HISTOPATHOLOGIC TECHNIQUES

Cornelius Celsus: First to recognize 4 cardinal

signs of inflammation.
Pathology Basics 1. Rubor – redness
2. Tumor – swelling
3. Calor – heat
4. Dolor – pain
Review of Histology 5. Function Laesa – loss of function
Four main tissue types (Rudolf Virchow)
A. Epithelial
B. Connective Inflammation according to duration:
C. Muscular • Acute – Vascular, exudative
D. Nervous Hallmark: increased PMNs and vascular
permeability
Epithelial Tissues • Subchronic – intermediate between
• Covers and lines surfaces acute and chronic
• Cells are close to each other • Chronic – Vascular, fibroblastic
• Avascular Hallmark: increased monocytes and
Classification based on: macrophages
-Cell shape (columnar, cuboidal, squamous)
- Arrangement (simple, stratified, Inflammation according to exudates:
pseudostratified) • Serous – Secretions of mesothelial cells
example:
Connective tissues • Fibrinous – increased fibrinogen
• Most abundant Example:
• Main functions: bind, protect and • Catarrhal – hypersecretion of mucosa
support • Hemorrhagic – mixture of blood and
exudate
Muscular Tissues • Purulent – increased pus (creamy,
• Highly specialized debris of tissue necrosis and increased
• Function: Movement polymorphonuclear cells)
• Types: Skeletal, Smooth, Cardiac
CHANGES IN CELLULAR GROWTH PATTERNS
Nervous Tissue
• Function: Rapid Communication RETROGRESSIVE CHANGES – smaller than
• Neuron – receives stimuli; conducts normal
waves of excitability
• Neuroglia; nerve glue – support, A. Developmental Defects
protection and insulation • Aplasia – Incomplete or
defective organ/tissue damage.
Pathology – Latin “pathos” which means There is no resemblance to
suffering adult structure
• Agenesia – non-appearance of
Inflammation- Latin “inflammare”- to set a fire an organ
It is a protective response to irritation or injury. • Hypoplasia – an organ or part
remains the same as of the
normal size or in an immature
state.
HISTOPATHOLOGIC TECHNIQUES
• Atresia – absence of closure of
an opening of the body (failure
to form an opening)
B. Atrophy
• Reduced size of an organ or tissue
resulting from a decrease in cell size
and number
Physiologic Atrophy: development of
notochord and thyroglossal ducts (fetal
development), and uterus (decrease in
size after parturition)
Pathologic Atrophy:
• Decrease Workload (Atrophy of
disuse)
• Loss of innervation (denervation
atrophy)
• Diminished blood supply
• Inadequate nutrition
• Loss of endocrine stimulation
• Pressure
PROGRESSIVE CHANGES
A. Hypertrophy – increase in the size of
cells, resulting in an increase in the size
of the organ.
B. Hyperplasia – increase in the number of
cells in an organ or tissue, usually
resulting in increases mass of the organ
or tissue.
Physiologic hyperplasia: hormonal
hyperplasia and compensatory
hyperplasia
Pathologic hyperplasia: endometrial
hyperplasia and benign prostatic
hyperplasia.
DEGENERATIVE CHANGES
A. Metaplasia- reversible change in which
one differentiated cell type (epithelial
or mesenchymal) is replaced by another
cell type.
Most Common: columnar to squamous
B. Dysplasia – “atypical hyperplasia”,
reversible and some changes in
structural components
C. Anaplasia – “dedifferentiation”, back to
primitive cell line, irreversible, and a
criterion towards malignancy
D. Neoplasia – pathologic overgrowth of
the tissue. Neoplasm- Greek – “
Differentiation – extent to which neoplastic cells
are comparable to normal cells, both
morphologically and functionally.
Metastasis
• Tumor implants discontinuous with the
primary tumor
• most realiable feature of malignancy
• All neoplasms metastasize except:
Glial cells and basal cell carcinoma
Manner of Dissemination of Malignant
Neoplasms:
• Seeding within body cavities:
neoplasms penetrates into a “natural
field”. Most often in the peritoneal
cavity
• Lymphatic Spread: Most Common
Pathway for carcinomas
• Hematogenous Spread: Most common
Pathway for sarcomas
• Liver and lungs are frequently
involved.
Types of Tumors:
A. According to capacity to produce death:
• Malignant – deadly, suffix (sarcoma,
carcinoma), leukemia/lymphoma
• Benign – not deadly, suffix (“oma”),
nevus, moles
B. According to histologic characteristics
• Medullary – cells > CT
• Serous – CT > cells
Grading of tumors – aggressiveness based on
cytologic difference of tumor cells and number
of mitoses within the tumor.
Broder’s Classification:
• Larger number of differentiated cells –
good prognosis
• Lower grades – amenable to surgery
• Higher grades – amenable to radiation
TNM Classification
A “T” score is based upon the size and extent of
invasion
HISTOPATHOLOGIC TECHNIQUES

The “N” score indicates the extent of lymph amorphous appearance in H&E stains
node involvement. called “fibrinoid” (fibrin-like)
The “M” score indicates whether distant
metastases are present SOMATIC DEATH
Secondary changes after death:
A – igor Mortis
TERATOMAS R – igor Mortis
“monstrous tumor” L – igor Mortis
• May contain hair, teeth, bones, P – ost Mortem Clot
eyeballs Dessication
Putrefaction
CELL DEATH Autolysis
Two Patterns of Cell Death:
1. Necrosis – (irreversible injury) LIVOR MORTIS ECCHYMOSIS
• Changes produced by enzymatic Disappearance of Opposite of livor
digestion of dead cellular elements pressure <12 hours mortis
• Protein denaturation Oozing of blood No oozing of blood
• Nuclear changes : Karyolysis
(fading), pyknosis (shrinkage), Algor Mortis – cooling 7°F/hr
karyorhexis ( fragmentation) Rigor Mortis – stiffening of skeletal muscles
2. Apoptosis: Programmed cell death 2-3 hours (Head and Neck)
• Vital process that helps eliminated Livor Mortis – post mortem lividity
unwanted cells Purplish discoloration
• An internally programmed series of After 10-12 hours; does not blanch on pressure
events effected by dedicated gene or shift when the body is moved.
products. Occurs in the dependent parts of the body.
• Unlike necrosis there’s no
inflammatory reaction POST MORTEM CLOT ANTEMORTEM CLOT
Patterns of Necrosis in Tissues or Organs Settling/ Separation Granular/friable
1. Coagulative – the architecture of dead of RBC
tissues is preserved for a span of at Chicken fat Not readily
least some days. The affected tissues appearance detachable from
exhibit a firm texture. -areas where RBCs vessels
are cleared
2. Liquefactive – digestion of the dead Currant Jelly- where
cells, resulting in transformation of the RBCs sediment
tissue into a liquid viscous mass.
3. Gangrenous – necrosis usually with Assumes vessel Seldom assumes the
superimposed infection shape vessel shape
4. Caseous – encountered in foci of rubbery
tuberculous infection “cheeselike
appearance” Dessication- drying and wrinkling of the cornea
5. Fat Necrosis – focal areas of fat and anterior chamber
destruction due to release of pancreatic Putrefaction – invasion of intestinal
lipases. “Chalky-white appearance” microorganisms
6. Fibrinoid – seen in immune reactions Autolysis – self-digestion of cells, Lysozyme
involving blood vessels. Bright pion and
 HISTOPATHOLOGIC TECHNIQUES

