Histopath

Histopathology
Fresh Tissue Examination

Teasing/Dissociation Immersed in a watch glass containing isotonic salt solution carefully dissected or separated
Phase contrast or Bright field microscopy
Squash Preparation (Crushing) Small pieces not more than 1mm in diameter
Forcibly compressed with another slide
Smear Preparation Cellular materials are spread lightly over a slide by wire loop or applicator stick
Cytologic: cancer diagnosis
Streaking Applied in a direct/zigzag line to obtain a relatively uniform distribution
Spreading Teasing the mucous strands apart with an applicator stick
Fresh sputum and bronchial aspirates
Pull-apart Two slides are pulled apart with a single uninterrupted motion
Thick secretions such as serous fluid, concentrated sputum and enzymatic lavage samples
Touch Preparation (Impression Freshly cut piece of tissue is brought into contact
smear) Phase contrast and light microscopy
Frozen Section Rapid diagnosis for lipids and nervous tissue elements
10-15µ in thickness Microtome with CO 2
Cryostat (-10° to 20°C)
Methods of freezing Liquid nitrogen
Isopentane cooled by liquid nitrogen
Carbon dioxide gas
Aerosol sprays
Tissue Processing 1. Fixation
2. Decalcification
3. Dehydration
4. Clearing
5. Infiltration (Impregnation)
6. Embedding
7. Trimming
8. Sectioning
9. Staining
10. Mounting
11. Labelling
Fixation
Fixation Preserve the morphologic and chemical integrity of the cell
Harden and protect the tissue from the trauma or further handling
Factors involved
Hydrogen Ion Concentration pH 6 and 8
Temperature Room temperature (40°C)
Electron Microscopy: 0-4°C
Formalin heated at 60°C: urgent biopsy
Formalin at 100°C: tuberculosis
Thickness Small:
1-2mm2 for Electron microscopy
2cm2 for light microscopy
Thin: 0.4cm for light microscopy
Note:
Brain: 10% buffered formalin for 2-3 weeks
Osmolality Hypertonic: 400-450 mOsm
Isotonic: 340 mOsm
Note:
Sucrose is added to osmium tetroxide fixative for electron microscopy
Glutaraldehyde fixatives – 300mOsm
Concentration 10% Formaldehyde
3% Glutaraldehyde
Duration of Fixation 2-6hrs
3hrs for electron microscopy
Considerations Speed
Penetration
Volume
Duration of fixation
Effects of fixative Harden soft and friable tissues
Make cells resistant to distortion and damage
Inhibit bacterial decomposition
Darla Ysavel V. Delos Reyes

Increase the optical differentiation
Act as mordants or accentuators
Reduce risk of infections
Microanatomical Fixative 10% Formol Saline
10% NBF
Heidenhain’s Susa
Formol Sublimate ( Formal corrosive)
Zenker’s solution
Zenker-formol (Helly’s)
Bouin’s
Brasil’s
Nuclear Fixative Flemming’s
Carnoy’s
Bouin’s
Newcomer’s
Heidenhain’s Susa
Cytoplasmic Fixative Flemming’s fluid with AA
Kelly’s
Formalin with post-chroming
Regaud’s (Muller’s)
Orth’s
Histochemical Fixative 10% Formol Saline
Absolute ethanol
Acetone
Newcomer’s
Lipid fixation Cryostat or frozen sections
Mercuric chloride and potassium dichromate
Note:
Phospholipids - Baker’s formol-calcium
Cholesterol - digitonin
Carbohydrate fixation Glycogen – alcoholic fixative
Human skin – Alcoholic formaldehyde
Protein fixation NBF – amino acid histochemistry
Glycogen Rossman’s fluid or cold absolute alcohol
Mixture of fixatives Karnovsky’s parafor-gluataraldehyde
Acrolein – surgical biopsies
Heat Fixation Secondary fixation
Post-chromatization: 2.5-3% potassium dichromate
Washing out:
1. Tap water – chromates from tissues; kelly’s, zenker’s and flemming’s
2. 50-70% alcohol – picric acid (bouin’s solution)
3. Alcoholic ioding – excessive mercuric fixatives
Factors that affect fixation
Retarded by: Size and thickness of the tissue specimen
Presence of mucus
Presence of fat
Presence of blood
Cold temperature
Enhanced by: Size and thickness of tissues
Agitation
Principles and Precautions in Handling and Fixation of Specimens in General
Autopsy materials Fixed ASAP
If not possible mortuary refrigerator (4’C) or arterial embalming
Surgical specimens Fixed ASAP
If not possible refrigerate
NSS during operation Autolysis may occur before fixation
Tissues are refrigerated Avoid slow freezing (ice crystal formation)
Repeated freezing & thawing destroy organelles, release enzymes
<5mm thick Size of tissues
Except lung edema: 1-2 cm thick
20:1 Ratio of fixative to tissue
Except osmium tetroxide (expensive) = ratio is 5-10:1
50-100:1 Ratio of fixative to tissue in prolonged fixation (ex. museum preparation)
Avoid drying of small tissue To prevent: place in a petri dish w/ moistened filter paper
biopsies

Hollow organs Stomach, intestines
Packed w/ co
tton soaked fixative or completely opened before being immersed in adequate fixing solution
Air-filled lungs Float on fixative
To prevent: cover w/ several layers of gauze to maintain it under surface
Human brains Suspended by a cord tied under the Circle of Willis to prevent flattening
Avoid Ringer’s lactate for washing out of blood intravascular perfusion
Fixation time: 2 weeks
Eyes Not dissected before fixation tissue collapse & wrinkling (escape of vitreous humor)
Inject formol-alcohol before immersing the organ in the fixative
Glycogen-containing tissues Do not use water
Glycogen is water-soluble
Hard tissues Cervix, uterus, fibroids, hyperkeratotic skin, fingernails
Wash in running water overnight immerse in 4% aqueous phenol for 1-3 days (Lendrum’s method)
Large specimens “bread loafing”
Improper Fixation
Problem Failure to arrest early cell autolysis
Removal of substances soluble in fixing agent
Presence of artifact pigments on tissue sections
Tissues are soft & feather-like in consistency
Loss/inactivation of enzymes needed for study
Shrinkage & swelling of cells & tissue structure
Tissue blocks are brittle & hard
Cause Failure to fix immediately (tissue was allowed to dry before fixing)
Insufficient fixative
Wrong choice of fixative
Incomplete washing of fixative
Incomplete fixation
Wrong choice of fixative
Over fixation
Prolonged fixation
Decalcification
Decalcification Calcium or lime salts are removed from tissues following fixation
37’C Impaired nuclear stain by Van Gieson’s stain
55’C Tissue Digestion (24-48 hrs)
Optimum temperature RT (18-30’C)
Time 24-48 hrs
HNO3 Most common
a. Perenyi’s = tissue softener & decalcifying agent
b. Phloroglucin-HNO3 = most rapid
- Disadvantage: Yellow color on tissue (neutralize w/ sodium thiosulfate)
5% Formic acid Both fixative & decalcifying agent
Best general decalcifying agent
For small pcs of bones & teeth
HCl (Von Ebner’s) For small pcs of bones & teeth
For surface decalcification (HCl)
EDTA For EM, IHC, & enzyme staining
Specimens Bones
Teeth
Tuberculous lungs
Removal of calcium Acids
Chelating agents
Ion exchange resins
Electrical ionization (electrophoresis): small bone fragments
Acid decalcifying agents Nitric acid
Hydrochloric acid
Formic acid
Trichloroacetic acid
Sulfurous acid
Chromic acid
Citric acid
Extent of decalcification
Physical or mechanical Touching or bending the tissue with fingers
x-ray or Radiologic Ability to detect smallest focus of calcium
Most ideal, sensitive and reliable

Chemical (Calcium oxalate) Routine
Tissue softeners Perenyi’s fluid
4% Aq. Phenol
Molliflex
2% HCl in 70% Alcohol
Dehydration
Dehydration Removing intercellular and extracellular water from tissue
Ratio 10:1
Commonly used dehydrating Alcohol – most common
agents Acetone – fixative and dehydrating
Dioxane 4 -cellosolve; dehydrating and clearing
Triethyl phosphate
tetrahydrofuran
Ethyl alcohol Routine dehydration of tissues
Methyl alcohol Toxic dehydrating agent; blood and tissue films and for smear preparation
Butyl alcohol Plant and animal micro-tehcniques
Acetone Urgent biopsies
Dioxane (Diethylene oxide) Readily miscible in water, melted paraffin, alcohol and xylol
Cellosolve Ethylene glycol monoethyl ether
Dehydrates rapidly
Note:
Combustible at 110-120°F
Toxic by inhalation, skin contact and ingestion
Triethyl phosphate Removes water readily
Minimum shrinkage
Tetrahydrofuran Demixing, clearing and dehydrating paraffin sections
Dehydration w/ dioxane Graupner’s method
Weiseberger’s method
Additives to dehydrating a. 4% phenol + 95% ETOH = softener
agents b. Anhydrous CuSO4 (Last ETOH bath)
- both dehydrating agent & indicator of H2O content of 100% ETOH
- (+) H2O = White Blue
incomplete dehydration 1. Anhydrous CuSO4 method
2. Xylene Milky
Clearing
Clearing Dehydrating agent is removed from the tissue and replaced with a substance that will dissolve the
wax
Common Clearing agents Xylene – most common
Toluene
Benzene
Chloroform
Cedarwood oil
Aniline oil
Clove oil
Carbon tetrachloride
Xylene Most commonly used
Toluene Substitute for xylene and benzene
Benzene Urgent biopsies and routine purposes
Chloroform Slower than xylene; recommended for routine work
Cedarwood oil CNS, smooth muscle and skin
Aniline oil Embryos, insects and very delicate specimens
Clove oil Minimum shrinkage of tissues
Carbon tetrachloride Dangerous to inhale on prolonged exposure
Methyl benzene and methyl Double-embedding techniques
salicylate
Impregnation and Embedding
Impregnation/Infiltration Clearing agent is completely removed giving a firm consistency to the specimen
Embedding/Casting/Blocking Precisely arranged position in mold containing a medium which is allowed to solidify
Types of media Paraffin wax
Celloidin
Gelatin
Plastic
Paraffin Most common and best embedding medium
Introduced by Bütschlii

Not recommended for fatty tissues
Note:
2 or more changes
Paraffin oven – 55-60°C
Routine - 56°C
Low MP = paraffin is soft
High MP = paraffin is hard
Paraffin filters Filtration at 2’C above the MP of wax
Ex. Green’s no. 904 (coarse filter paper)
Manual processing 4 changes of wax at 15 minutes
3 hrs to insure complete embedding
Automatic processing 2-3 changes of wax
Elliot Bench-type processor
Vacuum embedding Wax impregnation under negative atmospheric pressure
Hasten removal of air bubbles
Recommended for urgent biopsies and delicate tissues
Precautions Overheated paraffin (above 60°C) – shrinkage and hardening
Paraffin oven: Maintained at 2-5°C above melting point
Fresh wax: 2°C higher than its melting point
Substitutes Paraplast: 56-57°C
Embeddol: 56-58°C
Ester wax: 46-48°C
Water soluble waxes (Carbowax): 38-42°C or 45-56°C
Paraplast Highly purified paraffin and synthetic plastic polymers
Large dense tissue blocks: bone and brain
More elastic & resilient
Bones & brain
Note:
Embeddol
Bioloid
Tissue mat
Embeddol Synthetic wax
Less brittle and less compressible
Bioloid Semisynthetic wax for eyes
Tissue mat Product of paraffin containing rubber
Ester wax Harder than paraffin
Soluble in 95% ethyl alcohol
Note:
Cellosolve or xylene: clearing agents
3 – 4 changes of wax
Water soluble waxes Polyethylene glycol
Carbowax
Carbowax Doesn’t remove neutral fats and lipids
For enzyme histochemical studies
Easily dissolved in water
Celloidin impregnation
Celloidin (colloidon) Purified form of nitrocellulose
Specimens:
Large hollow cavities
Bones and teeth
Whole embryo
Wet celloidin Bones, teeth, large brain sections and whole organs
Equal parts of ether and alcohol for 12 – 24hrs
Dry celloidin Whole eye sections
Gilson’s mixture: equal parts of chloroform and cedarwood oil
Nitrocellulose method Forms harder tissue block
Thinner sections
Note:
Crack maybe prevented by adding plasticizers (oleum ricini or castor oil)
Gelatin impregnation When dehydration is to be avoided
Delicate specimens and frozen tissue sections
Prevents fragmentation of tough and friable tissues
Tissue: 2 – 3mm thick
Waterbath 6-10’C below the MP of wax (45-50’C)
Autotechnicon Fixes, dehydrates, clears & infiltrates tissues

