module 2 Histopathology

I. Procedures involved in Basic Tissue Processing.

• Fixation
• Decalcification (optional) – process of removing calcium from decalcified tissue, making
suitable for section cutting.
• Dehydration
• Clearing
• Infiltration (Impregnation)
• Embedding
• Trimming
• Section-Cutting (microtomy)
• Staining
• Mounting
• Labelling
Fixation

• 1st and most critical step in histotechnology

• 10% Neutral buffered formalin - mostly widely used fixative
• Physical characteristics to note: color, consistency, texture and size of tissue
to be processed - Size: 1 x 1 x 0.5
• Primary aim: preserve the morphological and chemical integrity of the cell in
as life-like manner. - Shape, structure, intercellular relationship and chemical
constituents of tissues are preserved. Prevents degeneration, decomposition,
putrefaction, and distortion of tissues after removal from the body.
• Secondary goal: harden and protect the tissue from the trauma of further
handling Stabilizing proteins: most important reactions for maintaining
morphology in the fixation of tissues for routine histopathology. They are fixed
to structural proteins and thus rendered insoluble.
Dehydration

• Process of removing IC and EC water from the tissue following

fixation and prior to wax impregnation
• Dehydration Agents:
• Alcohol - most common
• Acetone - provides a rapid method “stat” method
• Dioxane - a rapid dehydrating agent but its fumes are highly toxic
• General Rule: Whatever the dehydrating agent is used, the amount
in each stage should not be less than 10 times the volume of the
tissue in order to ensure complete penetration of the tissue by the
dehydrating solution.
Clearing (de-alcoholization)

• Alcohol or dehydrating agent is removed from the tissue and

replaced with a substance that will dissolve the wax with
which the tissue is to be impregnated.
• XYLENE (XYLOL) - Routine histologic processing ; suitable for
urgent biopsies
Infiltration/Impregnation

• is the complete removal of the clearing agent by

substitution as the medium (paraffin) penetrates the tissue
with the use of no less than two, and preferably three
baths of paraffin.
• Infiltrating Mediums are paraffin wax, celloidin, gelatin and
plastic
Embedding

• orientation of the tissue in melted paraffin, which when

solidified, provides firm medium for keeping intact all the
parts of the tissues when sections are cut.
Trimming

• Process where excess paraffin is removed from the block to expose

the tissue to be cut by the microtome.
Section-cutting

• technique of making the very thin slices of tissue specimens for the
microscopic examination.
• Microtome
Staining

• used to highlight important features of the tissue as well as to

enhance the tissue contrast.
• H&E staining
Mounting and labelling

• Mounting – process of putting the specimen on the slide by a

mounting medium and covered with a coverslip.
• Labelling - slide is labelled with patient’s fullname, Specimen
number, or by barcodes.

• S-20-0001
• P-20-0001
II. Histochemistry

• Chemistry in the context of biological tissue—is an invaluable set

of techniques used to visualize biological structures. This field lies
at the interface of organic chemistry, biochemistry, and biology. 
• Essentially, identification and distribution of chemical constituents
of tissues is achieved through the exploitation of unique chemical
environments in cells, heterologous expression techniques as well
as enzymatic activities.
Basic principles of Histochemistry

• Histochemistry combines the methods of histology with those of chemistry or biochemistry, to

reveal the biochemical composition of tissues and cells beyond the acid-base distribution shown by
standard staining methods (Hx & E), without disrupting the normal distribution of the chemicals.
• All the immunochemical methods/techniques depend on a highly specific and sensitive
reaction between antigens and antibodies. 
• Particle methods - This is the technique where the antigen-antibody interaction is
observed. It includes a number of methods such as Immunoprecipitation,
Immunoelectrophoresis, Immunofixation.
•  
• Label methods - With label methods, either the antigen or the antibody is labeled allowing
for the antigen-antibody reaction to be observed. Immunoassay and competitive binding
are examples of label methods.