• Autopsy Report

INTRACELLULAR ACCUMULATIONS
• Manifestation of metabolic B. Signatories
derangement
• Request form: Physician
1. Lipids – includes all major classes of
lipids • Result form: Pathologist
Steatosis (Fatty change) – abnormal C. Specimen Handling
accumulations of triglycerides within -Fix first, then label
parenchymal cells D. Turn-over of results
Atherosclerosis – accumulation of • Surgical Patho & Cyto: 3-5 Days
cholesterol or cholesterol esters. • Frozen Section: 5-15 mins
Xanthomas – accumulation of • Autopsy: 1 week
cholesterol within macrophages E. Storage of Specimen, Tissue blocks,
2. Proteins- appear as rounded, slides
eosinophilic droplets, vacuoles or
-Specimen: 3-4 weeks
aggregates seen in the cytoplasm
-Tissue blocks:10 years
Causes: Renal Disease, Plasma Cells
-Slides: Indefinitely
actively secreting Ig (Russel Bodies)
3. Glycogen – seen in patients with Labelling
derangement in glucose or glycogen
metabolism. Appear as clear vacuoles S- Year- Specimen #
within the cytoplasm.
4. Pigments: colored substances C-Year-Specimen #
• Exogenous A-Year-Specimen #
-Carbon, coal dust, tattoo
- does not evoke an inflammatory
reaction
-Anthracosis (lungs) II. Fresh Tissue Examination
• Endogenous 1. Teasing/Dissociation- watch glass with
Lipofuscin – Brown, wear and tear isotonic solution Bright field or
pigment Phase contrast
Hemosiderin – golden yellow to 2. Crushing/Squash Preparation – slice
brown, iron excess tissue (<1mm) – between 2 slides- vital
Melanin – Brown black, stain
Alkaptonuria, Onchronosis 3. Smear Preparation
-Streaking
-Spreading
-Pull apart
-Touch/impression smear
HISTOPATHOLOGY 4. Frozen Section – when rapid diagnosis
of tissue is required
Application: Enzyme histochemistry,
I. Quality Assurance and Documentation lipids and protein
A. Histopath Records – 3 copies each Fixative: NO FIXATIVE
• Surgical Pathology To localize hydrolytic enzyme and other
• Cytopath Report antigens
HISTOPATHOLOGIC TECHNIQUES

Cryostat temp: -18°C to -20°C FIXATION

First and most critical step
Special Processing technique
Process of preserving cells and tissue
1. Freeeze Drying constituents in a condition identical to that
• w/o chemical fixative existing during life
• rapid freezing/quenching: -160°C Primary aim: TO PRESERVE THE CELLS
Secondary aim: harden and preserve tissue for
(Freezing)
further handling
• vacuum the ice molecules: -40°C Stabilization of Proteins: Most important
(Drying) reaction in maintaining tissue morphology
2. Freeze Substition
• Frozen Tissue- Rossman’s/ 1% Two Mechanisms of Action:
Acetone – dehydrated in absolute • Additive Fixation
alcohol -the fixative becomes part of the
tissue by formation of cross links or
Stains for Frozen Section complexes
-Progressive H%E Ex: Formalin, Hg, Osmium Tetroxide
-Thionine • Non-Additive Fixation
-Polychrome Methylene Blue -fixative NOT incorporated into the
-alcoholic pinocyanol method tissue
Freezing methods -stabilizes the tissue by removing of
• Liquid Nitrogen the bound water
• Isopentane cooled by Liquid Ex: alcoholic fixatives
Nitrogen
• Carbon Dioxide gas Factors involved in Fixation
• Aerosol Sprays 1. PH: 6-8
2. Temperature: Tissue processors (40°C)
Solid structures and tissues must be preserved Traditionally at Room Temp
and carefully processed in the following order: EM and Histochem: 0-4°C
1. Fixation Rapid Fixation: 60°C
2. Dehydration For tissues with tuberculosis: 100°C
3. Clearing 3. Thickness
4. Infiltration EM (1-2mm2)
5. Embedding LM (2cm2)
6. Trimming No more than 0.4cm for LM
7. Section-Cutting 4. Osmolality: Slightly hypertonic solution
8. Staining around 400-450 mOsm
9. Mounting 5. Concentration:
10. Labeling 10% Formalin
*Decalcification and Numbering 3% Glutaraldehyde
0.25% Glutaraldehyde for Electron
Microscopy
6. Time Duration: Primary Fixation (2-6
hours)
EM Fixation: 3 hours
HISTOPATHOLOGIC TECHNIQUES

Considerations: -Flemmings without acetic acid

1. Speed -Orth’s Fluid
2. Penetration – 1mm per hour for -Regaud
formalin -Helly’s
3. Volume: 10-20 times the volume of -Formalin with post chroming
tissue to be fixed • Histochemical Fixatives
4. Duration of Fixation -Formol saline
-Absolute Alcohol
TYPES OF FIXATIVES ACCORDING TO -Newcomer’s
COMPOSITION -Acetone
1. Simple Fixatives – one compound
2. Compound Fixatives – two or more
Ex: Zenker’s- glacial acetic acid – ALDEHYDE FIXATIVES
swelling of tissues A. Formaldehyde (Formalin) – must never
Mercuric Chloride – shrinking of tissues be neutralized – violent explosions
Stock solution: 37%-40%
TYPES OF FIXATIVE ACCORDING TO ACTION Working solution: 10%

