume
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 skin, CNS tissue
o Wet Celloidin Method (bones,
teeth, large brain sections, large
section) EMBEDDING/CASTING/
o Dry Celloidin Method ( whole BLOCKING
eyeball)
chloroform + Embedding Tissue in Paraffin Wax
cedarwood oil
wax dispenser
LVN (low viscosity nitrocellulose)
cold plate
can be used in higher
heated storage area for molds
concentrations
penetrates well
Embedding molds
explosive when dry 1. Leukharts 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 Orientation of Tissues
- Bioloid (embedding eyes) starts during gross time
- Tissue mat ( paraffin + rubber Most tissues are cut from the largest
2. Ester wax (46 48 C) area.
- harder than paraffin
Tubular structures are cut in cross
- soluble in 95% alcohol
section
- sliding or sledge microtome
Skin and other epithelial biopsies are
3. Water soluble waxes (polyethylene
cut in a plane at right angles to the
glycols)
surface and oriented so the surface is
- 38-42 C to 45 56 C
cut first.
- Carbowax
Muscle biopsies are sectioned in both
- Does not require dehydration
transverse and longitudinal planes.
and clearing
When a particular tissue feature is
present on one aspect only.
Vacuum impregnation
*melt wax to temperature 5 10 C above the
speed up impregnation and remove any
melting point of wax
residual air bubbles
500 mmHg max
Other embedding methods Resin Embedding Media
Celloidin or nitrocellulose method Areas Where Paraffin is Unsuitable
Double embedding method (Peterfis Unsuitable for ultrastructural studies.
technique) o does not offer support for ultra
Resin embedding thin sections (30-80 nm)
o paraffin wax is unable to
Tissue Softeners withstand the high energy
For unduly hard tissues that may electron beam
damage the microtome knives. It doesnt 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
2% HCl and lymph node biopsies.
1% HCl in 70% alcohol It doesnt provide support for extremely
hard tissue for cutting.
Tissue Processing Methods
Manual Resin Media
o Can be accelerated by: Epoxy resins
- Microwaves o widely used for electron
- Ultrasonics microscopy
Automated Processing o allows sections as thin as 30
o fixes, dehydrates, clears and 40 nm
infiltrates o 3 Types
Two Main Types Bisphenol A (Araldites)
Tissue Transfer (Dip & Dunk) Type Hlycerol (Epons)
o Carousel type (Elliott Bench- Cyclohexene dioxide
Type) (Spurrs)
Fluid-transfer (enclosed pump fluid) Acrylic resins
Type o earliest resin used
o Autotechnicon, Hypercenter 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 can support 1-2 um
4. Less toxic fumes emitted sections
Factors Influencing the Rate of Processing o provides support for cutting
1. Agitation - 30% reduction undecalcified bone
2. Heat 45 C ideal o Types
3. Viscosity Butyl methacrylates
4. Vacuum Methyl methacrylates
 Glycol methacrylates 5. Ultrathin microtome
6. Vibrotome
for unfixed, unfrozen sections
Microtomy enzyme studies