Constant tissue agitation
Elliott Bench-Type processor
Wax bath: 3’C above the MP of wax
Embedding Melted paraffin at 5-10°C above its melting point
Cooled rapidly in a refrigerator at -5°C or immersed in cold water to solidify
Types:
Leuckhart’s embedding mold
Coumpounc embedding unit
Plastic embedding rings and base mold
Disposable embedding molds:
Peel away
Plastic ice trays
Paper boats
Orientation Tissue is arranged in precise positions
Leuckhart’s embedding mold Two L-shaped strips of heavy brass or metal arranged on a flat metal plate
Blocks produced are even with parallel sides
Recommended for routine use
Compound embedding unit Interlocking plates resting on a flat metal base
Embedding more specimens at a time
Plastic embedding rings and Stainless steel base mold fitted with a plastic embedding ring
base mold Tissue-tek
- warm plate to manage the impregnated specimen
- cold plate at -5°C for rapid solidification of the block
- white plastic cassette mold with detachable, perforated stainless steel hinge and snap-on lid
Peel away Disposable thin plastic embedding molds
perfect even block w/o trimming
Plastic ice trays Busy routine laboratories
ordinary refrigerators
Paper boats Embedding celloidin blocks
Rapid embedding of small or large volume of individual specimen
cheap & easy to make
Other embedding methods Celloidin or nitrocellulose method
Double-embedding method
Celloidin or nitrocellulose Hard tissues: bones and teeth
method Whole organs: eye
Note:
Bell jars: control the rate of evaporation of the solvent
Double – embedding method Tissues are first infiltrated with celloidin and subsequently embedded in paraffin mass
Firm tissues: brain
Recommended for small sections of celloidin blocks
Plastic (resin) embedding Light microscopic studies: Undecalcified bone
High resolution light microscopy: renal biopsies and bone marrow biopsies
Classification:
Epoxy
Polyester
acrylic
Epoxy Epoxy plastics:
Bisphenol A (Araldite) – slow
Glycerol (Epon) – lower viscosity
Cyclohexene dioxide ( Spurr) – obtained pure and infiltrate fastest
Disadvantage:
Hydrophobic
Compromise the result of IHC staining
*vinlycyclohexane dioxide - carcinogenic
Polyester Electron microscopy
Acrylic Polyglycol methacrylate (GMA) - Popular embedding medium
Methyl methacrylate (MMA) – undecalcified bone
Benzoyl peroxide – catalyst that decomposes to form phenyl radicals acting as active site for the
polymerization of acrylics
Recall Temperatures
Cold knife procedure Knife = -40 to -60’C
Tissue = 5 to -10’C
Environment = 0 to -10’C
Cryostat (Cold microtome) -18 to -20’C
Fixation Surgical specimen: room temp

HC & EM: 0-4’C
Freeze-drying Quenching: -160’C
Sublimation: -40’C
Algor mortis 7’F/hr (3.89’C/hr)
Formol calcium fixation 4’C
Impregnation Manual = 2-5’C above MP of wax (55-60’C)
Automated = 3’C above MP of wax
Vacuum = 2-4’C above MP of wax
Embedding 5-10’C above MP of wax
Flotation water bath 6-10’C below MP of wax (45-50’C)
Microtomy
Microtomy Trimmed and cut into uniformly thin slices or “sections”
Rocking
Rotary
Sliding
Freezing
Ultrathin
Block holder Tissue is held in position
Knife carrier and knife Actual cutting of tissue sections
Pawl, rachet feed wheel and Proper position
adjustment screws
Rocking Paldwell trefall (1881)
Serial sections of large blocks of paraffin embedded tissues
Slightly curved plane: 10 - 12µ thickness
*Cambridge rocking microtome
Rotary (minot) Minot (1885-1886)
Most common
Produce ribbons to serials sections
Sliding Adams (1789)
Base-sledge microtome: Hard tissue blocks
Standard sliding microtome: block remains stationary
Most dangerous type due to movable exposed knife
Freezing Queckett (1848)
Hollow and perforated
Cut undehydrated tissue in a frozen state
Rapid diagnosis
Demonstration of fat
Sensitive tissue constituents
Cryostat or cold -5°C to -30°C (ave. -20°C)
Capable of freezing fresh tissues within 2 – 3 minutes
Fluorescent antibody staining technique
Rapid preparation of urgent biopsies for intraoperative diagnosis
Ultrathin 0.5µ for electron microscopy
Specimen used is small, fixed in osmium tetroxide, and embedded in plastic
Microtome knives Plane-concave: 25mm
Biconcave knife: 120mm
Plane-wedge knife: 100m
Plane-concave One side of the knife is flat while the other is concave
Less concave: cutting celloidin-embedded tissue blocks
More concave: paraffin sections on base-sledge, rotary or rocking
Biconcave Paraffin embedded sections on a rotary microtome
Plane-wedge Frozen sections
Cutting extremely hard and tough specimens embedded in paraffin blocks
Base-sledge type or sliding microtome
Bevel angle 27° to 32°
Paraffin wax block 2-3µ thick
Honing Edge first, with a HEEL to TOE direction
10 – 20 strokes
*mechanical honing: 30 strokes
Precautions during honing Hone: 8”x3”
Coarse honing Removal of gross nicks on the knife edge
Honing proper Remove blelmishes, and grinding the cutting edge of the knife on a stone
Honing (hard sharpening) Carborundum: natural sharpening stone
Belgium yellow Manual sharpening; best result

Cutting edge has been rendered blunt or nicked
Arkansas More polishing effect
Fine carborundum For badly knicked knives
Lubrication Mineral oil and clove oil
Xylene
Liquid paraffin
Soapy water
Stropping Edge last; TOE to HEEL
“burr” formed during honing is removed
Polish abd sharpen the cutting edge
40 – 120 double strokes
Disposable blades 2-4µ thick
Magnetic knives: suitable for cryostat
Glass knives Electron microscopy
40x2.5cm plate glass strips
Diamond knives Resin block for electron microscopy
Paraffin sections
Sectioning Tissues are cut into uniformly thin slices or “sections”
Paraffin
Celloidin
Frozen
Trimming Expose the tissue surface in preparation for actual cutting
Paraffin section Paraffin embedded tissue blocks
Rocking or rotary
Sections: 4-6µ
Clearance angle: 0-15°
Water bath: 45-50°C; 6-10°C lower than the melting point of the wax
Paraffin oven: 2-5°C above the melting point regulated at 37°C and at 45-55°C
Celloidin section Celloidin embedded tissue
Sliding
Sections: 10-15µ
Wet medthod: kept moist with 70% alcohol during cutting
Frozen section Fixed and frozen with CO 2 or for fresh or fixed tissues frozen with the cryostat
Frozen sections
Frozen section Rapid diagnosis of tissue
Cold knife procedure
Cryostat procedure (cold microtome)
Applications 1. Rapid diagnosis
2. Enzymhistochemistry
3. Lipids and carbohydrates
4. IF and Immunocytochemical staining
5. Sliver stains in neuropathology
Cold knife procedure Tissue: 3 – 5mm thick
Knife: 40° to -60°C
Tissue: 5° to -10°C
Environment: 0° to -10°C
Cryostat Maintained at temperatures near -20°C
Optimum working temperature: -18° to -20°C
Methods of freezing Liquid nitrogen
Isopentane cooled by nitrogen
Carbon dioxide gas
Aerosol sprays
Liquid nitrogen Intra-operative procedures
Most rapid
-70°C  overcools urgent biopsy blocks  damage to both block and blade
Non-fatty unfixed tissue: -10°Cto -25°C
Note:
Vapor phase can be overcome by freezing the tissue in isopentane, O.C.T, or Freon 2.2 that has a
high thermal conductivity
Isopentane Liquid at room temperature
Temperature: -170°C
Affixed in a cork disc, aluminum foil or cryostat chuck
Aerosol sprays Freezing small pieces of tissue except muscle
Quick-freezing spray can of fluorinated hydrocarbons: freezing blocks rapidly
Mounting of tissue block Synthetic water-soluble glycols and resins

Tissue block: 2-4mm thick
Microtome knife and the tissue block are left in the cryostat for 15-30mins at -20°C
-5 to -15°C: brain, lymph nodes, liver, spleen, uterine curetting, soft cellular tumors
-15 to -25°C: non-fatty breast tissue, ovary, prostate, tongue and GIT
-35°C: fatty breast and omental tissue
Note:
Cryostat: -18 to -20°C
Mounting cryostat sections Cut only individual sections, and do not form ribbons, as in paraffin blocks
Special processing techniques Freeze-drying
Freeze-substitution
Freeze drying Quenching: rapid freezing of fresh tissue at -160°C
Dessication: subsequently removing ice water water molecules
Sublimation: -40°C
Time consuming and expensive; minimum tissue shrinkage
Note:
Tissue 2mm thick is plunged into isopentane or propane-isopentane mixture chilled to -160°C to -
180°C with liquid nitrogen  solidify the tissue in 2-3 seconds
High vacuum apparatus maintained at a temperature of -30°C to -40°C
Complete dessication  24-48hrs
Special studies (freeze-drying) 1. Immunocytochemistry
2. Fluorescent antibody studies of polypeptide and polypeptide hormones
3. Autoradiography
4. Microspectrofluorimetry of autofluorescent substances
5. Formaldehyde-induced fluorescence of biogenic amines
6. Scanning electron microscopy
Freeze substitution Rapid freezing of tissue
Fixed in Rossman’s formula or in 1% Acetone and dehydrated in absolute alcohol
Alternative to freeze Rapid freezing of tissue to -160°C in isopentane supercooled in liquid nitrogen
substitution Cryostat sections: 8 - 10µm  water-free acetone  cooled to -70°C for 12hrs
Principles of staining
Staining Applying dyes on the sections to see and study the architectural pattern of the tissue and physical
characteristics of the cell
Histologic
Histochemical (histochemistry
Immunohistochemical
Histologic staining Direct interaction with a dye or staining solution
Microanatomic stains, bacterial stains and specific tissue stains
Demonstrate general relationship of tissues and cells with differentiation of nucleus and cytoplasm
Histochemical staining Chemical reactions that will permit microscopic localization of a specific tissue substance
Perl’s Prussian blue – hemoglobin
Periodic acid Schiff – carbohydrates
IHC staining Immunologic and histochemical techniques
Polyclonal or monoclonal, fluorescent labeled or enzyme labeled antibodies
Methods of staining Direct staining: giving color to the sections by using aqueous or alcoholic dye solutions
Indirect staining: action of the dye is intensified
1. Mordant – link or bridge between the tissue and the dye (ex. Potassium alum with
hematoxylin in Ehrlich’s hematoxylin and iron in Weigert’s hematoxylin
2. Accentuator – accelerate or hastens the speed of the staining reaction (ex. KOH in
Loeffler’s methylene blue and phenol in carbol thionine and carbol fuschin)
Progressive staining Staining solution is applied for specific periods of time until desired intensity of coloring of the
different tissue elements is attained
Less favored
Regressive staining Overstained
Excess stain is removed or decolorized
Differentiation (decolorization) Selective removal of excess stain
*mordant can act as a differentiating agent
Metachromatic staining Use of specific dyes which differentiate particular substances by staining them with a color that is
different from the stain itselt
Basic dyes:
- Methyl violet or crystal violet
- Cresyl blue (reticulocytes)
- Safranin
- Bismarck brown
- Basic fuschin
- Methylene blue