• Some of the other methods include: Immunofluorescence, Immunoelectron Microscopy

 Importance of Histochemistry in the
diagnosis of diseases
• Immunohistochemistry (IHC) is an important application of monoclonal as well as polyclonal
antibodies to determine the tissue distribution of an antigen of interest in health and disease.
• It is widely used for diagnosis of cancers because specific tumor antigens are expressed de
novo or up-regulated in certain cancers.
• IHC methods have brought about a revolution in approach to diagnosis of tumors of uncertain
origin, primary as well as metastatic from unknown primary tumor. A panel of antibodies is
chosen to resolve such diagnostic problem cases. 
• Prediction of response to Therapy.
• In autopsy pathology while basic histologic examination of tissue is considered a useful and
necessary component IHC may provide a greater insight.
• IHC is also used in drug development to test drug efficacy by detecting either the activity or
the up- or down-regulation of disease targets.
• IHC can also be used to determine the function of specific gene products in
fundamental biological processes such as development and apoptosis. 
• Immunohistochemical methods are also being applied to confirm
infectious agent in tissues by use of specific antibodies against microbial
DNA or RNA, e.g. in Cytomegalo virus, Hepatitis B virus, Hepatitis C
virus, etc. 
• It has played an increasingly important role in the subclassification of
neurodegenerative disorders and the development of consensus criteria for
their diagnosis.
 Goal of Histochemistry

• To provide color and contrast to microscopic images. The field uses disparate techniques to accomplish
the specific labeling of biological structures. Histochemists pioneered the use of small-molecule
cellular stains, labeled molecules such as antibodies, and enzyme-mediated detection and signal
amplification.
• Presentation of Normal Chemical Distribution: The substance being analyzed must not diffuse away
from its original site.
• Presentation of Normal Chemical Composition: The procedure must not block or denature the reactive
chemical groups being analyzed, or change normally non-reactive groups into reactive groups.
• Specificity of the Reaction: The method should be highly specific for the substance or chemical groups
being analyzed, to avoid false-positive results.
• Detectability of the Reaction Product: The reaction product should be colored or electron scattering,
so that it can be visualized easily with a light or electron microscope.
• Insolubility of the Reaction Product: The reaction product should be insoluble, so that it remains in
close proximity to the substance it marks
Iron : PRUSSIAN BLUE REACTION - MALLORY'S METHOD

• Principle: The reaction occurs with the treatment of sections in acid solutions of
ferrocyanides. Any ferric ion (+3) in the tissue combines with the ferrocyanide and results
in the formation of a bright blue pigment called 'Prussian blue" or ferric ferrocyanide.
• Procedures: 1)Deparaffinize and hydrate to distilled water 2) *Working solution, *
microwave, 30 seconds. Allow slides to stand in
solution for 5 minutes, in the fume hood 3)Rinse in distilled water 4) Nuclear-fast red,
5 minutes 5) Wash in tap water 6) Dehydrate, clear, and coverslip.
*Conventional method: room temperature for 30 minutes.
RESULTS: Iron (hemosiderin) blue/Nuclei red/Background pink
• Reagents:
• 5% Potassium Ferrocyanide: Potassium ferrocyanide 25.0 gm, Distilled water 500.0
ml
• 5% Hydrochloric Acid: Hydrochloric acid, conc. 25.0 ml, Distilled water 475.0 ml
• Working Solution: 5% potassium ferrocyanide 25.0 ml, 5% hydrochloric acid 25.0
ml
• Stains: Perl’s prussian blue stain, Lillie’s Method for ferric and ferrous iron,
gomori’s PB stain, Turnbull’s stain
• Clinical significance:To demonstrate ferric iron in tissue sections. Small amounts
of iron are found normally in spleen and bone marrow. Excessive amounts are
present in hemochromatosis, with deposits found in the liver and pancreas,
hemosiderosis, with deposits in the liver, spleen, and lymph nodes.
• to indicate levels of iron storage and may provide reliable evidence of iron deficiency.
Copper

• Principle: The reaction utilizes the property of copper to bind with high affinity to protein copper deposits in tissue
sections.
• Procedures: 1) Take sections to water 2) Incubate in the rhodanine working solution for 3 hours at 56°C or
overnight in a 37°C oven 3) Rinse well in several changes of distilled water 4) Stain in dilute Mayer
hematoxylin for 10 minutes 5) Briefly rinse in distilled water and place immediately in borax solution for l
0 seconds 6) Rinse well in distilled water 7) Mount with Apathy's mounting media.
• Results: Copper and copper associated protein red to orange-red Nuclei blue, Bile green
• Reagents: Rhodanine Stock Solution: 5-p-Dimethylaminobenzidine rhodanine 0.05 gm, Absolute ethanol 25
ml
• Prepare fresh and filter prior to use.
• Rhodanine Working Solution: Rhodanine stock solution 5 ml, 2% Sodium acetate trihydrate 45 ml
• Borax Solution: Disodium tetraborate 0.5 gm, Distilled water 100 ml
• Stains: Lindquist's rhodanine method, Timm’s silver. Orcein, rubeanic acid and counterstain: Mayer or
Lillie- Mayer hematoxyli, and Harris hematoxylin
• Clinical significance: Wilson’s dse, primary biliary cirrhosis, other liver dse.
 Calcium salts