Microtome Cutting
tissue is advanced for a predetermined Microtome Knives
distance then slide the object to the Knife materials
cutting tool, usually a steel knife. 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 o (alloy of cobalt, chromium and
Freezing microtome tungsten)
Ultrathin microtom o Non-rusting
Vibrotome o For cryostat
o Not recommended for paraffin
1. Rocking microtome (Cambridge) embedded tissues
most popular from 19th century to 6. Disposable blades
1950s 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 o replaced the conventional steel
also used in cryostats knives
o Classification:
2. Rotary microtome (Minot) 1. A. Low-profile blades
commonly used today, even in cryostats 2. B. High-profile blades
heavier and stable
Profiles of Steel Knives
provides a flat face to the tissue block
1. Plane wedge
retracting mechanism
2. Planoconcave
- sliding microtome
3. Sliding microtome
- celloidin embedded
Adams (1789)
3. Biconcave
Base sledge/Standard sliding
- very keen edge
for celloidin embedded tissues
- rigidity is sacrificed
4. Tool edge
4. Freezing microtome
- used with the hardest tissue
Queckett (1848)
and embedding media
Cryostat (cold microtome)
Cutting edge profiles Floating Out & Fishing Out
1. Bevel angle 27 to 32 Sections
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 for embedding the tissue.
2. Stropping toe to heel 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
MOUNTING
Types of Tissue Sections & Thickness Desirable Characteristics
1. Paraffin Sections 4 to 6 u, 3 to 4 u Refractive index approaching
2. Celloidin sections 10 to 15 u glass(1.518)
3. Frozen sections It does not interfere with the stain and
Trimming: distort tissues.
o to expose the tissue It should set hard
o apply ice over the surface after Types:
trimming o Aqueous Media
Cutting Sections: o Resinous Media
o Softer tissue, slower stroke.
o Gentle exhaling breath on the Aqueous Media
block surface. Generally 1.40 to 1.45
o Sections should stick to stick For water miscible prep if xylene or
(ribboning) alcohol dissolves the stain.
o Ribbons are separated for every Usually for enzyme histochemistry
6 to 8 sections Most use:
Floating out sections o gum arabic & gelatin as
o Water temp 6 to 10 C lower solidifiers
than the melting point of the o glycerol to prevent drying,
wax increase refractive index,
preservative
Adhesives
1. Mayers Egg Albumin Aqueous Mounting Media
2. Dried Albumin Water
3. Gelatin
Farrants medium (gum arabic)
4. Starch paste
Apathys mountant (gum arabic)
5. Plasma
Kaisers glycerol jelly (gelatin)
Polyvinyl pyrrolidone
 Resinous Mounting Media
Canada balsam (1.54)
natural, Abus Balsamea
stains fade after sometime
turns dark yellow after sometime
DPX (1.52)
neutral colorless solution
good for small tissue mounts
XAM (1.52)
pale yellow or colorless solution
preserves stain
dries w/o tissue retraction
Clarite (1.54)
Ringing
Sealing the margins of the coverslip :
prevent escape of mountant
usually for aqueous mountants
prevent evaporation
immobilize the coverslip
prevent sticking of slides during
storage.
Kronig cement (Paraffin + resin)
Durofix (cellulose)
STAINING
4 Major Applications of Stains
Routine staining procedures
Special stains
Histochemical stains
Immunohistochemical stains
Mechanisms of tissue STAINING
Chemical Staining Dye-to-Tissue
Mechanisms
A. Chemical stain
1. Ionic or Electrostatic bonding
2. Hydrogen bonding
3. Van der Waals Forces
4. Covalent bonding
B. Pure Physical stain
Dyes used in histopathology
1. Natural Dyes
2. Synthetic (Artificial) Dyes
Natural Dyes
Hematoxylin
Cochineal dyes
o Cochineal bug (Coccus cacti)
o Carmine dyes
o Powerful chromatin and
nuclear stain
Orcein dyes
o vegetable dye (lichens)
o litmus
Saffron
o Crocus
Synthetic (Artificial) Dyes
Coal Tar Dyes (Hydrocarbon benzene)
Aniline dyes
Basic Principles (Synthetic Dyes)
Chromophores are substances capable
of producing colors.
Chromogens are substances containing
chromophores.
Auxochrome alters the chromogens to
form salts to allow retention of the dye
to the tissue.
o (-NH2, -COOH, -S03H, -OH)
Classification of Dyes
Acid dyes (Anionic dyes)
o react with cationic or basic
components in cells
Basic dyes (Cationic dyes)
o react with anionic or acidic
components in cells
General Methods of Staining Staining
Direct Staining
Indirect staining Haematoxylin & Eosin
o mordant
Progressive staining Haematoxylin
Regressive staining most widely used natural dye in