1. Microanatomical – general microscopic B. 10% Formol Saline

study of tissues. -Fixation of CNS tissues and general
• 10% Formol Saline: CNS tissues Post mortem tissues for histochemical
• 10% NBF: Best general tissue examination
fixative C. 10% Neutral Buffered Formalin
• Heidenhain’s Susa -Best Fixative for iron containing
• Formol Sublimate (Formol pigments and elastic fibers
corrosive) -Inert to lipids
• Zenker’s solution D. Formol Corrosive (Formol Sublimate)
• Zenker-Formol -contains Mercuric Chloride
• Bouins -Excellent for stains like Silver Reticulin
• Brasil Solution -No washing out
-Fixes lipids
2. Cytological E. Gendre’s (Alcoholic Formalin)
• Nuclear Fixatives – must contain -has 95% EtOH with Picric acid and
glacial acetic acid which fixes Glacial acetic acid
nucleoprotein -good for glycogen, microincineration
Usual PH ≤ 4.6 techniques, and it fixes SPUTUM
-Carnoy’s = most rapid tissue fixative (1-3 F. Glutaraldehyde
hours), for chromosomes and preservation of -2 formalin residues linked by 3 carbon
brain tissue, in diagnosis of rabies chains
-Newcomer’s = both nuclear and histochemical -2.5% (small fragments) 4% (large tissue
fixative fragments)
-Flemmings -recommended for enzyme
-Bouins histochemistry and electron microscopy
-Heidenhain’s Susa -specimen vial should be refrigerated
• Cytoplasmic Fixatives – must NOT
contain glacial acetic acid
Usual Ph ≥ 4.6
HISTOPATHOLOGIC TECHNIQUES
G. Paraformaldehyde (4%)
-polymer of formalin and in white
powder form
- for thin and ultrathin section for
plastic embedding
METALLIC FIXATIVES
A. Mercuric Chloride – most common
metallic fixative
-may produce black granular deposits
except Heidenhain (REMOVAL CAN BE
DONE BY ADDITION OF SATURATED
IODINE SOLUTION OF 96% ALCOHOL)
-corrode all metals except for monel
(nickel alloy)
-routine fixative of choice for
preservation of Cell Detail in Tissue
Photography
1. Zenker’s: Contains Glacial Acetic
Acid
-recommended for small pieces of liver,
spleen, CT Fibers, and Nuclei
-recommended for Trichrome staining
Dezenkerization: REMOVAL OF
MERCURY DEPOSITS USING LUGOL’S
IODINE AND NA THIOSULFATE
2. Zenker Formol (Helly Solution)
- contains Potassium Dichromate and
Formalin
-preserves cytoplasmic granules
-produces brown pigments (remove
using picric acid or NaOH)
3. Heidenhain Susa
Su – sublimate (HgCl)
Sa – Saure (acid)
- has TCA, Glacia HoAc, Formalin
-recommended for skin tumor biopsies
4. B5 Fixative
-has anhydrous Na Acetate
-recommended for BM biopsies
B. Chromate Fixatives
-strong oxidizing agents and should not
be combined with reducing agents
-preserves carbohydrates
-fine yellow brown deposits
1. Potassium Dichromate
-preserves lipids
2. Regaud’s (Muller’s)
-chromatin, mitochondria, mitotic figures, Golgi
bodies, RBC and colloid containing tissues
3. Orth’s Fluid
-for early degenerative processes and tissue
necrosis
-for demonstration of RICKETTSIA
-preserves myelin
C. Lead Fixatives
-recommended for acid
mucopolysaccharides
PICRIC ACID FIXATIVES
-Explosive when dry
-excessive yellow staining
-excellent for glycogen
1. Bouin’s
-recommended for embryos and pituitary
biopsies
-excellent for preserving soft and delicate
structures
-preferred fixative for tissues to be stained
by Masson’s trichrome, Not for kidney
fixation
2. Brasil’s Alcoholic Picroformol Fixative
-Has TCA
-better and less messy than Bouin’s
-fixes glycogen
GLACIAL ACETIC ACID FIXATIVES
-solidifies at 17°C
-precipitates Nucleoproteins, Chromatin
materials
-NOT for Cytoplasmic Fixation
ALCOHOL FIXATIVES
-Acts as a fixative and dehydrating agent
-disadvantage: Polarization
-rapidly denatures and precipitates proteins
1. Methanol (100%)
-for fixing dry and wet smears, blood
smears and BM tissues
HISTOPATHOLOGIC TECHNIQUES

2. Ethanol (70-100%) -for frozen tissue sections and preparations of

95%- most common in cytology bacteriologic smears

3. Isopropyl Alcohol Microwave Technique

-fixes touch preparations • Physical agent similar to heat (oven)
4. Carnoy’s Fixative • Increases movement of molecule
-most rapid fixative and accelerate fixation, staining,
-contains absolute alcohol, chloroform, decalcification, EM, and
and glacial acetic acid immunohistochem
-fixes Nissl granules, Cytoplasmic • Optimum temp: 45-55C
granules
-chromosomes and RABIES Fixatives for Enzyme Histochem
5. Gendre’s Alcoholic Fixative -4% formalin or formol saline
-recommended for SPUTUM -acetone
6. Newcomer’s
-act as both nuclear and histochemical Fixatives for EM
fixative -osmium tetroxide
-glutaraldehyde
OSMIUM TETROXIDE -paraformaldehyde
-kept in amber bottle, avoid evaporation
-produces black prepicipitate (osmic oxide) – KARNOVSKY’S PARAFORMALDEHYDE
remove by COLD WATER GLUTARALDEHYDE: fixative for electron
-causes conjunctivitis/blindness histochemistry and electron
-fats are stained black immunocytochemistry
-fixation for ultrathin and sectioning in EM
-Expensive and Inhibits HEMATOXYLIN Terms related to fixation:
1. Secondary Fixation: Placing an already
1. Flemming’s fixed tissue into another fixative for:
-most common chrome-osmium acetic -special staining
acid fixative -complete hardening and preservation
-excellent fixative for nuclear structures -demonstration of substances
2. Flemming’s without Acetic Acid 2. Post-Chromatization: secondary
-for cytoplasmic structures especially fixation using 2.5-3% Potassium
mitochondria Dichromate for 1 day to act as mordant
3. Washing out: removal to excess fixative
TCA FIXATIVES in order to improve staining and
-precipitates proteins remove artefacts
-weak decalcifying agent also -tap water
-has a softening effect on dense tissues -50-70% alcohol
-alcoholic iodine
ACETONE FIXATIVES
-use at Ice cold temperature from -5°C to 4°C
-for diffusible enzymes such as phosphatase and ARTIFACT APPEARANCE REMOVED BY
lipases Acid Brown-black Sat. Picric Acid
-for fixing brain tissues (Rabies Diagnosis) Formaldehyde granules, Alcoholic KOH
Hematin extracellular Kardasewitch
HEAT FIXATION Lillies
-thermal coagulation of tissue proteins
HISTOPATHOLOGIC TECHNIQUES
HgCl Black granular Alc.iodine
deposits
Chromate Fine yellow Acid alcohol
brown
Osmic oxide Black deposit Cold water
DECALCIFICATION
-Removal of calcium or lime salts from bones or
calcified tissues following FIXATION
Ratio is 20:1
Heat and agitation hastens decalcification
Duration: 1-2 days
Temp:
Types of decalcifying agents
1. ACID DECALCIFYING AGENTS
A. Nitric Acid (5-10%)
• Most common
• Rapid decalcifying agent and can be
removed by 70% alcohol
• Imparts yellow color with nitrous
acid
✓ Formol Nitric Acid – rapid, for
urgent biopsies
✓ Perenyi’s Fluid – decalcifies and
softens tissues at the same time
✓ Phloroglucin Nitric Acid – most
rapid decalcifying agent
B. HCL
-Inferior to HNO3
✓ Von Ebner’s – contains 36% HCL, for
teeth and small pieces of bone
C. Formic Acid
-moderate acting decalcifying agent
-for post-mortem research tissues
-addition of CITRATE hastens
decalcification
-both a fixative and decalcifying agent
✓ Formic acid Sodium Citrate Solution
– has better nuclear staining, for
autopsy materials, BM, Cartilage
and tissues for research
D. TCA
-weak decalcifying agent and fixative
-suitable for only small bone spicules
E. Sulfurous Acid
-weak decalcifying agent and only for
minute pieces of bone
F. Chromic Acid
-AKA flemming’s fluid
-both a fixative and a decalcifying agent
-chromic acid is carcinogenic
2. CHELATING AGENTS
-Combine with calcium salts and other salts
to form complexes and to facilitate removal
of calcium
-commonly used is EDTA (Versene)
-Duration: 1-3 weeks for small specimens
6-8 weeks for dense bones
pH is adjusted to 7-7.4
-excellent for immunohistochem or enzyme
staining
3. ION EXCHANGE RESIN
-Hastens decalcification by using formic acid
containing decalcifying solutions.
4. ELECTROPHORESIS
-makes use of 88% formic acid
Measurement of Decalcification
A. Physical or mechanical test
B. Xray method
-expensive but most ideal
C. Chemical Method (CALCIUM OXALATE)
POST DECALCIFICATION
-immersing in saturated Li2CO3 or 5-10%
NaHCO3
-rinsing in running tap water
-storing in formol saline containing 15% sucrose
or PBS with 15-20% sucrose at 4°C
Tissue Softeners
1. Perenyi’s
2. Lendrum’s: 4% Phenol
3. Molliflex
4. 2% HCl
5. 1% HCl in 70% alcohol
HISTOPATHOLOGIC TECHNIQUES