- Thionine
- Toluidine blue
- Azure A, B, C
Counterstaining Provide contrast and background
Cytoplasmic stains
Nuclear stains
Cytoplasmic Red: eosin Y, eosin B, Phloxiine B
Yellow: Picric acid, Orange G, Rose Bengal
Green: Light green SF, Lissamine green
Nuclear Red: neutral red, Safranin 0, Carmine, Hematoxylin
Blue: methylene blue
Toluidine blue
Celestine blue
Metallic impregnation Tissue elements are demonstrated by colorless solution of metallic salts
Not absorbed by the tissues
Vital staining Selective staining of living cell constituents
Demonstrate cytoplasmic structures
Trypan blue: RES
Janus Green: mitochondria
Intravital staining Injecting the dye into any part of the animal body
Common dyes: lithium, carmine and India Ink
Supravital staining Stain living cells
Common dyes used Neutral red – best vital dye
Janus green – mitochondria
Trypan blue
Nile blue
Thionine
Toluidine blue
“sections to water” Alcohol is replaced with water before actual staining of section is performed
“sections to alcohol” Subjected to decreasing grades of alcohol
Materials for staining Coplin jar – slotted jar holding from 5 to 9 slides
Slotted staining dishes – holding from 5 to 19 slides places singly or in staggered fashion
Metal or glass staining racks or carriers – 10 to 30 slides upright
H&E staining technique Microanatomical stures of tissues
Methods:
1. Hematoxylin-eosin method
2. Thionine method
3. Polychrome methylene blue
4. Alcoholic pinacyanol methdod (supravital staining of mitochondira and color sensitization
in photography)
Regressive staining results Nuclei – blue to black
Karyosome – dark blue
Cytoplasm, proteins in edema fluid – pale pink
RBCs, eosinophilic granules, keratin – bright orange red
Basophil cytoplasm, plasma cells and osteoblast – purplish pink
Cartilage – pink or ligh blue to dark blue
Calcium and calcified bone – purplish blue
Decalcified bone matrix, collagen and osteoid – pink
Muscle fibers – deep pink
Heidenhain’s susa results Cells nuceli, cytoplasmic inclusions and muscle striations – black
Celestine blue results Cell nuclei – blue
Mallory’s phloxine methylene Sharp nuclear stain fixed with Zenker’s fluid
blue
Collodionization of sections Firmly attached by coating the slide with dilute (thin) celloidin sections
Staining of celloidin sections Cellulose nitrate (celloidin) is soluble in alcohol
Re-staining of old sections Immersed in xylene for 24hrs
Gently heated until the mounting medium begins to bubble
Note:
Sections is placed xylene for 30 minutes ro remove remaining balsam
0.5 potassium permanganate solution for 5 – 10 minutes
Immersed in 5% oxalic acid for 5 minutes
Tap water for 5 minutes
Broken slides Mounting to another clean xylene-moist slide with a drop of mounting media (Clarite or Permount)
Covered with a mixture of 6 parts butyl acetate and 1 part durofix  incubator (30 mins)
Stains and staining solutions

Natural dyes Hematoxylin
Cochineal dyes and its derivatives
Orcein
Saffron
Hematoxylin Mexican tree “hematoxylin campechianum”
Powerful nuclear and chromatin staining capacity
Hematin: active coloring agent
“ripening” – oxidation of hematoxylin
“artificial ripening” – chemical oxidation
“over-ripening” – excessive oxidation
Cochineal dyes Female cochineal bug (coccus cacti)
Treated with alum to produce “carmine”
Demonstration of glycogen
Orcein Vegetable dye extracted from lichens
Produce blue or violet colors
*litmus – obtained from lichens, treated with lime and soda and exposed to ammonia and air
Used as indicator
Synthetic dyes “coal tar dye”
Derived from hydrocarbon benzene
Known as “Aniline dyes”
Chromophores Produce visible colors
Chromogen Impart to the tissue is not permanent
Auxochrome Altering the shade of the dye
Groups:
Acid dyes
Basic dyes
Neutral dyes
Acid dye Picric acid – fix, differentiate or stain
Counterstain:
Basic cytoplasmic stains
Acid fuschin in van gieson’s connective tissue staining
Crystal violet for microscopic study of fungi
*trichloroacetic acid, picric acid, chromium-fixed tissues
Basic dye Combines with acid radicals
Methylene blue – bacterial staining
*tissue fixed with mercuric chloride and formaldehyde
Neutral dye Combining aqueous solutions of acid and basic dye
Examples:
Romanowsky dye – hematology
Giemsa and Irishman’s stain – leukocyte differentiation
Chief solvents used for stain Water – distilled
Alcohol – ethyl alcohol (various concentrations); methyl alcohol (absolute)
Aniline water – 10mL to every ½ to 1L of hot distilled water
Phenol – aq. Solution of 0.5 – 5%
Dye modifiers Attached on benzene ring
a. Ethyl group
b. Methyl group
c. Sulphonic acid
Van der Waals forces Alum hematoxylin
Sudanophilia Tissue stained w/ fat or oil-soluble dyes
Hematoxylin Hematoxylin capechianum/ Hematoxylon campechianum
Nuclear/basic/1’ stain
Waldeyer: 1st to use hematoxylin
Hematoxylin ---(Ripening)---> Hematein (active coloring substance)
Lake Tissue-Mordant-Dye complex
Oxidizing agents H2O2
HgO2 = Harris’
K2MnO4
Na perborate
Na iodate = Mayer’s, Ehrlich’s, Gill’s
Alum hematoxylin Routine H & E = Red
Mordant: K Alum
“MEGDH”
Mayer’s = Na iodate (ripening agent)
Ehrlich’s = Na iodate (ripening agent)

Gill’s
Delafield’s
Harris’ = HgO2 (ripening agent)
Iron hematoxylin Mordant = oxidizing/ripening agent = Iron
a. Weigert’s
- Mordant: FeCl3
- Weigert’s + Van Gieson’s = CT & E. histolytica
b. Heidenhain’s
- Mordant: Ferric ammonium sulfate
Tungsten hematoxylin a. Mallory’s PTAH
- Mordant = sunlight/K+
- Stain fibrin
Copper hematoxylin Spermatogenesis
Eosin (Eosin Y) Cytoplasmic/acidic/2’ stain
Counterstain
a. Eosin Y (Yellowish) = most commonly used
b. Eosin B (Bluish) = deep red
c. Ethyl eosin/Eosin S/Eosin alcohol soluble
H & E staining steps 1. Xylol (2) = deparaffinization
2. Descending grade of alcohol = rehydration
3. H2O
4. Remove fixative artifact pigments after rehydration & before staining
5. Stain: Nucleus = light blue
6. H2O
7. Acid alcohol (differentiator): Nucleus = light blue
8. Ammonia water (blueing agent): Nucleus = blue
- NH4OH
- LiCO3
- Scott’s tap H2O
9. Wash
10. Stain: Eosin Y
11. Ascending grade of alcohol = dehydration
12. Xylene = dealcoholization/clearing
13. Mount & label
Nuclei: blue to blue black
Cytoplasm: pale pink
Pap Smear staining Hematoxylin = nuclear stain
OG-6 = cytoplasmic stain (mature superficial cells)
EA (Eosin Azure) = cytoplasmic stain (immature cells: parabasal/intermediate)
EA 65 = for body fluids
EA 36/50 = for gynecologic smears
Other Stains
Benzidine Hgb
Acridine orange RNA (red fluorescence)
DNA (green fluorescence)
Gentian violet Crystal violet + methyl violet + dextrin
Congo red Elastic tissue, amyloid, myelin
Iodine Oldest stain
Malachite green Ascaris eggs
Janus green Mitochondria (intravital stain)
Night blue Substitute for carbolfuchsin
Victoria blue Neuroglia
Lysochromes Oil soluble dyes
a. SBB = Black (most sensitive)
b. Sudan III = orange
c. Sudan IV (Scharlach R) = red
Methods of Staining
According to the presence of a a. Direct staining = w/o mordant
mordant b. Indirect staining = w/ mordant: serves as a link/bridge between the tissue & the dye
According to the presence of a. Progressive = w/o differentiator/decolorizer
differentiator b. Regressive = w/ differentiator/decolorizer
*1’ stain = acidic (decolorizer: basic)
*2’ stain = basic (decolorizer: acidic)
According to the resultant color a. Orthochromatic = “ortho”: correct/same | same color = dye & tissue
b. Metachromatic = “meta”: after/change | different color = dye & tissue

Vital staining Selective staining of living cells
a. Intravital stain = injection of dye animal body
- Ex. Lithium, Carmine, India ink
b. Supravital stain = staining of cells immediately after removal from the animal body. Examples
are:
- Neutral red = Best vital dye
- Janus green = mitochondria
- Trypan blue
- Nile blue
- Thionine
- Toluidine blue
Neurons Functional cells of the CNS
Nerve fibers:
a. Dendrites (Greek: “Tree”) = conduct impulses to the cell body
b. Axons = conduct impulses away from the cell body
Criteria for the diagnosis of Marked progesterone effect
normal pregnancy At least 50% of intermediate cells in clusters
At least some typical pregnancy cells present
<30% of matured superficial cells
Doderlein-filled dirty BG
Staining solutions used in a. Hematoxylin = dark purple to black
Pap’s staining method b. OG-6 = orange w/ a hint of green
(Macroscopic) c. EA 36/50 = olive green w/ a hint of brown & red
EA 36/50 Components:
a. Eosin Y
b. Bismarck brown
c. Light green SF
# in EA designates the proportion of SF
OG-6 Affinity:
Matured superficial cells
Keratinizing malignant cells
Cytoplasmic:
Bright orange to yellow orange
EA 36/50 Affinity:
Immature vaginal cells (parabasal, intermediate)
Cytoplasm color:
Transparent blue to gray to brown hue
Adhesives and Mounting media
Fixing slides 37°C incubator overnight
56° to 60°C wax oven for 2hrs
45° to 55°C hot plate for 45mins
*CNS tissue/Brain - 37°C for 24hrs
Adhesives Mayer’s egg albumin
Dried albumin
1% gelatin
Gelatin – formaldehyde mixture
Starch paste
Plasma
Poly-L-Lysine
APES (3-aminopropylthriethoxysilane)
Mayer’s egg albumin Most commonly used
Egg white
Glycerin
Note: transferred from 95% alcohol bath dried and stored in 70% alcohol
Dried albumin Dried albumin
Sodium chloride
1% gelatin Gelatin
Distilled water
Glycerol
Phenol crystals
Gelatin – formaldehyde mixture 1% gelatin
2% formaldehyde
Starch paste Powdered starch
Distilled water
N/I hydrochloric acid

Plasma Outdated blood stored in blood banks, dispense into sterile tubes
Poly-L-Lysine 0.1% solution diluted 1:10 with distilled water
APES (3- Useful in cytology
aminopropylthriethoxysilane) For cytospin preparation of proteinaceous or bloody material
Mounting medium Syrupy fluid applied between the section and the coverslip after staining
Incubated at 37°C for 12 – 24hrs after mounting
*setting may be hastened in a hot oven at 50°C for 2hrs
Groups:
Aqueous media
Resinous media
Good mounting medium 1. Avoid distortion of the image
2. Freely miscible with xylene and toluene
3. Not dry quickly
4. Not crack or produce artefactual granularity
5. Not dissolve out or fade
6. Not cause shrinkage or distortion
7. Not leach out any stain
8. Not change color in pH
9. Set hard
Aqueous media Composition:
Mount water-miscible preparations
Gelatin, glycerin jelly or gum Arabic – solidify the medium
Glycerol – prevent cracking and drying
Sugar – increase the refractive index
Preservative solutions
Water Low refractive index
Temporary mounting
Glycerin Refractive index 1.46
Greater visibility if slightly diluted with water
*Glycerin jelly (Kaiser’s 1880) Refractive index 1.47
Glycerin jell – standard mounting media
Formula:
Gelatin
Glycerol
Distilled water
Phenol crystals
Farrant’s medium Refractive index 1.43
Formula:
Gum Arabic
Distilled water
Glycerol
Sodium merthiolate
Note:
Arsenic trioxide – substitute of sodium merthiolate
Raise refractive index 1.44
Apathy’s medium Refractive index 1.52
Used for methylene blue-stained nerve preparations
Formula:
Pure gum Arabic (crystals)
Pure cane sugar or sucrose
Distilled water
Thymol crystals
Bruns’s fluid Recommended for mounting frozen sections from water
Formula:
Glucose
Glycerine
Spirits of camphor
Distilled water
Resinous mounting media Used for preparations that have been dehydrated and cleared wit xylene or toluene
Canada balsam
DPX
XAM
Clarite
Canada balsam Refractive index 1.524
Canadian tree “Abus Balsamea”