• Principle: Calcium salts of phosphates and carbonates in tissues are stained deep purplish blue in routine
hematoxylin and eosin. Because calcium oxalate is birefringent, it can be identified easily by polarization. The
other calcium salts are not birefringent and will not polarize.
• Procedures: 1. Deparaffinize and hydrate to distilled water. Rinse well in distilled water 2) Place in Silver
nitrate solution and expose to strong light for 10 to 20 minutes. If the day is overcast longer incubation
will be necessary. Checkthe slides periodically and stop the reaction when the calcium salts are brown-
black 3) Rinse sections in distilled water 4) Place slides in 5% sodium thiosulfate for 2 to 3 minutes 5)
Wash slides in distilled water 6) Counterstain in nuclear fast red for 5 minutes 7) Wash sections well in
water. 8) Dehydrate and clear in 2 changes each of 95% alcohol, absolute alcohol, and xylene 9) Mount
with synthetic resin.
• Results: Mineralized bone Black, Osteoid Red, Nuclei blue
• Reagents: 5 % aqueous silver nitrate, 5 % Sodium thiosulfate, 1 % Neutral Fast Red, Neutral Fast Red 0.5
gm
• Stains:Von kossa’s, Alizarin red S, Calcium dye lake rxn, Azan stain
• Clinical significance: Abnormal deposits of calcium phosphate or carbonate can be associated with
necrotic tissue in lesions of atherosclerosis, hyperparathyroidism, nephrocalcinosis, sarcoidosis,
tuberculosis, and in some tumors.
 Urates

• Principle: Sodium urate crystals can be visualized on a hematoxylin & eosin (H&E) stained slide under a polarized light
with a red compensator. The urates will demonstrate a negative yellow or blue birefringence, based on the alignment of
the urate crystals.
• Procedures: 1) Deparaffinize to Xylene and rinse with 3 changes of absolute alcohol.
• 2. Place slides in preheated working methenamine silver.
• 3. Incubate for 30 minutes at 60ºC.
• 4. Rinse sections is distilled water.
• 5. Tone with 0.1% Gold Chloride for 5 minutes.
• 6. Give sections 4 or 5 rinses of distilled water.
• 7. 3% Sodium Thiosulfate for 5 minutes.
• 8. Wash in tap water for 5 minutes.
• 9. Rinse in distilled water.
• Reagents: Methenamine-Silver Nitrate Stock Solution, Methenamine-Silver Nitrate Working Solution
• Stains: Gomori’s Methenamine silver stain, deGalantha stain
• Clinical significance: gout, poor kidney function, kidney stones and arthritis, Chondrocalcinosis
Argentaffin granules

• Principle: cells can reduce the silver salts to metallic silver (brownish-black) color without the aid of reducing agent.
• Procedures: 1. Deparaffinize and hydrate to distilled water.
• 2. *Working Silver Solution, microwave 20 power, 2 minutes. Check
• slides microscopically for adequate intensity, place in microwave a
• few seconds longer if needed.
• 3. Rinse in distilled water.
• 4. 0.1% Gold chloride, 10 minutes.
• 5. Rinse in distilled water.
• 6. 5% Hypo, five minutes.
• 7. Wash in tap water, rinse in distilled.
• 8. Nuclear-fast red, 5 minutes.
• 9. Wash in tap water.
• 10. Dehydrate, clear, and coverslip. Conventional Method: Place in 60°C oven for 1 hour.
• Reagents: Ammoniacal Silver Stock Solution, Ammoniacal Silver Working Solution, 10% silver nitrate, 0.1% gold chloride, 5% hypo
• Stains: azo (diazonium salts), Fontana-Masson, Schmorl’s, Autofluorescence
• Clinical significance: Carcinoid Tumours
 Dehydrogenases

• Principle: demonstrated by the transferring the released hydrogen ions into

• tetrazolium salt to produce formazan deposits.
• Procedures: The tissue need not be absolutely fresh; refrigeration for 4 hours at
4°C does not cause any noticeable loss of activity. Fixation in chilled acetone for 4
hours causes only 40 percent inactivation of the enzyme. Use frozen sections 25-
50 µ thick because thinner sections often fail to stain. Incubation time at 37°C.
Range from 20 minutes to 3 hours. Elementary tellurium is black or brown-black
• Reagents: stock incubating solution, incubating medium.
• Stains: counterstained with hematoxylin or carmine; they should be mounted in
glycerol or glycerol-jelly. Methylene blue, tellurite, tetrazolium method
• Clinical significance: structural abnormality in the sarcoplasmic reticulum network
of the fiber, as well as mitochondrial abnormalities.
Cytochrome oxidase