Weiseberger’s: Tissue is wrapped in gauze and

DEHYDRATION suspended in bottle containing dioxane and
-intercellular and extracellular water from the anhydrous calcium oxide
tissue are removed after fixation and prior to
wax infiltration CELLOSOLVE
-usually occurs by placing in ascending grades of -ethylene glycol monoethyl ether
alcohol -rapid dehydrating agent
-30% ethanol for delicate tissues
Amount of dehydrating agent: 10 times the TRI-ETHYL PHOSPHATE
volume or more -used to dehydrate sections and smears
following certain stains
ALCOHOLS
A. Ethanol TETRAHYDROFURAN
• Recommended for routine tissue -both a dehydrating and clearing agent
dehydration -offensive odor
• Considered to be the best -can cause conjunctival irritation
dehydrating agent
B. Methanol
• For blood and tissue films and for ADDITIVES
1. 4% Phenol + 95% Etoh – Tissue
smear preparations
softeners
C. Butanol
2. Anhydrous CuSO4- bothe dehydrating
• Utilized in plant and animal
agent and indicator of water content at
microtechniques
100% ethanol
Original color: white
Important reminders when using alcohol
Water + Anhydrous: blue (water
• The strength of initial alcohol
positive)
required in each concentration will
depend upon the size, and nature
of the tissue and fixative used
• Temperature: hastens at 37C CLEARING
• Anhydrous copper sulfate: Serves as -dealcoholization
an indicator that will accelerate and -makes tissues transparent
ensure complete dehydration. Duration of Clearing: Over clearing may cause
• Xylene: If it becomes milky tissue brittleness
For frozen sections: glycering and gum syrup are
ACETONE used. No need for dealcoholization
-both fixative and dehydrating agent
-for most urgent biopsies (dehydrates in half to XYLENE
2 hours) but can cause considerable shrinkage -AKA xylol
and brittleness. -most commonly used in routine procedures
-rapid clearing time: 15-30 minutes to 1 hour
DIOXANE -for tissue sections with a thickness of less than
-Diethylene Dioxide 5 mm
-both dehydrating and clearing agent -not for nervous tissues and lymph nodes
Gaupner’s: Uses pure dioxane and paraffin -it turns milky when tissues are incompletely
dehydrated
HISTOPATHOLOGIC TECHNIQUES

TOLUENE WAX IMPREGNATION AND

-Substitute for xylene or benzene
EMBEDDING
BENZENE
Wax Impregnation/Infiltration: Replacing the
-Recommended for urgent biopsies and for
clearing agent with the infiltrating medium to
routine purposes
fill all tissue cavities
-carcinogenic; toxic to man
Ratio: 25:1
-can cause aplastic anemia
Embedding (Casting/Blocking): Impregnated
CHLOROFORM
tissue is placed into a precisely arranged
-can be used for tissue blocks (up to 1 cm)
position in a mold containing an embedding
-recommended for tough tissues
medium and allowed to solidify.
-“hepatotoxic”
Types of Impregnating Medium
CEDARWOOD OIL
A. PARAFFIN
-recommended for CNS tissues and cytology
-Simplest, most common and best
such as smooth muscles and skin
embedding medium
-requires 2 changes of clearing agent
Melting point for routine work: 56°C
- If the lab temp is between 20-24C
ANILINE OIL
54-58C
-recommended for embryos, insects and very
- If the lab temp is between 15-18C
delicate tissues
50-54C
-not recommended for fatty tissues
CLOVE OIL
-recommended for skin and smooth muscle
Paraffin Wax Substitutes
1. Paraplast
CARBON TETRACHLORIDE
2. Embeddol
-similar to chloroform but cheaper
3. Bioloid
-causes tissue hardening
4. Tissue Mat
5. Ester Wax
METHYL BENZOATE AND METHYL SALICYLATE
6. Water Soluble (Carbowax)
-for double embedding technique
B. CELLOIDIN
-AKA collodion
-purified form of nitrocellulose
Other clearing agents
-for spx with large hollow cavities w/c tend
Oil of Bergamot: skin and smooth muscle
to collapse, hard and dense tissues
Oil of Origanum: skin
-not requires heat during processing
Oil of Wintergreen: artificial oil, for delicate
Wet celloidin: for bones, brain sections and
tissues
teeth.
Carbon Disulfide: smooth muscle
Dry Celloidin: for processing whole eye
Carbon xylene: friable tissues
sections, uses Gilson’s mixture for storage
Terpineol: delicate material like eyes
Nitrocellulose Method (Low Viscosity
Phenol: smooth muscles
Nitrocellulose): soluble in equal
High Test Aviation Lead Free Gasoline: excellent
concentration of ether and alcohol
clearing agent
-preferred since it produces a harder tissue
block and thinner sections
HISTOPATHOLOGIC TECHNIQUES

-add plasticizer (castor oil) to prevent tissue SECTIONING

cracking
Paraffin sections = 4-6 um sections
C. GELATIN Celloidin sections= 10-15 um sections
-for histochemical and enzyme studies and EM=0.5um
frozen sections Frozen Sections
-tissue should be less than 2-3mm Two methods:
-add 1% phenol to prevent molds ✓ Cold knife – use of conventional
-used when dehydration is to be avoided freezing microtome (carbon
-not a wax dioxide)
-water soluble -tissue block: 3-5mm
Dew line: point in which sections
may be cut at 10um
EMBEDDING ✓ Cryostat: -18 to -20C
-done after wax impregnation Methods of Freezing
-temperature of paraffin: 5-10°C higher than - Liquid Nitrogen= most rapid
MP of Paraffin wax - Isopentane= liquid at room temp
Molds used - Aerosol Sprays (Cryokwik)
- Leuckhart’s Embeddingh mold: 2L - Carbon Dioxide
shadped strips of heavy brass or metal
- Compound embedding unit Mounting= use of synthetic water soluble
- Plastic embedding rings and Bae Molds glycols and resins
- Tissue Tek - OCT (optimal cutting temperature
- Disposable Molds(Peel Away, Ice tray, compound) -recommended
Paper Boat) - Tissue block- 2-4mm
Other Embedding Methods
-Celloidin or Nitrocellulose Method: For hard MICROTOMY
tissues and large sections of whole organs
-Double Embedding Method: 1st infiltration with
1. Rocking Microtome (Paldwell Trefall)
celloidin and subsequently with embedded with
-simplest microtome
paraffin
-10-12um sections
-for large paraffin embedded blocks
2. Rotary Microtome (Minot)
-Plastic (Resin) Embedding:
-routinely used microtome
Classified into:
3. Sliding Microtome (Adams)
Epoxy- reduces antigenicity, toxic and damages
-for celloidin sections and hard rough
tissue
tissue block
Polyester- not often used
2 types: Base Sledge and Standard
Acrylic- used extensively for light microscopy
Sliding
4. Freezing Microtome (Queckett)
TRIMMING -for frozen section
-four sided prism/truncated pyramid -CO2 as propellant
-at least 2mm of wax should surround the tissue 5. Ultrathin Microtome
-as thin as 0.5um
-EM
-tissues are embedded in Plastic
-Special knife: Diamond knife
HISTOPATHOLOGIC TECHNIQUES