Recommended for whole mounts and for thick sections
Note:
Dissolved in 37°C incubator or 58°C paraffin oven
Miscible with xylene
Benzene – substituted for xylene
DPX Refractive index 1.532
Recommended for small tissue secions
XAM Refractive index 1.52
Synthetic mixture in xylene
Pale yellow or colorless solution
Sections are quickly mounted from xylene
Clarite Refractive index 1.544
Soluble in xylene
Preferred over DPX
Other synthetic mouting media Permount (fisher scientific)
HSR (harleco synthetic resin)
Clearmount (Gurr)
Ringing Process of sealing the margins of the cover-slip
Prevent escape of fluid or semi-fluid mounts
*kronig cement – 2 parts paraffin wax mixed with 4-9 parts powdered colophonium resin
Cellulose adhesive Durofix
Immunohistochemical Techniques
Immunohistochemistry Detect organisms in cytologic preparations
Selective cellular epitopes or antigens in frozen or paraffin-embedded tissues
Antigen-antibody interactions
IgG Most commonly used
Epitope Structural part of the antigen that reacts with an antibody
Polyclonal antibodies Immunizing an animal with an immunogen that contains antigen of interest
Rabbit, goat, pig, horse, guinea pig
Non specific staining
Monoclonal antibodies Individual clones of plasma cells
mice
Preparing tissue for IHC 1. Proteolytic enzyme digestion
2. Microwave antigen retrieval
3. Microwave and trypsin antigen retrieval
4. Pressure cooker antigen retrieval
Proteolytic enzyme digestion Demonstrate heavy chain immunoglobulins, complement and specific antigen
Destroy exogenous peroxidase Treated with 0.5% methanolic hydrogen peroxide for 10-15 minutes
Trypsin method 0.1% trypsin in 0.1% calcium chloride in distilled water
Note:
Cold running water: terminate enzyme digestion
Protease method 0.05 – 0.1% protease in distilled water
Faster rate of enzyme digestion
Microwave antigen retrieval Boiling of formalin-fixed deparaffinized sections
1. 0.01 M-citrate buffer: pH 6.0
2. EDTA: pH 8.0
3. Tris EDTA: pH 9.9-10
Proliferation markers Ki67
MIB-1
Hormone receptors ER
PR
Growth factor receptors HER-2/neu
Optimal length of exposure 10-60 minutes
Vectabond Strong adhesive
Pressure cooking antigen Less time consuming
retrieval More consistent recovery of many antigens
Subjected to “hot spots” and “cold spots”
Inconsistent antigen recovery
Epithelial tumor markers Keratin

EMA (Epithelial membrane antigen)
CEA (Carcinoembryonic antigen)
TTF-1 (Thyroid transcription factor-1)
PSA (Prostate specific antigen)
Keratin Carcinoma – epithelial tumors
Mesotheliomas – non-epithelial tumors
CK7 (Cytokeratin-7): lung, breast, uterus and ovaries (serous tumors); negative for CK20
CK20 (Cytokeratin-20): colon and stomach; negative for CK7
CK7 & CK20 positive: Transitional cell carcinoma of the bladder and mucinous ovarian tumor
CK7 & Ck20 negative: renal cell carcinomas, hepatocellular carcinomas, prostatic
adenocarcinomas, thyroid carcinomas and squamous cell carcinomas (skin, lung and espophagus)
EMA (Epithelial membrane Site of tumor
antigen) Positive: adenocarcinoma of the breast, lung and kidneys
Nonreactive: hepatocellular carcinomas or embryonal carcinomas
Negative: non-epithelial tumors (sarcomas, lymphomas, melanoma
Other tumors: meningiomas, mesotheliomas, anaplastic large cell lymphomas
CEA (Carcinoembryonic Oncofetal antigen
antigen) Carcinoma: GIT, pancreas, lung, breast, ovary, uterus, cervix
CEA +: adenocarcinoma
CEA -: mesothelioma
Nonreactive: prostate, thyroid and renal carcinomas
TTF-1 (Thyroid transcription Lung adenocarcinomas from mesotheliomas
factor-1) + : thyroid, lung and neuroendocrine tumors ( medullary thyroid carcinomas, carcinoid tumors and
small cell tumors of the lungs)
PSA (Prostate specific antigen) Prostatic adenocarcinoma
+ : pancreatic and salivary gland tumors
Intermediate Filament markers Actin
Vimentin
Desmin
GFAP (Glial Fibrillary acidic protein)
NF (Neurofilament)
S100 protein
Actin Sensitive marker for muscle differentiation
Identify tumors derived from smooth, skeletal and cardiac muscle
Vimentin 57kD IF
Present: normal mesenchymal cells and their neoplastic counterparts (sarcoma, melanoma,
lymphoma, leukemia, seminoma and some neural tumors)
+ : Melanoma and Schwannomas
Desmin 53kD IF
Specific for myogenic tumors:
Leiomyoma: smooth muscle
Rhabdomyosarcoma: skeletal muscle
Mixed tumors: carcinosarcomas or malignant mixed mesodermal tumors
GFAP (Glial Fibrillary acidic 51kD IF
protein) Expressed by astrocytes  astrocytoma
Certain cases: Ependymomas, oligodedrogliomas, medulloblastomas
Negative: meningiomas, metastatic carcinomas and lymphomas
NF (Neurofilament) + : Neuronal or neuroendocrine differentiation
Tumors: neuroblastoma, ganglioneuromas, neuromas, chemodectomas, pheochromocytoma
S100 protein Low molecular weight calcium-binding protein
Expressed in:
CNS glial cells
Schwann cells
Melanocytes
Histiocytes
Chondrocytes
Skeletal and cardiac muscle
Myeoepithelial cells
Epithelial cells of the breast, salivary and sweat gland epithelium
Neuroendocrine markers NSE (Neuron-specific enolase)
Chromogranin
Synaptophysin
NSE (Neuron-specific enolase) Isoenzyme marker
Neural or neuroendocrine differentiation
Chromogranin Neural secretory granules

Neuroendocrine differentiation
Keratin & chromogranin + : neuroendocrine carcinoma
Keratin - & chromogranin + : paraganglioma
Synaptophysin 38kD transmembrane protein
Presynaptic vesicles of neuron
Germ cell tumor markers HCG (Human Chorionic Gonadotropin)
AFP (Alpha-fetoprotein)
PLAP ( Placenta-like alkaline phosphatase)
HCG (Human Chorionic Placental syncytiotrophoblasts
Gonadotropin) Marker for choriocarcimoa
AFP (Alpha-fetoprotein) Normal liver hepatocytes
Marker for endodermal sinus tumors: yolk sac differentiation
Positive: embryonal carcinomas and teratomas; hepatocellular carcinomas
PLAP ( Placenta-like alkaline Placental syncytiotrophoblasts
phosphatase) Marker for germ cell tumors  germinomas
Positive: embryonal carcinomas, chorio-carcinomas and endoderma sinus tumors
NOTE: positive in majority of seminomas
Mesenchymal tumor markers Myogenic tumors
Fibrohistiocytic tumors
Vascular tumors
Melanomas
Lymphomas
Myogenic tumors Skeletal muscle
Positive: muscle-specific actin and desmin
Muscke markers: myo-D1, myoglobin and myogenin
Fibrohistiocytic tumors Malignant fibrohistiocytic sarcomas
Histiocytic markers: CD68, FAM 56
Enzymes: alpha-1-antitrypsin and alpha-1-antichymotrypsin
Vascular tumors Angiosarcomas
Endothelial markers: Factor VII-related antigen, CD31 and Ulex Europaeus I (UEA)
Melanomas Melanocytes: derived from neural crest
Reactive: S100 protein
Intensity: inversely proportional to the melanin content
Melanosome (HMB-45): highly sensitive and specific marker
Melan-A (MART-1): melanoma-specific antigen
Lymphomas Best screening marker: LCA (Leukocyte common antigen) aka as CD45
Markers:
T-cells – CD3, CD4, CD8
B-cells – CD19, CD20, CD23
Reed-sternberg cells – CD15 and CD30
Cell proliferation markers Ki-67 (MIB-1) and PCNA (Proliferating cell nuclear antigen)
Assess proliferation of tumor cells
Cancer-associated genes Related to abnormalities of structure or activity of proto-oncogenes and/or mutation of tumor
suppression genes
Cellular oncogenes: activated in cancer  breast
p53
c-erb B-2
c-myc
ras
Infectious agent markers HAV
HbsAg & HbcAg
HCV
HPV
CMV
EBV
Toxoplasma
P. carinii
H. pylori
Cryptosporidium
C. neoformans
Histoplasma
E. histolytica
Mycobacteria
Enzyme labeling Horseradish peroxidase
Diaminobenzidine (DAB)  insoluble dark brown end product

Optimal incubation time: 30-60 mins
Direct technique Fluorochrome or horseradish peroxidase
Advantage – simple and quick
Note:
EPOS (enhanced polymer one-step staining)
- “baclbone” or “spine moleculre”
- Reduced number of incubation steps
- 70 enzyne, 10 antibody
- Suitable for frozen section immunohistochemistry
Indirect technique Application of the unconjugated antibody followed by a labeled antibody directed against the first
antibody
Horseradish peroxidase: most common
Two-step indirect techniques Unconjugated primary antibody first binds to the antigen
Three-step indirect techniques Second enzyme-conjugated antibody is added
Soluble enzyme immune Unlabeled antibody tech.
complex tech. Preformed soluble enzyme-anti-enzyme immune complex
Peroxidase-antiperoxidase Soluble peroxidase-antiperoxidase complex is bound to unconjugated primary antibody
(PAP) tech. “bridging”  binds to both the primary antibody and the rabbit PAP complex
Recommended for use on blood and bone marrow smears
Note:
horseradish perpxodase – most widely used enzyme for labeling
diaminobenzidine – insoluble dark brown reaction
Lack of interference from endogenous peroxidase activity
Endogenous alkaline phosphatase activity is usually blocked by adding levamisole to the substrate
solution
Avidin-Biotin complex (ABC) Derived from egg white
techniques Variants: peroxidase, and alkaline phosphatase
*streptavidin – culture broth of the bacterium streptomyces avididin
Labeled streptavidin Avidin 4 to 8x more sensitive
Biotin techniques (LSAB)
Direct immunofluorescence For solid tissue biopsies
technique Performed on then (2 to 5µm) cryostat sections of fresh unfixed material
Tissue is reacted directly with a fluorescein-conjugated antibody specific for the material being
south within the tissue
Results:
Fluorescein: apple-green fluorescence
Rhodamine: orange-red fluorescence
Indirect immunofluorescence Used for detection of autoantibodies:
Anti-nuclear antibody (ANA)
Antimitochondrial antibody (AMA)
Liver-kidney microsomal antibody
Frozen section Identify antigens in fresh frozen sections
immunocytochemistry Acetone – fixative to preserve the antigen  destroy harmful infective agents
Note:
Cold acetone – best preserved (5mins at 4°C)
Tris buffered saline (TBS) – prevent one reagent from contaminating another
Frozen section Used to detect antibodies
immunofluorescence Diagnosis of glomerular disease in frozen sections of renal biopsies
Applied to skin biopsies of patients with systemic lupus and vasculitis  examine the pattern of
deposition of immunoglobulins
Direct immunoperoxidase Solutions
method for frozen sections Tris-buffered saline wash:
- Distilled water
- Sodium chloride
- TRIS (hydroxymethyl methylamine)
- M HCl
Tris solution
Substrate
- DAB (diaminobenzidine tetrahydrochloride)
- Tris-HCl buffer
- 0.2M Tris
- 0.1M HCl
- Distilled water
DAB solution
- DAB