• Principle: copper-yielding cytochrome complex that catalyzes the oxidation of ferrocytochrome c to produce
ferricytochrome c and 2H2O.
• Procedures:
• 1. Place coverslips in the incubating solution in a columbia staining dish (Thomas Scientific for 60 minutes at room
temperature.

2. Wash with three exchanges of tap or deionized H2O.

3. Dehydrate in ascending alcohols (50%,70%,80%,95% x 2, 100% x 2) in columbia staining dish(jar)

4. Clear with at least 2 changes of xylene columbia staining dish(jar)

5. Mount with PERMOUNT or another synthetic organic mounting medium.

• Reagents: 0.2 M Phosphate Buffer, pH 7.6, Incubating Solution, Alcohol (50%, 70%, 80%, 95%), catalase, cytochrome
C, xylene, deionized water, permount, DAB, etc.
• Stains: modified gomori stain, burstone’s method, lugol’s iodine
• Clinical significance: results in neuronal loss in the brain leading to psychomotor retardation and neurodegenerative
disease.
Tyrosinase

• Principle: copper containing monooxygenases that catalyze the

• production of melanin and other pigments from tyrosine by oxidation.
• Procedures: Cryostat sections (4, 6 and 8 microns thick) were
• studied from the following tissues: 1. tongues of 6 week old ACI/mai rats; 2.
two spontaneous ca- nine mastocytomas; 3. lesions from a case of nodular
human mastocytosis; 4. Harding-Passey mouse melanomas.
• Sections were incubated both with and without 15 minutes prefixation in
27% formalin at 370 C. for 3
• Reagents: L-tyrosine, phosphate uffer, DL-dopa
• Stains: giemsa stain, W-G, astrablau stain
• Clinical significance: albinism, melanoma
Peroxidase

• Principle: heme-containing enzymes that use hydrogen peroxide as the

electron acceptor to catalyze a number of oxidative reactions. Enzymes of
the peroxidase group catalyze the reduction or transfer of oxygen from
hydrogen peroxide and other peroxides to a variety of substrates.

• Stains: peroxidase stain

• Clinical significance: HCC
Hydrolytic enzymes

• Principle: The acids are demonstrated by their regular precipitation

reactions with metal ions, most often calcium, lead; cobalt, iron, and
copper. The precipitate formed is usually colorless and not easily seen under
the microscope.
• Procedures:
• Reagents:
• Stains:
• Clinical significance: brain tumour
Non-specific esterase

• Principle: In one staining method, α-naphthyl acetate is enzymatically hydrolyzed, liberating a free naphthol
compound. This then couples with a diazonium compound, forming dark brown-red or black colored deposits at
sites of non-specific esterase activity.
• Procedures: α- Napththyl acetate method
• 1. After suitable fixation, bring sections to water.
• 2. Incubate sections in incubating medium at 37°C for 2 to 20 minutes.
• 3. Wash in running water.
• 4. Counterstain in 2% methyl green (chloroform extracted).
• 5. Wash well in tap water.
• 6. Dehydrate rapidly through fresh alcohol to xylene and mount in DPX.
• Results: Esterase reddish brown, Nuclei green
• Reagents: sodium nitrite, buffer sol’n, incubating medium, Pararosanillin HCL stock, subs sol'n
• Stains: NSE
• Clinical significance: myophagocytosis, Denerveted myofibers, Macrophages in inflammatory myositis,
Myofasciitis.
 Chloro-acetate esterase