-temp: 10C below MP of wax

MICROTOME KNIVES ✓ Drying Oven
1. Plane Concave (25mm) -temp: 5C higher than MP of wax
-less concave side- ✓ Forceps
-more concave side- ✓ Clean Slide
-for base sledge, rotary or rocking
microtome TISSUE ADHESIVES
2. Biconcave Knife (120 mm) ✓ Albumin (Mayer’s Egg albumin)
-Paraffin sections -may add phenol crystals to prevent
-rotary microtome growth of molds
3. Plane Wedge (100mm) -glycerol is also added to increase
-for frozen sections or very hard tissues viscosity and prevent complete drying
-base sledge or sliding microtome ✓ Gelatin
Bevel: cutting facet -provides firmer attachment than
-found on the tapered edge of a knife albumin
Bevel angle: angle formed between the cutting -added to the flotation water bath
edge 27-32 degrees ✓ Cellulose
Cutting angle:15 degrees’ optimum, there is -in the form of 1% methyl cellulose
maximum penetration of tissue and less Advantage: not staining to any
distortion appreciable extent with commonly
Clearance angle: 0-15 degrees used stains of histochemical reagents
-angle formed between the surface of the block Poly-L-lysine: use as a general purpose
and the cutting edge of the knife section adhesive
✓ Sodium Silicate
Honing – knife sharpening -1:10 Dilution
-heel to toe 20-30 times ✓ Resin
-removal of nicks -greatest adhesion
Hones (8inch x 3 inch) ✓ 3-aminopropyltriethoxysilane
• Belgium Yellow (Best Result) -cytology
• Arkansas- more polishing -best adhesive
• Fine Carborandum (Badly nicked
knives) STAINING
Lubricants: Soapy water, mineral oil, clove oil, -application of dyes on tissue sections to study
xylene or liquid paraffin the architectural patterns and physical
Knife sharpeners: Mechanical knife sharpener characteristics of cells
and Flat circular glass plate with powdered
aluminum oxide Affinity
Acidic (Nucleus) – basic stains
Stropping – polishing Basic (cytoplasm) – acidic stains
-removal of burrs Groups of Histologic Staining
-toe to heel 1. Histologic Staining
- use paddle strop made of horse leather 2. Histochemical Staining
-strops should be oiled on the back (Don’t use 3. Immunohistochemical staining
mineral oil)
-40-120 double strokes

OTHER EQUIPMENT USED IN SECTIONING

✓ Flotation Water Bath
HISTOPATHOLOGIC TECHNIQUES
METHODS OF STAINING
A. According to presence of mordant
1. Direct Staining
-uses aqueous or alcoholic dye solutions
to produce a color
2. Indirect Staining
-uses a mordant or another agent to
intensify the action the dye used
Mordant: serves as a link or bridge between the
tissue and the dye
Dye + Mordant =insoluble tissue mordant dye
complex e.g Potassium alum with hematoxylin
in Ehrlich’s hematoxylin
Accentuator: not essential and does not
participate to the chemical reaction of the
tissue and the dye. It hastens the staining
reaction by increasing the staining power and
selectivity of the dye
B. Presence of Differentiator
PROGRESSIVE STAINING
-tissue elements are stained in sequence
-no decolorization is applied
REGRESSIVE STAINING
-overstaining is done
-excess stain is removed or decolorized from
unwanted parts of the tissue and until the
desired color is obtained
DECOLORIZER/DIFFERENTIATION
-selective removal of excess stain so that a
specific substance may stain distinctly from the
surrounding tissue
-usually done by washing the section in simple
solution or use of acids and oxidizing agents
C. According to resultant color
METACHROMATIC STAINING
-staining with a color that is different from that
of the stain itself
-water is important in metachromatic staining,
alcohol tends to lose the metachromatic stain
-formalin is used to fix tissues for this type of
staining
ORTHOCHROMATIC STAINING
-color of dye same with the tissue
COUNTER STAIN
-for contrast and background
-stain with a different color that of the primary
stain
METALLIC IMPREGNATION
-demonstration of tissue elements using
solutions of metallic salts that are deposited on
the tissue surface
-it is not absorbed by the tissues, could be a
precipitate or a reduction product on certain
tissues
VITAL STAINS
-the selective staining of living cell constituents
-demonstrates cytoplasmic structures
- NUCLEUS is resistant to vital stains
Two types of Vital Staining:
A. Intravital Staining
-by injecting the dye into any part of the
animal body
Ex: Lithium, Carmine, India ink
B. Supravital Staining
-used immediately after removal of cells
from the living body
Ex: Neutral Red – best vital dye
Janus Green – mitochondria
Thionine, Trypan blue, toluidine blue, nile blue
ROUTINE HEMATOXYLIN AND EOSIN
-most common
-uses the regressive staini0ng which consists of
overstaining the nuclei, removal of superfluous
and excessive color of the tissue constituent by
acid differentiation
HEMATOXYLIN (Waldeyer, 1862)
-primary stain, nuclear stain and basic stain
-Hematoxylin campechianum
-it is not a stain
-Active coloring Hematin
-ripening=oxidation of hematoxylin
Ripening
• Natural ripening
• Artificial ripening
 HISTOPATHOLOGIC TECHNIQUES

I. Alum Hematoxylin -with picric acid

-used in routine H&E -Picrocarmine
-mordant: Alum (Potassium Alum/Potassium -neuropathological stain
aluminum sulfate) -with aluminum chloride
Ehrlich’s – sodium iodate -Best’s Carmine
Harris – mercuric chloride -demonstrates glycogen

II. Iron Hematoxylin ORCEIN

-iron as oxidizing and mordant
Weigert’s: FeCl3, standard Iron Hematoxylin. In
combination with Van Gieson= demonstrates CT
elements and E. hystolytica
Heidenhain’s = FeNH3SO4, for muscle striations
III. Mallory’s PTAH
-fibrin and muscle striations
IV. Copper Hematoxylin
-Spermatogenesis
-vegetable dye, from lichens
-used for staining elastic fibers
SYNTHETIC DYES
-AKA “coal tar dyes” or aniline dyes
Chromophore – substances that are capable of
producing visible color but not permanent
Auxochrome – substances that are added to a
chromogen