- Tris-HCl buffer (pH 7.6)
- H2O 2
Paraffin wax section Standard method among most laboratories
immunoperoxidase technique In combination with frozen section processing for immunofluorescence of renal and skin biopsies
Note:
Best achieved by using trypsin, chymotrypsin or protease
Improve cytological activity – formol sublimate and B5
In-situ hybridization Thermodynamically stable
Interaction of a probe with the target nucleic acid
“hybridization reaction” – process of searching a sample for specific nuclei acid sequence
Cytology
Diagnostic cytology Microscopic examination of cells from different body sites
Exfoliative cytology and fine needle aspiration (FNA)
Exfoliative cytology Des-quamated from epithelial cells
*spontaneous exfoliation – constant growth and replacement with new cells
Recommended for:
1. Malignant cells  staging cancer
2. Precancerous cervical lesions
3. Female hormonal status
4. Genetic sex
5. Infectious agents
Specimen Cervicovaginal smear (Pap smear)
Nipple discharge
Gastric or bronchial secretions
Pleural and peritoneal fluids
Sputum
Urine sediment
CSF
Fixation 50% alcohol for all types of effusions
Common:
Equal parts of 95% ethyl alcohol and ether
95% ethyl alcohol
*can be replaced by Saccomano preservative (50% alcohol and carbowax)
Note:
Time : 10 – 15mins.
Cytologic collection and Endocervical brush – endocervical cana;
preparation Vaginal scrape – hysterectomy
Lateral vaginal scrape – hormonal evaluation
Four quadrant vaginal scrape – vaginal adenosis
Vulvar scrape – herpetic lesions or carcinoma
Papanicolau method (Pap Detect human uterine and cervical cancers
smear) Advantages:
1. Transparent blue – cytoplasm
2. Excellent nuclear stain
3. Good differential coloring
4. Comparing cellular appearances
Modified Pap staining Provides optimum nuclear detail information
procedure Examination of Pap smears, non-gynecological and FNA cytologic specimens
Gynecological specimen Provide a standardized method for staining gynecologic specimens
staining procedure Equipment:Shandon varistain 24-3
Cervico-vaginal smears Mature superficial cells
Intermediate cells
Parabasal cells
Navicular cells
Pregnancy cells
Endometrial cells
Endocervical glandular celles
Mature superficial cells 45-50µm
Polyglonal squamous cells
Pale, pink-staining cytoplasm and dark pyknotic nculei
Note:
True acidophilia – superficial vaginal cells under estrogen influence
Pseudo-acidophilia – drying of smears
Intermediate cells Polyhedral or elongated cells
Basophilic vacuolated cytoplasm

Parabasal cells 15-30µm
Round to oval
Fried eggs w/ sunny side up
Small dense basophilic cytoplasm
Large vesicular nucleus
Found from:
Two weeks of age to puberty
After childbirth
With abortions
After menopause
Navicular cells Boat-shaped intermediate cells
Combined estrogen progesterone effect
Found in:
Latter half of the menstrual cycle
During pregnancy
Menopause
Pregnancy cells Round, oval or boat-shaped cells
Translucent basophilic cytoplasm
Glycogen accumulation
Endometrial cells Small cells and slightly cylindrical
Less basophilic cytoplasm
Found during:
1-10 days after menstruation
Endocervical glandular cells Large groups or small sheets
Cytoplasm: pale blue/gray (finely vacuolated)
Honeycomb appearance
Respiratory tract specimen Exclude the possibility of malignancy or infectious agents (px with AIDS)
Sputum 3 consecutive morning sputum specimens
*wide-mouthed jar containing Saccomano fluid (50% ethyl alcohol and 2% carbowax)
Note:
Induced sputum – inhalation of an aerosol solution for 20mins to produce deep cough
“down deep” – ensure that the specimen is a true representative of the sputum
Bronchoscopy specimen Bronchial brushing
Bronchial washing
Bronchial aspirate
Bronchial brushing 2 labeled slides by pull technique
Fixed by a spray fixative or immediately immersed in 95% alcohol
Bronchial washing Freshly collected in the bronchoscopy collection container
Bronchial aspirate Aspiration into a glass suction or by washing the bronchi with 1-2cc. of saline
Cell suspensions Optimum amount: 20 to 30mL
Note:
Cells can remain viable for up to 4 days if the specimen is kept refrigerated at 4°C
Cytospin slide 1000 RPM for 1min
Slide previously coated with a thin layer of egg albumin
Fixed in 95% ethyl alcohol
Transbrochial FNA Two-slide pull method and fixed immediately
Staining Harris hematoxylin – nuclear stain
OG-6 and EA 50 – green, blue and pink hues
Gastrointestinal specimen Malignant tumors
Type:
Gastric lavage
Gastric brush
FNA (submucosal lesions)
Transudates Peritoneal, pleural and pericardial fluids
*jelly – like clots: prevented by adding 300 units of Heparin for every 100mL of aspirate
Breast secretion Hormonal imbalance
Bloody smear – intraductal papilloma
*drop slide in a bottle of 95% isopropanol or use spray fixative
Urinary tract specimen Malignancy
Types:
Voided urine
Catheterized specimen
Washings from bladder or renal pelvis
Urinary sediment 50mL
Body cavity effusion History of cancer

(+) effusion: malignancy
Type:
Pleural fluid
Ascitic/abdominal fluid
Peritoneal washing
Pericardial
CSF
Fine needle aspiration (FNA) Diagnosis of cancer
cytology
Palpable masses Needle: 22-23 gauge
Breast
Thyroid
Soft tissue
Lymph nodes
Non-palpable masses Fluoroscopy
Fixed with 95% alcohol
Slide preparation: 4 slides (side pull techniques)
Rapid tissue processing
Microwave technique Fixation
Processing
Antigen retrieval
Staining
immunohistochemistry
Microwave fixation Non-formaldehyde
Alcohol based
*permit recovery of DNA, RNA, and proteins for molecular analyses
Note:
UMFIX – mixture of methanol and polyethylene glycol
Microwave processing Using microwave energy to speed up the diffusion of the alcohols
Boiling pt - 82°C for ethyl alcohol; 78°C for isopropanol
Microwave antigen retrieval Optimum temperature:
75°C to 95°C – metallic stain
55°C to 60°C – non-metallic stains
Microwave Microwave irradiation
immunohistochemistry Temperature: 37°C for 2-3mins followed by room temperature incubation
Faults During Tissue Processing
Brittle/hard tissue
Clearing agent Milky Incomplete dehydration
tissue smells of clearing agent Insufficient impregnation
Tissue is opaque Insufficient clearing
Tissue shrinks away from wax Insufficient dehydration
Incomplete clearing & impregnation
Tissue is soft Incomplete fixation
Air holes on tissue Incomplete impregnation
Wax appears crystalline Contaminated wax
Block not cooled rapidly enough
block is moist & crumbles Insufficient paraffin impregnation
Sections fail to form ribbons Surface & edges of block not parallel
Wax too hard
Knife tilted too much
Thick sections
Dull knife
Sections roll up on cutting; Blunt knife
adhere & get broken against Dirty knife edge
the knife edge
Ribbon is curved, crooked, or Irregular knife edge
uneven Edges of block are not parallel
Knife not parallel to the block
Impure paraffin
Sections are compressed, Blunt/dull knife
wrinkled or jammed Block is warm & soft
Knife edge coated w/ paraffin
Thin sections
Microtome screw is loose
Tilt: vertical

Sections are squashed Bevel of knife is lost
Incorrect sharpening
Hole in section Bubble/dirt
Hard spot in the tissue (Ca2+)
Sections of unequal thickness Screw/holder is loose
Large & hard blocks
Sections adhere to knife or Static electricity
other parts of the microtome Dirty knife edge
Dull knife edge
Ribbon is split Nicks/damage on knife
Dirty embedding
Dirty knife
Chatters are seen Knife vibrates (hard tissue)
Section: sometimes thin & Blunt knife
thick Knife/block holder is loose
Frozen tissue crumbles & Inadequate freezing
comes off when the block
holder when cut
Frozen tissue chips into Tissue is frozen too hard
fragments

Inflammation
Rubor Redness
Blood flow injury
Calor Heat
Blood and heat
Tumor Swelling
Capillary permeability
Fluid extravasation
Dolor Pain
Functio laesa Loss of function
Destruction of functional units
Acute inflammation Vascular & exudative
PMN---(Tissue)---> Microphages
Subchronic inflammation Intergrade between acute & chronic
Chronic inflammation Vascular & fibroblastic
Monocyte---(Tissue)---> Macrophages
Inflammation according to Characterisics of Exudate
Serous inflammation Serum/secretions from serosal mesothelial cells (3P’s)
Pulmonary TB
Fibrinous inflammation Fibrinogen
Diphtheria, rheumatoid pericarditis
Early stage of pneumonia
Catarrhal inflammation Hypersecretion of mucosa
Hemorrhagic inflammation Blood + exudates
Bacterial infections & other infections
Suppurative/purulent Pus: creamy filled fluid component of PMNs and necrotic tissue debris
inflammation
Retrogressive Changes = Organ/Tissue smaller than normal

Developmental defects Aplasia Incomplete/defective development of a tissue/organ
Ex. amastia (breast aplasia)
Atresia Failure to form an opening
Hypoplasia Failure of an organ to reach its matured size
Agenesia Complete non-appearance of an organ
Atrophy Physiologic atrophy Natural
Thymus, brain, sex organs
Pathologic atrophy Vascular atrophy
Pressure atrophy
Atrophy of disuse
Exhaustion atrophy
Endocrine atrophy
Brown atrophy Lipofuscin
Progressive Changes = Organ/Tissue larger than normal
Hypertrophy Increased tissue size due to increased cell size
Physiologic: ásize of uterus
Pathologic: Systemic hypertension
Hyperplasia Increased tissue size due to increased cell number
Physiologic: Glandular proliferation of the female breast, ásize of uterus (preg.)
Pathologic: Skin warts due to HPV
Compensatory hyperplasia Ex. Enlargement of one kidney
Pathologic hyperplasia Ex. Endometrial hyperplasia
Congenital hypertrophy Phenytoin-induced
Degenerative Changes = Metaplasia Reversible
Tissues have abnormalities One adult cell type ↔ Another adult cell type
Dysplasia Reversible
One type of adult cell ↔ Changes in structural components
Anaplasia/ Dedifferentiation Irreversible
Criterion toward malignancy
Adult cell More primitive cells (release tumor markers)
Neoplasia/tumor Continuous abnormal proliferation of cells w/o control (no purpose/function)
Ex. Leukemia
Tumors
Parts of a tumor 1. Parenchyma = active elements (tumor cells)
2. Stroma = CT framework
Types of tumor 1. Capacity to produce death:
- Benign (Ex. mole)
- Malignant
2. Histologic characteristics:
- Medullary = cells (parenchyma) > supporting tissues (stroma)
- Scirrhous = supporting tissues (stroma) > cells (parenchyma)
Benign “-oma”
Malignant “SaMe CarE”
“-sarcoma” = mesenchymal/CT
“-carcinoma” = epithelial tissues
Leukemia Malignant
Lymphoma
Squamous cell papilloma Benign
Squamous cell carcinoma Malignant
Hepatoma/ hepatocarcinoma Malignant
Melanoma/ melanocarcinoma Malignant
Ectopic pregnancy Fallopian tube pregnancy
Grading Aggressiveness/level of malignancy
Differentiated cells = resemble normal cells
Undifferentiated cells = younger cells
Staging Size, extent of spread to lymph nodes, +/- metastases
UICC TNM classification
AJCS Grading + staging
TNM System
TNM system Applicable to all forms of neoplasia
T 1’ tumor
#: denotes the size of tumor and its local extent
Tis = carcinoma in situ
Ta = non-invasive
Tx = cannot be evaluated
T0 = free of tumor
T1 = lesion <2 cm (T1a = <0.5 cm | T1b = <1 cm | T1c = <2 cm)
T2 = lesion 2-5 cm (invasion in muscle)
T3 = skin and/or chest wall involved by invasion (T3a = deep muscle | T3b = through organ)
T4 = tumor invasion/fixation (T4a = adjacent organ | T4b = fixation to bladder or colonic wall, in
breast, edema)
N Regional lymph node involvement
High # denotes increasing extent of involvement
Nx = not evaluable
N0 = no axillary nodes involved
N1 = 1 mobile regional (axillary) node involved
N2 = multiple, mobile regional nodes involved
N3 = fixed regional lymph node involved
N4 = beyond regional lymph node involvement
M Metastasis
M0 = no evidence of metastases
M1 = distant metastases are present
Mx = distant metastases not evaluable
Teratomas Compound tumors
Greek: Monstrous tumors
May contain hair, teeth, bones
w/ heartbeat
Cellular Death Apoptosis Programmed cell death (cellular suicide)
Necrobiosis Physiologic cell death
Ex. normal sloughing off of skin cells
Necrosis Pathologic cell death
Types of Necrosis
Coagulation necrosis Most common
Tombstone formation
“MyLKS”
Myocardium
Lungs
Kidneys
Spleen
Liquefaction/colliquative Pus formation
necrosis Brain & spinal cord