o differentiation (decolorization) histotechnology
Metachromatic staining myelin, elastic, collagen, muscle
o staining of cartilage, connective striations, mitochondria, etc.
tissue, mucin, mast cell nuclear dye in the standard H & E,
granules and amyloid primary stain
o alcohol solvent usually takes Source
away the stain logwood, Haematoxylon campechianum
(order Leguminosae, genus
Metachromatic Dyes Eucaesalpinieae)
Methyl violet or Crystal violet reddish color of its heartwood and
Cresyl blue young leaves
Safranin indigenous to Central America
Bismarck brown (Campeche and Yucatan regions of
Basic fuchsin Mexico)
Methylene blue grown commercially in the West Indies
Thionine Extraction
Toluidine blue Crude product is obtained from chipped
Azure A, B, C heartwood by hot water or steam.
Purified by ether extraction.
Counterstaining Dried
Recrystallized from water or
Microanatomical staining precipitated with urea.
Cytoplasmic staining Alterations
Negative staining The Spanish recognized that the color of the
dye could be altered.
Metallic impregnation 1. iron alum black
Gold chloride & Silver nitrate 2. potash alum blue
3. salts of tin red
Vital staining
Intravital staining (lithium, carmine, Properties of Haematoxylin
India ink) It is important to realize that
Supravital staining (Neutral red, Janus haematoxylin itself is not a stain.
green, Trypan blue, Nile blue, thionine, It is its oxidation product, HAEMATIN,
toluidine blue) which is the natural dye.
 Haematin is anionic, having a poor similar features with Ehrlichs
affinity for tissue. 15 20 mins
Mordants are required to enable a 3. Mayers haematoxylin
binding to the tissue site, nucleus. chemically ripened with sodium
iodate
Haematoxylin used as regressive and
progressive stain
Oxidation methods: used as a nuclear counterstain
1. Natural oxidation (ripening) for glycogen demonstration
slow process, 3-4 mos 5 -10 mins
longer shelf life 4. Harriss haematoxylin
Ehrlichs and Delafields chemically ripened with
2. Chemical oxidation mercuric oxide, sodium or
conversion is almost instantaneously potassium iodate
shorter shelf life provides a clear nuclear staining
routinely used in exfoliative
Classification of Haematoxylin cytology staining
Alum haematoxylin (Papanicolaou)
Iron haematoxylin good for the staining of sex
Tungsten haematoxylin chromosomes
Molybdenum haematoxylin 5 20 mins
Lead haematoxylin 5. Coles haematoxylin
Haematoxylin w/o mordants chemically ripened with
alcoholic iodine solution
A. Alum Haematoxylins 5 mins
most are used for routine staining 6. Carazzis haematoxylin
aluminum potassium sulphate, chemically ripened using
aluminum ammonium sulphate potassium iodate
good for frozen section
stains the nucleus- red
1 min
addition of glycerin stabilizes and
7. Gills haematoxylin
prevents evaporation
chemically ripened with sodium
1. Ehrlichs haematoxylin
iodate
naturally ripened, 2 mos
good for regressive staining
generally used for regressive
5 15 mins
staining (routine H & E staining)
good for mucins of cartilage,
B. Iron Haematoxylins
bone
iron salts are both used as a mordant
NOT ideal for frozen sections
and oxidizing agent
15 40 mins
2. Delafields haematoxylin ferric chloride, ferric ammonium
naturally ripened sulphate
 over oxidation is a problem The Eosin
good for photomicrography most suitable stain to combine with an
alum haematoxylin as a counterstain.
1. Weigerts haematoxylin differing shades of red and pink
standard iron haematoxylin used xanthene dyes
routinely Classification
demonstration of muscle fibers and 1. Eosin Y (eosin yellowish, eosin
connective tissue water soluble)
2. Heidenhains Haematoxylin 2. Ethyl eosin ( eosin S, eosin
regressive staining alcohol-soluble)
demonstration of chromatin, 3. Eosin B (eosin bluish,
chromosomes, nucleoli, and erythrosine B)
mitochondria
muscle striations and myelin, TUMOR
BIOPSIES OF THE SKIN TRANS BY : PAPADARN
3. Loyez haematoxylin ( C ) : DOC BALDO
demonstration of myelin
can be used in paraffin, frozen and
celoidin embedded sections
4. Verhoeffs haematoxylin
elastic fibers

C. Tungsten Haematoxylins
Mallory PTAH
can be used unoxidized or oxidized
fibrin, muscle striations, cilia and glial
fibers

D. Molybdenum Haematoxylins
rarely used
Thomas technique
collagen and reticulin demonstration

E. Lead Haematoxylins
new addition
identification of endocrine cells in
malignant tumours

F. Haematoxylin w/o a mordant

demonstration of various minerals in
tissues ( lead, iron and copper)