3 Groups of synthetic dyes

EOSIN 1. Acid dyes
-secondary stain, counterstain, acid stain, 2. Basic Dyes
cytoplasmic and connective tissues stain 3. Neutral Dyes
differentially
3 forms OTHER STAINS
• Yellow (Eosin Y)
Most commonly used • Acid Fuchsin-Picric Acid (Van Gieson’s
Green yellow fluorescence Stain)
• Eosin B, Erythrosin B -connective tissues
Deeper red color • Acridine Orange (Masson Stain)
• Eosin S, Eosin alcohol-soluble -discriminates dead and living cells
Ethyl eosin DNA- green fluorescence
RNA- red fluorescence
STAINS • Acridine Red 3B
Two categories -calcium salt deposits and phosphatase
1. Natural dyes activities
2. Synthetic (Artificial) dyes • Alcian Blue
-connective tissue and epithelial mucin
NATURAL DYES • Aniline Blue
-derived from plants and animals -cytoplasmic stain and counterstaining
-e.g., Hematoxylin, Cochineal dyes, orcein, of epithelial sections
saffron • Basic Fuchsin
-plasma stain
COCHINEAL DYES -for staining acid-fast organisms, for
-extracted from coccus cacti mitochondria, and differentiation of
-with alum smooth muscles
-Carmine Dye A. Carbol Fuchsin
-Chromatin and nuclear stain for fresh B. Coleman’s Feulgen Rgt
And smear preparation
HISTOPATHOLOGIC TECHNIQUES
C. Schiff’ Rgt
D. Mallory’s fuchsin stain
E. Aldehyde Fuchsin (Gomori’s stain)
• Benzidine
-for hemoglobin
• Bismarck Brown
-counterstain for Gram’s technique,
acid fast and Papanicolau method
-used for DIPHTERIA ORGANISMS
• Carmine
-chromatin stain
• Celestine Blue
-routine staining of fixed sections
-resistant to strong acid dyes
• Congo Red
-best known as an indicator
-stains elastic tissues, amyloid, myelin
• Crystal Violet
-a nuclear or chromatin stain
-stains amyloid in frozen section
• Giemsa Stain
-used for staining blood to differentiate
WBCs
• Gold Sublimate
-stain used for metallic impregnation
• Iodine
-oldest stain, stains amyloidm
cellulose, starch, carotenes and
glycogen
-used to remove mercuric fixative
artifact pigments
Gram’s Iodine: microorganism and
fibrin
Lugol’s Iodine: test for glycogen,
amyloid and corpora amylacea
• Janus Green
-demonstates mitochondria during
intravital staining
• Malachite Green
-counterstain for ascaris eggs,
erythrocytes, bacterial spores,
• Methylene Blue
-common basic nuclear stain used with
eosin
• Neutral Red
-demonstrates cell granules and
vacuoles of phagocytic cells
• Orcein
-stains elastic fibers
-recommended for dermatological
studies (fibers in the skin)
• Osmium Tetroxide
-used to stain fat-black
• Picric Acid
-counterstain to for acid fuschin,
connective tissues, cytoplasmic stains
in contrast to basic dyes, counterstain
for crystal violet
• Prussian Blue
-colored salt of ferric ferrocyanide
• Rhodamine B
-used with osmic acid to fix and stain
blood and glandular tissues
• Toluidine Blue
-used as a nuclear stain in fixed tissues,
stains Nissl granules or chromophilic
bodies
OIL SOLUBLE DYES (LYSOCHROMES)
-not real dyes
-lack auxochrome
Sudan Black B
-most sensitive of the oil soluble dyes
-greater affinity for phospholipids, neutral fats
but not crystalline cholesterol, free fatty acids
Sudan IV (Scharlach R)
-has no secondary amino group
-most commonly used
-stains neutral fats, but not phospholipids or
fine lipid drop;ets
Benzoic acid – intensifies fat staining and
prevents rapid deterioration of the solution
Sudan III
-1st sudan dye
-stains CNS tissues
Oil Red O
-stains neutral fats and lipofuschin
HISTOPATHOLOGIC TECHNIQUES