Caseous/caseation necrosis Yellow, cheesy, crumbly material
TB, syphilis, tularemia, lymphogranuloma inguinale
Gangrenous necrosis Sulfide gas production
a. Dry gangrene = arterial occlusion
b. Wet gangrene = venous occlusion
Fat necrosis Chalky white precipitates
Pancreatic degeneration
Fatty degeneration Liver
Somatic death
1’ changes During somatic death
“CRC”: circulatory, respiratory, CNS failure
2’ changes After somatic death
“ARLP DPA”: Algor mortis, Rigor mortis, Livor mortis, Postmortem clotting, Dessication,
Putrefaction, Autolysis
Algor mortis (1st) Postmortem cooling
Cooling: 7’F/hr
Rigor mortis (2nd) Stiffening
1st: neck & head (2-3 hrs)
Persists for 3-4 days
Livor mortis Lividity/suggillations
Purplish discoloration
After 10-12 hrs, it does not blanch on pressure or shift when the body is moved
Postmortem Lividity Disappears on pressure (reappears when pressure is released
Oozing of blood (incision)
Ecchymosis Opposite of postmortem lividity
No oozing of blood (incision)
Postmortem Clot Settling of RBCs from plasma
Chicken fat
Currant jelly
Assumes the shape of the vessel
Rubbery consistency
Antemortem Clot Not readily detachable from the blood vessels
No chicken fat
No currant jelly
Seldom assumes the shape of blood vessels
Granular & friable
Dessication Drying & wrinkling of the anterior chamber of the eye
Putrefaction Invasion of intestinal microorganisms
Autolysis Self digestion of cells
Lysosome: suicide sac of the cell, releases lysozyme
Organ Weights
Liver 1,100 – 1,600g
Brain 1,150 – 1,450g
Right lung 300-400g
Left lung 250-350g
Heart 250-300g
Spleen 60-300g
Thyroid 10-50g
Adrenals 4g or so each
Exfoliative cytology Desquamated cells
Pap smear stain method Method of choice
Barr body (+) for females
Quality Assurance
3 copies/report 1. Doctor
2. Patient = original copy
3. File
Reports Surgical pathology report
Cytopathology report
Autopsy report
Signatories Request forms = patient’s doctor
Result forms = pathologist
Turnover of results Surgical pathology & cytology = 24 hrs
Frozen section = 5-15 mins
Autopsy report = 1 week (Autopsy procedure: 24 hrs)

Storage Specimen (tissue) = 1 month to 1 year
Tissue blocks (paraffin) = 3 to 10 years
Slides = indefinite
Suggested Guidelines for Record and Specimen Retention
Records Requisitions 2 years
QC 2 years
Instrument maintenance 2 years
BB QC 5 years
BB employee signatures 10 years
BB donor/recipient records Indefinitely
Reports Clinical pathology lab reports 2 years
Surgical pathology (and BM)
reports
10 years
Cytogenetics reports 20 years
Autopsy forensic reports Indefinitely
Specimens Serum/other body fluids 48 hours
Blood smears (routine) 7 days
Microbiology smears 7 days
BB donor/recipient
specimens
7 days post-transfusion
Pathology/BM slides 10 years
Pathology blocks 10 years
Cytogenetic slides 3 years
Cytogenetics diagnostic
images
20 years
Forensic Cases
Body fluids 1 year
Tissue for toxicology 1 year
Wet tissues 3 years
Paraffin blocks Indefinitely
Slides Indefinitely
Reports Indefinitely
Gross photographs/
negatives
Indefinitely
Dried blood films Indefinitely
Frozen tissue for DNA indefinitely
Autopsy (Postmortem Examination)
Autopsy Gold standard for confirmation of a medical disease

Wherever scientific medicine of high quality is practiced, postmortem exams are performed
Whenever a conscientious physician knows why he lost his patient, a postmortem exam has been
performed
Whenever criminal law is enforced
Whenever a death certificate shows accurately the causes of death & confirmed medical diagnosis
for the assembling of vital statistics, a postmortem has been performed
Whenever there is medical research on the causes & nature of diseases such as cancer, heart
diseases & stroke, the investigative method is the postmortem exam
An informed society requires a postmortem exam in human death for the good of medical science,
for the public’s health & for the future care of the living patient
Types of autopsy 1. Complete autopsy

- Requires consent
- Complete examination of all organs, including the brain
2. Partial autopsy
- Part of the anatomy
3. Selective autopsy
- Restricted to at least a single organ (Ex. MI – heart)
Preliminaries for PME 1. Written consent
- next kin-abide by the extent or restrictions allowed
- Relative: oriented by the attending physician, not the pathologist
2. Death certificate (Old: Blue form | New: Blue border/frame)

- Signed by:
a. Physician
b. Pathologist (back): will sign when PME has been performed
3. Medical abstract or clinical data
4. Medico-legal clearance
- Suspicious evidence of foul play
- Ex. physical injury
Other Uses of Death Certificate Burial & cremation purposes
Transport of body from hospital funeral cemetery
Medical insurance claiming
- If suicide: (-) insurance
- Acts of God (lightning, flood): (-) insurance
- Civil war: (-) insurance
PME is permitted w/o consent 1. When it is ordered by the police or coroner (NBI)
in the following circumstances 2. When it is necessary to complete the death certificate
3. When the deceased himself has given consent before he died (advanced directive)
- Stipulate that in the event you will die, you will be giving out a consent for autopsy
- Donate your organs for medical purposes or for transplantations
4. Deceased military personnel who dies in active services/training duty or military services
If pathologist is not available The medico-legal examiner or the coroner has jurisdiction in medico-legal cases & may authorize
the pathologist to proceed w/ an autopsy
The coroner has authority in the 1. All natural deaths occurring in the hospital w/in 24 hrs of admission, unless the case was
following cases attended by a private physician w/in 36 hrs of death
2. Newborns in the 1st 24 hrs of life
3. All injury cases, old or recent
4. All deaths due to unknown cases
5. All deaths due to suspicious cases
6. All abortion, whether self-induced or otherwise
7. All violent deaths
8. All accidental deaths
9. All sudden deaths
10. All cases w/o medical attendance w/in 36 hrs prior to the hour of death
11. All deaths due to drowning, hanging or strangulation (asphyxia)
12. All deaths due to shooting, stab wounds, burns, electricity, lightning, tetanus, etc.
13. Homicides
14. All suicides
15. All poisoning
16. Stillborn = omission
17. Premature death
Somatic death Death of an organism
Cessation of circulation & respiration (1960’s)
Criteria for the pronouncement 1. Advanced resuscitation techniques that are capable of reviving effectively cases of clinical death
of death *Clinical death: cessation of heartbeat & respiration but the brain is still alive but injured
2. Advance life-sustaining equipment capable of maintaining cardiovascular & respiratory
functions despite severe brain injury
3. Redefinition from cessation to irreversible cessation of cardiorespiratory functions after
resuscitation attempts
4. Brain death: cannot be revived anymore [National institute of neurological diseases & stroke in
the US (1977)]
- Clinically dead & dead are the same
Criteria for brain death Brain death: perpetual state of deep sleed
a. Coma (patient will not respond) & cerebral unresponsiveness
b. Apnea
c. Absent cephalic (brainstem) reflexes
d. Electrocerebral silence
criteria should be present for 30 mins at least 6 hrs after onset of coma & apnea
American bar association & 1. irreversible cessation of circulation & respiratory functions
national conference of 2. Irreversible cessation of all functions of the entire brain, including the brainstem is dead
commission of uniform state
laws legislative definition of
death (1980)
American academy of Death:
neurology 1. Coma
2. Absence of the following:
- Motor response

- Pupillary response to light & pupils at mid-position
- Corneal reflexes
- Caloric responses
- Gag reflexes
- Coughing in response to tracheal suctioning
- Sucking & rooting reflexes
Postmortem changes 1. Algor mortis
- 1st demonstrable change after death is cooling of the body
- At room temp: 2’-2.5’F/hr (1st hr)
- 1.5-2’F/hr (next 12 hrs)
- 1’F/hr (next 12-18 hrs)
- As a rule, the body cools at 1.5’F/hr (50% of cases)
- Not a reliable indicator as to the time of death
2. Rigor mortis
- Rigidity of the body due to hardening of the skeletal muscles caused by a series of
physiochemical events after death
- This interlocking is fixed & produces rigor mortis w/o shortening of the muscles
- Sets w/in 2 hrs after death (head & neck)
- Complete w/ 12 hrs
- Persists about 3-4 days
3. Livor mortis (postmortem lividity/hypostasis)
- Blood supply gravitates to the skin vessels w/c becomes toneless & dilate after circulation
ceases
- Becomes evident as early as 20 mins after death
- Fully evident w/in 4-8 hrs
- Tardien spots: petechiae
4. Postmortem clotting of blood
5. Discoloration of tissue
- Abdomen: green
- Formation of sulfur gases (bacteria)
6. Putrefaction
7. Dessication (mummification)
Techniques of Autopsy
Technique of Virchow Organs removed & dissected individually in the body
Most widely used metohd
Technique of Rokitansky In-situ dissection in part combined w/ en bloc technique
En bloc:
- By cavity
- Interrelated to each other
- Systemic dissection
- Ex. thoracic cavity (lungs, heart, diaphragm), respiratory system
Technique of Ghon En bloc technique
Technique of Letulle En masse:
- All organs of thoracic, abdominal, & pelvic are removed at the same time
- Sweeping of all organs
Autopsy: Larynx Rectum Very popular, easy to do, convenient
Part of consent: organs should be retained completely or partially
Organs set aside later
Body undertaker of the body

TO NOTE!
Sputum Saccomanno’s fixative
3 specimen
(+) alveolar macrophage = sputum
(-) alveolar macrophage = saliva
BAL P. carinii/P. jiroveci
Jelly-like clots Prevent by adding 300U heparin/100mL aspirate
GI specimen If >½ hr delay of fixation digestion of cells
Fasting: 8 hrs
Urine 50mL = cytology
10-15mL = UA
2nd urine = preferred
Pap smear Dr. George Papanicolau (1940)
Smears: prepared by rotary motion
1. Mailing of specimens
- air drying after 2 hrs fixation
- glycerin technique
2. Egg albumin = not recommended as adhesive agent (intensifies stain by Light Green)
Adhesive agents Pooled human serum/plasma
Celloidin ether alcohol
Leuconostoc culture
3 primary materials used for 1. Speculum
obtaining specimen for Pap 2. Ayer’s spatula = rotate 3600
smear 3. Cytobrush = Os
Strawberry cervix T. vaginalis
Doderlein bacillus L. acidophilus
Pap’s stain: blue to lavender
C. albicans Diabetic patients
Sish kebab appearance
T. vaginalis Pear-shaped, blue-gray to blue-green
Pigs on a scruff appearance
Leptothrix Indicates T. vaginalis infection
G. vaginalis Clue cells
Koilocytes Wrinkled prune appearance
w/ perinuclear halo
HPV (LSIL)
Ferning Formation of salt crystals
Early pregnancy
Quantitative Evaluation: Cytohormonal Maturation Index (CHMI)
CHMI MI = P/I/S
Pregnancy MI = 0/90/10 (+ progesterone)
Newborn (8 weeks) MI = 0/90/10 (+ progesterone from mother)
Infancy (8 weeks-puberty) MI = 80/20/10 (+ estrogen)
Late menopausal MI = 100/0/0 (no estrogen)
75 y.o. woman w/ estrogen MI = 0/20/80
therapy