• Principle: Chloroacetate is enzymatically hydrolyzed by "specific esterase," liberating a free naphthol compound. This then couples with a
diazonium compound, forming highly colored deposits at sites of enzyme activity.
• Procedures:
• 1. Fix cryostat section in buffered formalin or take paraffin sections to water.
• 2. In a 50 ml beaker take 4 drops 4% Pararosanilin hydrochloride solution, add 4 drops freshly prepared 4% Na nitrite solution, and mix fo 1
min. The mixture should turn straw colored.
• 3. Add 20 ml Veronal acetate buffer solution pH 9.1 adjust to pH 6.3 using N HCl.
• 4. Weigh out 0.01g Naphthol AS-D chloroacetate and dissolve in 0.5 ml Dimethyl formamide.
• 5. Add this solution to the Pararosanilin/Buffer mixture, the resulting solution should turn flocculent pale pink in color.
• 6. Filter onto slides and leave 20-30 min at room temperature.
• 7. Wash in water.
• 8. Lightly counterstain with Hematoxylin.
• 9. Wash in water, do not differentiate, blue in Scott’s tap water.
• 10. Dehydrate clear and mount in Permount.
• Results: Chloroacetate esterase activity reddish brown, Nuclei blue
• Reagents: veronal acetate sock and buffer, basic fuchsin sol’n
• Stains: CE stain
• Clinical significance: Myelomonocytic leukemia
 Acetyl cholinesterase

• Principle: an enzyme present in nervous tissue, muscle, and red blood cells that catalyzes the hydrolysis of
acetylcholine to choline and acetic acid.
• Procedures:
• 1. Incubate in solution a. at 37ƒC for 15-120 minutes.
• 2. Rinse in distilled water.
• 3. Counterstain in Hematoxylin.
• 4. Dehydrate, clear. Mount sections in DPX
• Results: Cholinesterase activity Red/brown.
• Reagents: incubating sol’n: Acetyl thiocholine iodide 5mg, 0.1M acetate buffer (pH.6.0) 6.5ml, 0.1m
sodium citrate (2.94g/100ml) 0.5ml, 30mM copper sulphate (0.58g/100ml) 1.0ml, Distilled water 1.0ml,
5mM potassium ferricyanide
• Stains: AchE stain
• Clinical significance: neural tube defects, congenital absence of colonic ganglion cells, Hirschsprung’s
disease.
Mono amine oxidase

• Principle: This enzyme is involved in the breakdown of adrenaline and 5 -hydroxytryptamine. It is

demonstrated by the oxidation of tetra nitro- blue tetrazolium (TNBT).
• Procedures:
• 1. Place sections in incubating medium at 37°C for 45 minutes.
• 2. Wash in running tap water for 2 minutes.
• 3. Place sections in 10% formol-saline for 30 minutes.
• 4. Wash well in tap water for 2 minutes.
• 5. Mount in glycerin jelly.
• Results: Monoamine oxidase activity bluish black
• Reagents: Tryptamine hydrochloride 25 mg, Sodium sulfate 4 mg, Tetra nitro-blue tetrazolium
(TNBT) 5 mg.
• Stains: tetrazolium stain
• Clinical significance: MI
 Phosphorylase

• Principle: phosphorylase catalyzes the breakdown of glucose-1 phosphate to glucose and phosphate ions.
The glucose is subsequently stained with iodine to give a purple color.
• Procedures:
• 1. Incubate in solution at 37 oC for 90 minutes.
• 2. Wash in 40% ethanol for 5 seconds; air dry.
• 3. Fix in ethanol for 3 minutes; air dry.
• 4. Wash in 1:30 Lugol’s iodine for 5 minutes.
• 5. Mount in 9:1 glycerine jelly/Lugol’s iodine.
• Results: Phosphorylase activity blue/black
• Reagents: 0.1 M acetate buffer, pH 5.9 100 ml, 0.1 M magnesium chloride 10 ml, Glucose-1-
phosphate 1 gm, Glycogen (oyster/rabbit liver) 20 mg, ATP salt 50 mg, Sodium fluoride 1.8 gm,
Ethanol 20 ml, Polyvinyl pyrrolidine 9 gm
• Stains: phosphorylase stain
• Clinical significance: McArdle’s disease
Aldolase

• Principle: based on the fact that the enzyme splits hexose diphosphate into
two molecules of triose phosphate. The latter hydrolyzes spontaneously at an
alkaline reaction, and the phosphate liberated is visualized much as in the
regular method for alkaline phosphatase.
• Procedures:
• Reagents:lodoacetate and fluoride, etc.
• Stains:
• Clinical significance: muscle or Liver damage
Sulfatase

• Principle: demonstrates the sulfate ion by precipitating it with benzidine;

the latter is then demonstrated with y8-naphthoquinone sulfonate.
• Procedures: Incubation of fresh tissue in the presence of 6-bromo-2-
naphthylsulphate for 3 hours gives a very pale blue particulate
localization of the stain for aryl sulfatase.
• Reagents: benzidine, acetone, 6-bromo-2-naphthylsulphate
• Stains:
• Clinical significance: lysosomal storage d/o