Osmic Acid -useful for staining encapsulated fungi

-for unsaturated fats (Southgate’s mucicarmine technique)
Nile Blue Sulfate Method – differentiates two • Acridine Orange
lipid classes • Colloidal iron
Red Oxazone – dissolves neutral lipids -adsorbed into mucin at low pH,
Blue Oxazone – basic and reacts with subsequently staining with Prussian blue
phospholipids and free fatty acids -greater sensitivity than alcian blue but
more complex and time consuming
Solvents used for staining
1. Water STAINS FOR PROTEIN, ENZYMES AND NUCLEIC
2. Alcohol ACIDS
3. Aniline Water ✓ Histochemical Identification of Proteins
4. Phenol -detection of amino acids molecules
o Alkaline Fast Green Method-
CARBOHYDRATE STAINS Basic Proteins (Protamines and
• Periodic Acid Schiff Histones)
-Intensity of PAS proportional to sugar content o Peracetic Acid Alcian Blue-
• Schiff Reagent Cystine and cysteine
-Basic Fuchsin (rosanilin, pararosanilin, magenta
III) ✓ Enzyme Histochemistry
Methods of Preparation: -tissue requirement= -70C
Barger and de lamater: thionyle chloride o Gomori Calcium- ALP
De Tomasi-coleman: potassium metabisulfite o Gomori Lead – ACP
Itikawa and Oguru: sulfur dioxide gas o Lead method – 5-nucleotidase
o Metal Precipitation for ATPase
MUCOPROTEIN most common PAS positive o Alpha Naphtyl Acetate- Non
substance specific esterase
• PAS with Diastase o Indoxyl Acetate Method – non
-Diastase can digest glycogen specific esterase
• Best Carmine o Acetylthiocholine –
-selective and highly specific for glycogen acetylcholinesterase
o Tetrazolium – monoamine
ACID MUCOPOLYSACCHARIDES oxidase
• Alcian blue
-most popular method for acid mucins Nucleic Acid Stains
• Metachromatic Staining o FEULGEN TECHNIQUE for nuclear
Azure A – most useful DNA
Uranyl Acetate – excellent results for CT -most reliable and specific
mucins histochemical technique
• Combined Alcian blue PAS Technique for o Methyl Green Pyronin Method for
Acid and Neutral Mucins DNA and RNA
-demonstration of any mucins o Fluorescent stain for DNA and RNA
• Gomori Aldehyde Fuchsin Stain for sulfated -Fluorescein-widely used
mucin fluorochrome
• Mucicarmine -Rhodamine
-carmine with aluminum hydroxide to -Auramine- most common
improve mucin staining -Acriflavin
• In situ hybridization – “on site”
HISTOPATHOLOGIC TECHNIQUES
-most sensitive technique for DNA
Idenfication
• PCR
CONNECTIVE TISSUE STAINS
• Stains for reticulin
-PAS
-Gomori’s silver impregnation
• Stains for Collagen
-Van gieson _____________
-Masson’s trichrome stain
-Mallory’s Aniline Blue stain –stains
collagen, reticulin, hyaline, fibroglia,
smooth and striated muscle fibers
and amyloid (Excellent and colourful
method of CT fiber demonstration)
-Azocarmine – heidenhain’s
modification of Mallory’s
• Stains for Elastic Fiber
-Weigert’s Elastic Tissue stain
-Orcein (Tanzer Unna Orcein) = elastic
fibers in skin
-Krajians – rapid method for elastic
fibers, fibrin and amyloid
• Stains for basement membrane
-PAS – most common especially for
glomerular basement membrane
PATHOLOGIC CHANGES AND DEPOSITS IN CT
A. Fibrin
-insoluble fibrillar protein
-tissue damage, blood clots
Stains: MSB Technique ( Lendrum’s Martius,
Scarlet, Blue)
Mallory’s PTAH
B. Fibrinoid
-eosinophilic material and identical
staining reactions to fibrin
C. Hyalin
-degenerated collagen, hypertension,
atheroma
-PAS (Non specific)
D. Amyloid
-semi translucent, ground glass, or
hyaline eosinophilic substance
Methods for amyloid demonstration
1. Gram’s Iodine Stain
2. Congo Red method – highly selective;
sine qua non for amyloid]
3. Metachromatic staining
4. Induced fluorescence staining with
Thioflavine
MUSCLE AND BONE STAINS
• Modified Gomori’s Trichrome
• Rapid Gomori Trichrome for frozen
muscle tissue
• Mallory’s PTAH
• Heidenhain’s Iron Hematoxylin
Method
• Lissamine Fast Red Tartrazine
Method- for muscle and bone
Stains for Bone Tissue
• H&E – standard for bone biopsies
• Masson’s Trichrome stain – standard
popular method for Collagen fibers of
the bone
• Aniline Blue or light green fiber –
osteiod
• Schmorl’s Picro Thionin – stains lacuna,
matrix cells
STAINING FOR BONE MARROW AND BLOOD
ELEMENTS
Bone Marrow
- Zenker’s Solution = recommended
fixative
Stains
- Toluidine blue = most useful and
informative stain for plastic embedded
tissue sections
- Methyl green pyronin = plasma cells
- Perl’s = iron stores
CNS STAINS
• Bielschowky’s = neurons, axons and
neurofibrils
• Bodian’s = nerve fibers and nerve
endings, for ALZHEIMER’S DIAGNOSIS
• Sevier Munger Technique =
demonstration of neuritic plaques and
neurofibrillary tangles in brains of
Alzheimer’s diseases
HISTOPATHOLOGIC TECHNIQUES
• Cresyl Fast Violet = Nissl’s stain for
paraffin section
• Weigert Pal = Normal myelin sheath
• Kluver and Barrera Luxol fast blue =
myelin with nissl bodies
• Luxol Fast blue H&E stain for myelin
• Luxol fast blue PAS hematoxylin for
myelin
• Weil’s Method = myelin sheath
• Cajal’s Gold sublimate for astrocytes
• Modified PTAH for reactive astrocytes
• Modified Holzer’s method for
astrocytic processes
STAINING OF TISSUE PIGMENTS AND DEPOSITS
Endogenous pigments – pigments within the
tissue
• Hemosiderin – iron containing pigment of
Hb
• Hematoidin – iron free pigment of Hb
• Hematin – haemoglobin without the globin
• Hemozoin – malarial pigment
• Hemofucsin (Lipofuscin) wear and tear
pigment, iron free brownish pigment
• Perl’s Prussian blue – hemosiderin (ferric
iron)
• Turnbull’s blue reaction – ferrous iron
• Benzidine Nitroprusside stain for
haemoglobin and oxidase granules
• Modified Fouchet’s for liver bile pigments
• Gmelin technique for Bile and Hematoidin
(Diagnostic for Bile Pigments)
• Masson Fontana Technique – melanin and
argentaffin
Demonstration of Calcium deposits
- For soluble calcium salts
-gypsum method
-oxalate method
- Insoluble calcium salts
-calcium dye lake reactions = embryo
and fetus skeletal system
- Metal Substitution (Indirect Method)
VON KOSSA’S SILVER NITRATE METHOD
Copper – Lindquist Modified Rhodamine
Technique
Urates and Phosphates – birefringence, use of
lithium carbonate
Carbon – most common exogenous pigments
STAINING OF MICROORGANISMS
Bacteria
✓ Brown and Brenn – nocardia,
actinomyces
✓ Wade Fite – Leprosy and Nocardia
✓ Auramine Rhodamine – mycobacteria
✓ Toluidine Blue – helicobacter
✓ Cresyl Violet Acetate Method –
Helicobacter
✓ Dieterle - Legionella
✓ Levaditi’s- Spirochetes
✓ Modified steiner and steiner –
spirochetes
✓ Warthin Starry - spirochetes
Fungal
✓ Grocott Methanamine Silver – Fungi
Viral
✓ Lendrum’s Phloxine Tartrazine Method
= viral inclusions
✓ Orcein method- HEPA B SURFACE
ANTIGEN
Protozoan stains – dilute giemsa
IMMUNOHISTOCHEMISTRY
-identification of specific or selective tissue or
cellular antigen
-makes use of ag-ab reaction
Controls
Positive Control – a section known and proven
to have the antigen in question
Negative Control – omit the primary antibody
from the staining schedule
Internal tissue control - “built in control”,
contains the target antigen but not in the tissue
elements under investigation
 HISTOPATHOLOGIC TECHNIQUES