FIXATIVES
Fixative Function Fixation time Formula
Aldehyde 10% Formalin Most widely used Formaldehyde
10% Formol-saline CNS tissue & Post- 24hrs @ 35°C (95°F) 40% Formaldehyde
mortem tissue 48jrs @ 20-25°C (65-77° NaCl
F) Distilled Water
10% NBF/PBF Post-mortem & research 4-24hrs Anhydrous dihydrogen
specimens phosphate
Anhydrous Disodium hydrogen
phosphate
40% Formaldehyde
Distilled water
Formol-Corossive Routine post-mortem 3-24hrs Sat. Aq. Mercuric Chloride
(Formol-sublimate) tissue 40% Formaldehyde
Alcoholic Formalin Enhance 6 hrs 95% Ethyl alcohol sat. with
(Gendre’s) immunoperoxidase picric acid
studies Strong formaldehyde solution
Glacial acetic acid
Glutaraldehyde Small tissue fragments 2-4hrs 2.5% Glutaraldehyde
and needle biopsies 24 hrs 4% Glutaraldehyde
Larger tissues
Metallic
Mercuric Zenker’s Fluid Small pieces of liver, 12-24hrs Mercuric chloride SS
spleen, connective tissue Glacial Acetic acid
fibers and nuclei
Zenker-Formol Pituitary gland, bone 12-24hrs Mercuric chloride SS
(Helly’s) marrow, spleen and liver 40% Formaldehyde
Heidenhain’s Susa Tumor biopsies of the 3-12 hrs Mercuric Chloride
skin and excellent Sodium Chloride
cytologic fixative Trichloroacetic acid
Glacial Acetic acid
40% Formaldehyde
Distilled water
B-5 Fixative Bone marrow biopsies 1.5-2hrs Distilled water
Mercuric chloride
Sodium acetate (anhydrous)
Chromate Chromic acid Precipitates all proteins 1-2% Aq. solution
and preserve
carbohydrates
Potassium Preserves lipid and 3% Aq. solution
dichromate mitochondria
Regard’s (Muller) Demonstration of 12-48hrs 3% Potassium dichromate
chromatin mitochondria. 40% Strong Formaldehyde
Mitotic figures, golgi
bodies, RBC and Colloid-
containing tissues
Orth’s Early degenerative 36-72hrs 2.5% Potassium dichromate
processes and tissue Sodium sulfate
necrosis, rickettsiae and 40% strong formaldehyde
myelin
Lead Lead Acid 4% Aqueous solution of basic
mucopolysaccharides, lead acetate
mucin
Picric acid Bouin’s Embryos and pituitary 6-24hrs Sat. solution of picric acid
biopsies 40% strong formaldehyde
Brasil’s Alcoholic Glycogen 30mins-2hrs 37% Formaldehyde
Picroformol Picric acid
Ethanol/Isopropanol
Trichloroacetic acid
Glacial acetic acid Glacial acetic acid Nucleoproteins,
(solidifies at 17°C) chromosomes and
chromatin materials

Alcohol 100% Methanol Dry and wet smears,
blood smears and bone
marrow tissues
95% Isopropanol Touch preparations
70-100% Ethanol Nucleoproteins and 18-24hrs
nucleic acids, blood,
tissue films and smears
Carnoy’s Chromosomes, lymph 1-3hrs Absolute alcohol
glands and urgent Chloroform
biopsies Glacial acetic acid
Newcomer’s Mucopolysaccharides and 12-18hrs @ 3°C Isopropanol
nuclear proteins Propionic acid
Petroleum ether
Acetone
dioxane
Osmium Tetroxide Flemming’s Nuclear preparation 24-48hrs 1% Aq. Chromic acid
2% Aq. Osmium tetroxide
Glacial Acetic acid
Flemming’s w/o AA Cytoplasmic preparation 24-48hrs 1% Aq. Chromic acid
2% Aq. Osmium tetroxide
Tricholoracetic Tricholoracetic acid Precipitates proteins
acid
Acetone Acetone Brain tissues and rabies
Heat fixation Heat fixation Frozen tissue sections
and preparation of
bacteriologic smears
DECALCIFICATION
Agent Function Decalcification time Formula
Nitric acid 10% Aq. Nitric acid Urgent biopsy, needle and 12-24hrs Concentrated Nitric acid
small biopsy Distilled water
Large or heavily mineralized
cortical bone
Formol-Nitric acid Urgent biopsies 1-3 days Concentrated nitric acid
40% Strong Formaldehyde
Distilled Water
Perenyi’s Routine 2-7 days 10% Nitric acid
0.5% Chromic acid
Absolute Ethanol
Phlorogucin-Nitric Most rapid 12-24hrs Concentrated nitric acid
acid Phlorogucin
10% Nitric acid
Hydrochloric acid Von Ebner’s Teeth and small pieces of 36% Sat. Aq. Solution of
bone NaCl
Concentrated HCl
Distilled Water
Formic Acid Formic Acid Small pieces of bones and 2-7 days Formic Acid
teeth Formal Saline
Formic acid-sodium Autopsy materials, bone 3-14 days 20% Aq. Sodium Citrate
citrate marrow, cartilage and tissues 45% Formic acid
for research
Trichloroacetic Trichloroacetic acid Permits good nuclear staining 4-8 days Trichloroacetic acid
acid 10% Formal saline
Sulfurous acid Sulfurous acid Weak decalcifying agent
Minute pieces of bone
Chromic acid Flemming’s Minute bone spicules Chromic acid
2% Osmium tetroxide
Glacial Acetic acid
Citric acid Citric acid Permits excellent staining 6 days 7% Aq. Solution Citric acid
(monohydrate)
7.4% Aq. Solution
Ammonium citrate
1% Aq. Solution Zinc sulfate
Chloroform

Common staining solutions used
Stain Function Formula
Hematoxylin Aluminum Progressive staining: Lithium carbonate
hematoxylin Stain nuclei but leave the background tissue unstained Bicarbonate
Regressive staining: section is overstained, differentiated  “blueing” Potassium or sodium
Note: acetate
Blueing Scott’s tap water substitute
- insoluble blue aluminum hematin-tissue lake
- warm tap water (40° to 50°C)
cold water
- below 10°C may produce pink artifact discoloration
Ehrlich’s Regressive staining Hematoxylin
hematoxylin Mucopolysaccharide – cartilage and cement lines of bones Absolute ethyl alcohol
Suitable for tissues that have been subjected to acid decalcification Aluminum potassium
Not ideal for frozen sections Glycerin
Staining time: 15 – 40 mintues Distilled water
Glacial acetic acid
Harris Good regressive stain Hematoxylin
hematoxylin Routine nuclear staining in exfoliative cytology and sex chromosome Absolute ethyl alcohol
Staining time: 5 – 20 minutes Ammonium/potassium
alum
Distilled water
Mercurix oxide (red)
Glacial acetic acid
Cole’s Used in sequence with celestine blue Hematoxylin
hematoxylin Artificially ripened with an alcoholic iodine solution 1% iodine in 95% alcohol
Ready for immediate use Sat. aq. Ammonium alum
Distilled water
Mayer’s Chemically ripened with sodium iodate Hematoxylin
hematoxylin Progressive and regressive stain Sodium iodate
Used as nuclear counterstain – detect presence of cytoplasmic Potassium alum
glycogen Citric acid
Used in celestine blue hemalum method of nuclear staining Chloral hydrate
Distilled water
Iron Weigert’s Standard hematoxylin used Solution A:
hematoxylin hematoxylin Demonstrate muscle fibers and connective tissues Hematoxylin
Absolute ethyl alcohol
Solution B:
30% anhydrous ferric
chloride
Concentrated hydrochloric
acid
Distilled water
Heidenhain’s Regressive staining of thin sections Mordant differentiator:
hematoxylin Components are black or dark grey-black Ferric ammonium sulfate
Demonstration of both nuclear and cytoplasmic inclusions (chromatin, Distilled water
chromosomes, nucleoli, centrosomes, and mitochondria) Hematoxylin stain:
95% ethyl alcohol
Distilled water
Phosphotyngs Progressive staining Hematoxylin
tic acid Blue: nuclei, fibrin, muscle striations, myofibrils and fibroglia Phosphotungstic acid
hematoxylin Orange-red or brownish red to deep brick-red stain: cartilage Distilled water
Staining time: 12-24hrs
Eosin 5% aqueous Most commonly used Eosin Y
eosin y Green yellow fluorescence Distilled water
Bluish – deeper red color (eosin B, erythrosine B)
Ethyl eosin – eosin S, eosin alcohol-soluble
Eosin, stock Cytoplasmic stain Eosin Y
alcoholic Distilled water
solution 95% alcohol
Eosin- Rarely used 1% phloxine
phloxine B 1% eosin Y
solution 95% alcohol
Glacial acetic acid

Acid fuchsin- Picric acid Demonstration of connective tissue Acid fuchsin (Masson
picric acid (saturated stain):
(van gieson’s aqueous Acid fuchsin
stain) solution) Glacial acetic acid
Acid fuschin Distilled water
(1% aqueous Picro-fuchsin solution:
solution) 1% acid fuchsin
Saturated aqueous picric
acid
Acridine Discrimination between dead and living cells
orange DNA: green fluorescence
RNA: red fluorescence
Acridine red Demonstrate deposits of calcium salts and possible sites of phosphatase activities
3B
Alcian blue Water-soluble phthalocyanine dye Alcian blue
Produces a striking blue color Glacial acetic acid
Specific for connective tissue and epithelial mucin Distilled water
Aniline blue Cytoplasmic stain Aniline blue
Used for counterstaining of epithelial sections Distilled water
Glacial acetic acid
Basic fuschin Plasma stain for deep staining of acid-fast organisms, for mitochondria, for Main constituent: feulgen’s
differentiation of smooth muscles and schiff’s reagent
Carbol- Basic fuchsin
fuschin Phenol crystals
Absolute ethyl alcohol
Distilled water
Coleman’s Basic fuchsin
Feulgen Sodium betabisulphite
reagent 1N hydrochloric acid
Distilled water
Schiff’s Basic fuchsin
reagent Sodium metabisulfite
anhydrous
Distilled water
Normal hydrochloric acid
Mallory’s Basic fuchsin
fuchsin stain 95% ethyl alcohol
Distilled water
Aldehyde Concentrated hydrochloric
fuchsin acid
(gomori’s Paraldehyde
stain) 0.5% basic-fuchsin in
70% alcohol
Benzidine Stain hemoglobin Solution A:
Benzidine
Absolute alcohol
Sodium nitroprusside
Distilled water
Solution B:
Absolute alcohol
Glacial acetic acid
30% hydrogen peroxide
Distilled water
Bismarck Contrast stain for gram’s technique in acid fast and papanicolau method
brown Stain diphtheria organisms
Carmine Chromatin stain for fresh materials
Combined with aluminum chloride to stain glycogen
Best carmine Carmine
stain (stock Potassium carbonate
solution) Potassium chloride
Distilled water
Concentrated ammonia
Best carmine Best carmine (stock
stain (working solution)
solution) Concentrated ammonia