TUMOR MARKERS MOUNTING AND LABELLING

I. Epithelial Tumor Markers
1. Keratin (CK 7, CK20, CK 5/6) Mounting – use of a medium and a coverslip to
2. EMA facilitate the handling, storage, protection of
3. CEA the tissue section
4. TTF-1
5. PSA Aqueous Mounting Medium
6. ER/PR - Water (temporary)
- Glycerin (semi-permanent) sections
- Farrant’s Medium (gum Arabic
II. Intermediate Filament Markers medium)
1. Actin - Apathy’s Medium (Methylene blue
2. Vimentin stained nerve preparation)
3. Desmin - Brun’s fluid – mounting frozen sections
4. GFAP from water
5. NF Resinous Mounting Media
6. S100 - Canada Balsam – transparent and
III. Neuroendocrine Markers colorless but darkens and oxidizes with
1. NSE ages for whole mounts and thick
2. Chromogranin sections
3. Synaptophysin - DPX – small tissue sections
IV. Germ Cell tumor markers - XAM – synthetic resin mixture
1. HCG dissolved in xylene
2. AFP - Clarite – synthetic resin, preferred over
3. PLAP DPX
V. Mesenchymal Tumor marker - Permount, HSR, Clearmount
1. Myogenic Tumors – tumors of skeletal
muscle origin Ringing
2. Fibrohistiocytic tumors – malignant - Sealing of margins of the coverslips to
fibrohistiosarcoma prevent escape of fluid and
3. Vascular Tumors – endothelial markers evaporation; prevents sticking of slides
4. Melanoma – HMB45
5. Lymphoma Labelling – process of indicating the year and
+ LCA CD45 specimen number on one end of the prepared
T cells - + CD3, CD4, CD8 slide for proper identification
B cells - + CD19, CD20, CD23
Reed Sternberg = CD 30 + and CD15 +
VI. Cell Proliferation markers
1. Ki67, and PCNA EXFOLIATIVE CYTOLOGY
VII. Cancer-associated genes -Branch of general cytology that deals with the
1. P53 – overexpression uncommon or study of desquamated from the epithelial
absent in normal or benign cells surfaces.
Recommended for:
- Detection of malignant cells or
cancerous conditions in the body
- Detection of asymptomatic or
precancerous cervical lesions in women
HISTOPATHOLOGIC TECHNIQUES
Specimens for examination: body fluids,
sputum, gastric washings, urine sediment
Specimens that require adhesive agent
1. Urinary Sediment
2. Bronchial lavage
3. Specimens that utilizes proteolytic
enzymes during processing ( eg.
Sputum, GIT)
Fixation
-exfoliated cells decompose rapidly which may
destroy cellular and nuclear details, in turn will
give inadequate results for diagnosis.
1. Equal parts of 95% eTOH and ether
2. 95% eTOH
3. Carnoy’s fluid
4. Equal parts of tertiary butyl alcohol and
1 part 95% EtOH
5. SCHAUDINN’S FLUID – sat. Aq, hgCl2,
absolute acetic acid
6. Methanol – dried films
7. Saccomano’s – 50% alcohol and 2%
carbowax
PAPANICOLAU SMEAR AND STAIN
- Nicolas Papanicolau
- Screening test for cervical cancer
Anatomic Sites for Cytologic Samples
(Gynecological)
1. Upper (Proximal) third of the vaginal
wall
-ideal for studying the hormonal status
of, evaluation of inflammatory
conditions
2. Ectocervix
-most common for cancer screening
- use of Ayre’ s spatula
T zone where most malignancies arise
3. Endocervix
-for detection of endocervical lesions ,
intrauterine lesions
Histology: simple columnar epithelium
PAPS STAIN
1. Harris Hematoxylin – nuclear stain
2. OG-6 – strong affinity for mature cells
3. EA 50 – strong affinity for
_____________
EA 50 is comparable to EA 36
EA 65 differs from EA 50 or EA 36 only
with respect to the concentration of the
light green stock solution
HORMONAL CYTOLOGY
- Based on the specific response of the
vaginal epithelium to steroid hormones
ESTROGEN: produced by the
proliferating granulose theca cells of
the ovarian follicles. It acts upon the
SUPERFICIAL CELLS
PROGESTERONE: produced by corpus
luteum formed after ovulation. It acts
upon the INTERMEDIATE CELLS
Hormonal changes are best mirrored in the
upper third of the vagina.
CELLS
1. Superficial cells
-cytoplasm: may be acidophilic or
basophilic
-presence of small dark pyknotic nuclei
(less than 6u)
-true acidophilia (estrogen influence)
2. Intermediate Cells
-cytoplasm: basophilic with vacuoles
-vesicular nuclei (6-9u)
• Navicular cells – boat shaped
intermediate basal cells with a
strong tendency to fold and curl
their edges.
-expression of the combined
estrogen-progesterone effect
HISTOPATHOLOGIC TECHNIQUES
• Pregnancy cells – translucent
basophilic cytoplasm (glycogen
accumulation)
-cytoplasm stains deep blue or
green + cell membrane = a
double cell wall appearance
3. Parabasal Cells
-round to oval cells
-SUNNY SIDE UP LIKE CELLS
-have strong basophilic cytoplasm and
vesicular nuclei (6-9u)
• Basal cells – found in vaginal smears
only before pregnancy and after
menopause
CYTOHORMONAL SMEAR
-used to evaluate the hormonal status
based on the distribution of the cells
(superficial, intermediate, parabasals)
CHMI: Cytohormonal Maturation Index (%
per 100 cells)
CHMI = parabasal; intermediates; superficial
Example:
Left shift: increase parabasal: post
menopausal 100/0/0
Midzone shift – 10/90/0 – pregnant,
increase intermediate
Right shift – increase superficial 0/10/90
OTHER NORMAL CELLS THAT MAY BE SEEN IN A
PAP SMEAR
• Endometrial Cells
-Found during menstruation period
and 1-4 days after the cessation of
the period (single)
Endometrial stromal cells: seen in
tight clusters of small, oval dark
cells, glandular cells, slightly larger
-confused with parabasal cells
• Endocervical Cells
-slightly cylindrical appearance
-HONEYCOMB END
-occurs in groups and strips of three
or more cells
• Doderlein Bacilli
-“LACTOBACILLUS ACIDOPHILUS”
-predominant organism of the
vaginal normal flora: establishes the
low pH that inhibits the growth of
pathogens
• Neutrophils – normally increased
just before, during and shortly after
menstruation
• RBC
• Leptothrix sp – indirectly tells
possibility of infection
• Talcum – contaminant, ovoid bodies
ABNORMAL CELLULAR COMPONENTS OF A PAP
SMEAR
• Candida albicans: budding yeast,
diabetes, oral contraceptives
• Trichomonas vaginalis: STD
• Gardnerella vaginalis: tiny pleomorphic
coccobacilli. “clue cells”
• Koilocytes: squamous epithelial cells
that show cytopathic effects of HPV
-nucleus appears like a “WRINKLED
PRUNE APPEARANCE” that show
cytopathic effects of HPV
-presence of it is diagnosed as LOW
GRADE SQUAMOUS INTRAEPITHELIAL
LESION
• HSV II
• High Grade Squamous Intraepithelial
lesions: sever dysplasia, Carcinoma in
situ
• Invasive squamous cell carcinoma: most
common form of cervical malignancy
QUANTITATION IN VAGINAL CYTOLOGY
• Acidophilic index – percentage of cells
that stain pink-orange to red with Paps
and red in Shorr Mtd
• Pyknotic Index – “karyo-pyknotic index”
percentage of cells having shrunken,
dark, small structures nuclei
HISTOPATHOLOGIC TECHNIQUES

• Maturation Index – percentage -to rule out P. jirovecii

proportion of cells from the three layers • Sputum
of the vaginal epithelium -obtain at least 3 consecutive mornings
-alveolar MAC + for sputum
MANNER OF REPORTING PAP SMEARS • Gastrointestinal
Two Systems -delay of more than 30 minutes will
- Class System ( obsolete) automatically digest the cells
- Bethesda System -fasting : 8 hours
• Urine
Class system -dx of urothelial malignancy
- at least 50 ml
Class I – Negative for malignant cells -2nd morning urine
• Peritoneal, Pericardial, Pleural
Class II – atypical cells present, but negative for -add 300U of heparin/100ml of aspirate
malignancy to prevent formation of jelly-like clots.

Class III – suspicious for malignant cells

Class IV – strongly suggestive for malignant cells

-JAGEMD ☺
Class V – conclusive for malignant cells

Bethesda System
Report Format
-Specimen Adequacy
-General Categorization
-Descriptive Diagnosis

Adequacy
- Satisfactory
- Limited
- Unsatisfactory

Descriptive
- Normal
- Benign
- Epithelial cell abnormality
- Atypical squamous cells of unknown
significance ( ASCUS)
- Low grade squamous intraepithelial
lesion (LGSIL)
- High grade squamous intraepithelial
lesions (HGSIL)

NON GYNECOLOGICAL SPECIMENS

• Bronchoalveolar Lavage