Absolute methyl alcohol
Best’s Absolute methyl alcohol
differentiation Absolute ethyl alcohol
Distilled water
Mayer’s Mordant dye Carminic acid
carmalum Act as basic dye and staining acidic substances Potassium alum
solution Distilled water
Salicylic acid
Sodium salicylate
Celestine blue Routine staining of fixed sections Ferric ammonium sulfate
Good nuclear definition when used in conjunction with alum hematoxylin Distilled water
Celestine blue
Glycerin
Congo red Indicator
Stain for axis cylinders in embryos
4% aqueous solution in Krajian’s method for staining elastic tissues, amyloid and
myelin
Crystal violet Staining amyloid in frozen sections and platelets in blood Crystal violet
Methyl violet
Dextrin
Giemsa stain Staining blood to differentiate leukocytes Giemsa stain
Glycerin
Absolute methyl alcohol
Gold Stain for metallic impregnation Mercuric chloride
sublimate Distilled water
1% gold chloride (brown)
Iodine Oldest stain
Stains amyloid, cellulose, starch, carotenes and glycogen
Used for removal of mercuric fixative artefact pigments
Gram’s iodine Stain microorganisms and fibrin in tissue sections Iodine
Potassium iodide
Distilled water
Lugol’s iodine Test for glycogen, amyloid, and corpora amylacea Iodine
Potassium iodide
Distilled water
Janus green Demonstrate mitochondria
Malachite Stain ascaris eggs and erythrocytes, and as a bacterial spore stain 2% aqueous malachite
green Use as decolorizer and as a counterstain green
Glacial acetic acid
Glycerol, C.M.
Methyl green Stains chromatin green in the presence of an acid
Gives false positive reactions with mucin
Methylene “polychroming” – oxidation of methylene blue Methylene blue
blue Valuable stain for plasma cells Potassium carbonate
Cytological examinations of fresh sputum for malignant cells Distilled water
Bacterial stain for evaluation and differentiation
Diagnosis of diphtheria
Vital staining of the nervous tissue
Loeffler’s Rapid diagnosis Saturated solution of
polychrome Frozen section methylene blue in alcohol
methylene 1:10,000 KOH
blue
Methylene Color nuclei reddish-purple
violet
Neutral red Observing cell granules and vacuoles of phagocytic cell 1:10,000 up to 1:100,000
Night blue Substitute for carbol fuchsin in acid fast staining
Orcein Excellent stain for elastic fibers
Recommended for dermatological studies
Osmium Stain fat (black)
tetroxide
Picric acid Contrast stain to acid fuchsin
Demonstration of connective tissues
Cytoplasmic stain in contrast to basic dyes
Counterstain to crystal violet
Tissue fixative

Decalcifying agent
Prussian blue Manufacture of paints
Microanatomical color contrast of specimens
Demonstration of the circulatory system (intravital staining)
Rhodamine B Stain blood and glandular tissues
Siler nitrate Identification of spirochetes, reticulum and other fiber stains 10% aqueous solution
Toluidine blue Nuclear stain for fixed tissues
Substitute for thionine in fresh frozen tissues sections
Recommended for staining of Nissl granules in chromophilic bodies
Victoria blue Demonstration of neuroglia in frozen sections
Oil soluble Sudan black Most sensitive
dyes B Black color - intracellular lipids
Recommended for paraffin sections
Stains phospholipid and neutral fats
Note:
It does not stain crystalline cholesterol
Sudan IV Scharlach R 2% benzoic acid
70% alcohol
Sudan III Introduced to histochemistry
Stain for CNS tissues (light orange)

Stains
Stain Substance Stained (+) Color/Result Comments
PAS CHO, Glycogen, Mucins, PAS (+):Magenta red Basic fuchsin: essential component of
Bacteria & Fungi, basement Schiff reagent
membrane
PAS w/ diastase ctrl Glycogen Red Method of choice for glycogen staining
Best Carmine Glycogen Bright red Selective & highly specific for glycogen
Langhan’s iodine method Glycogen Mahogany brown Obsolete
Not specific for glycogen
Alcian blue Acid mucins Blue Avoid celloidinization of slides
Alcian Blue-PAS Any mucins (acid/neutral) Acid mucin: blue Avoid Ehrlich’s
Neutral mucin: magenta
Gomori’s aldehyde fuchsin Acid MPS Sulfated mucins: purple
stain Sulfated mucins Carboxylated mucins: blue
Carboxylated mucins
Mucicarmine stain Cryptococcus neoformans Mucin: red Avoid Ehrlich’s hematoxylin
Mucins
Colloidal (Dialyzed) iron Acid mucins Dark blue
technique
Acridine orange Acid mucins/MPS Acid MPS: black Lasts for only 2 hrs
Fungi Fungi: greenish red
fluorescence
Sudan black Lipids Blue black
Sudan IV (Scharlach R) Lipids (TAG) red Most commonly used stain
Oil red O Lipids Brilliant red
Osmium tetroxide Lipids Black
Nile blue sulfate method Neutral fat = Pinkish red Nile blue: preliminary indicator of the
Cholesterin esters = Light red type of lipid present
Cholesterin fatty acids = Light red -Red oxazone (dissol. neutral lipids)
Fatty acids & soap = Deep blue to violet -Blue oxazone (reacts w/ PL and FFA)
Cerebrosides = Light blue
Toluidine blue-acetone mtd Sulfatide Metachromatic red-brown
or yellow
Borohydride-Periodic-Schiff Gangliosides Red
method
Alkaline fast-green method Histones Green Fast green stains basic groups in
Protamines tissues
Peracetic acid-Alcian blue Cystine Blue-green
Cysteine
Sakaguchi’s test Arginine Orange-red Uses Milton reagen
Gomori calcium method Alkaline phosphatase Brownish-black
Gomori lead method Acid phosphatase Black Substrate: β-glyceroPO4
Lead method 5’-nucleotidase Blackish brown deposits
Metal precipitation ATPase Dark brownish-black ppt For skel. muscle biopsies
Calcium cobalt method ATPase Cobalt phosphate ppt For skel. muscle biopsies
α-naphthyl acetate method Nonspecific esterase Reddish brown α-naphthyl acetate method
Indoxyl acetate method Nonspecific esterase Blue
Tetrazolium method Monoamine oxidase Bluish black
Feulgen technique DNA Red-purple Most reliable & specific histochemical
staining technique for DNA
Contains Schiff’s reagent
Methyl green-pyronin RNA RNA (nucleoli): red
DNA DNA (chromatin): green
Gomori’s silver impregnation Reticulin fibers black Reticulin = Argyrophilic (silver stain)
stain
Van Gieson’s stain Collagen = Pink/deep red Contains acid fuchsin & picric acid
Muscle, cytoplasm, RBC, = Yellow
fibrin
Masson’s trichrome stain Collagen & mucus = Blue = Red
Muscle, RBC & keratin
Mallory’s aniline blue Collagen fibers, cytoplasm, = Red (-) Fuchsin: Excellent & colorful
fibroglia fibrils, axon = Pale pink/yellow method of demonstrating CT fibers
cylinders, neuroglia = Yellow

Elastic fibers
RBCs, myelin sheets
Azocarmine CT = Deep blue Heidenhain’s modification of Mallory’s
Glomerular basement aniline blue stain
membrane
Amyloid & mucous colloid
Weigert’s Elastic fibers Dark-blue/blue-black
Verhoeff’s Elastic fibers Black
Taenzer-Unna-Orcein mtd Elastic fibers Dark-brown
Krajian’s technique Elastic fibers = Bright red Rapid method
Fibrin & CT = Dark blue
RBC = Orange-yellow
Martius-Scarlet-Blue RBCs = Yellow Early fibrin = yellow
Muscle = Red Old fibrin = blue
Collagen = Blue
Fibrin = Red
Mallory’s PTAH Fibrin, muscle striations, = Dark blue
neuroglia, amoeba = Blue
RBCs = Lighter blue
Myelin = Deep brownish-red
Collagen, osteoid,
cartilage, elastic fibers
Congo red Amyloid Red
Methyl violet-crystal violet Amyloid Purplish red
method
Thioflavin-T fluorescent Amyloid Yellow fluorescence
staining
Modified Gomori’s Muscle fibers = Red
Trichrome stain Collagen = Green
Lissamine fast red Muscles, RBC = Red
Collagen = Yellow
Schmorl’s Picro-Thionin Lacunae & canaliculi = Dark brown-black
method Bone matrix = Yellow/brownish-yellow
Bielschowsky’s technique Neurofibril, axons & = Black on a grayish BG
dendrites = Lightly stained
Neuroglia & collagen
Bodian’s stain Nerve fibers & nerve Diagnosis of Alzheimer’s disease
endings
Sevier-Munger technique Peripheral neuritis = Black
Axons = Black
Myelin sheath = Light brown
Neuritic plaques & tangles = Black
Argentaffin granules = Black
Toluidine blue Nissl granules & nucleoli Deep blue
Polychrome methylene blue Nissl granules & nucleoli Deep blue
Thionine Nissl granuls & nucleoli Purple
Cresyl fast violet Nissl substance = Purple-dark blue Nissl granules: a.k.a. Tigroid
Neurons = Pale purple blue substances
Weigert-Pal technique Myelin sheath Blue black Weigert-Pal technique
Luxol fast blue Myelin Blue-green Luxol fast blue
Weil’s method Myelin Black Weil’s method
Cajal’s gold sublimate Astrocytes Black on a light brownish Cajal’s gold sublimate method
method BG
Perl’s Prussian blue Hemosiderin Deep blue Perl’s Prussian blue
Gomori’s Prussian blue Iron pigments Bright blue Gomori’s Prussian blue
Turnbull’s blue Ferrous iron (Hemosiderin) Blue Turnbull’s blue
Benzidine-nitroprusside Hemoglobin & oxidase Dark blue Benzidine-nitroprusside stain
stain granules
Mod. Fouchet’s technique Bile pigments Emerald to blue green Mod. Fouchet’s technique
Gmelin technique Bile & hematoidin Blue-purple then green Gmelin technique
Stein’s iodine test Bile pigments Depend on the oxidation of the
pigment to green biliverdin by iodine

Schmorl’s ferric ferricyanide Bile, lipofuscins, melanin, Dark blue
method argentaffin cells,
chromaffin, thyroid colloid
Gomori’s aldehyde fuchsin Lipofuscin Purple
Mallory’s fuchsin stain Hemofuscin Red
Masson Fontana technique Melanin = Black Argentaffin reaction: melanin reduces
Argentaffin cell granules = Black ammoniacal silver solutions w/o use of
a reducer
Von Kossa’s silver nitrate Calcium Black
Lindquist modified Copper Red to orange-red
rhodanine technique
Gram-Twort stain Gram (+) organisms = Blue-black
Gram (-) organisms = Pink-red
RBCs = Green
Elastic fibers = Black
Brown & Brenn method Gram (+) bacteria = Blue
Gram (-) bacteria = Red
Wade-Fite technique M. leprae Golden yellow
Toluidine blue H. pylori Dark blue against blue BG
Cresyl violet acetate mtd H. pylori Blue-violet
Dieterle method L. pneumophila & Dark brown to black
spirochetes
Levaditi’s method Spirochetes Black on a yellowish BG
Modified Steiner & Steiner Spirochetes, Donovan Black
technique bodies, fungi, bacteria
Warthin-Starry method Spirochetes Black
Grocott Methenamine Silver Fungi = Sharply outlined I black
Mucin & glycogen = Gray-black
Mycelia & hyphae = Old rose
RBCs = Yellow
Lendrum’s phloxine- Viral inclusions Bright red
tartrazine method
Orcein method HBsAg Brown-black
Giemsa stain Bacteria = Blue Recommended for blood and BM
Mast cell granules = Deep blue parasites, inclusion conjunctivitis,
Eosinophilic granules = Red Toxoplasma, spirochetes & other
Nuclei = Blue bacteria
Cytoplasm = Pink

Histopath

Uploaded by

Copyright:

Available Formats

Histopath

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Histopath

Uploaded by

Copyright:

Available Formats

Histopathology

Fresh Tissue Examination

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Retrogressive Changes = Organ/Tissue smaller than normal

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Autopsy Gold standard for confirmation of a medical disease

Types of autopsy 1. Complete autopsy

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

Darla Ysavel V. Delos Reyes

You might also like