Role of Histochemistry and Immunohistory in Diagnostic Patho

8/21/2019 Role of Histochemistry and Immunohistory in Diagnostic Patho

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ROLE OF HISTOCHEMISTRY AND

IMMUNOHISTORY IN DIAGNOSTIC PATHOLOGY

• The mainstay in surgical pathology is the

examination of the specimen after fixation informalin, embedding in paraffin and staining with

hematoxylin and eosin.

• Special techniques are used for difficult cases.This includes histochemistry, immunohistoche-

mistry, electron microscopy, etc.

• Histochemistry is time and cost effective.


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MUCIN STAINS

• There are a variety of mucin stains todemonstrate different types of mucopoly-

saccharides in tissue, example colloidal iron,

Alcian blue, PAS and mucicarmine.


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TYPES OF MUCIN

1. Neutral

* found in glands of GIT e.g. stomach, lung and

prostate.

* will stain by PAS stain BUT NOT by Alcian blue,

colloidal iron or mucicarmine.2. Acid mucin (simple, non-sulfated)

* found in epithelial cells containing sialic acid e.g.

goblet cells of small intestine.

* stain with PAS, Alcian blue at pH 2.5 and colloid

iron and resist digestion by hyaluronidase.


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Type of Mucin (contd)

3. Acid (simple, mesenchymal). These contain hyaluronic acidand are found in tissue stroma.

* stain with Alcian blue at pH 2.5 and colloidal iron BUT

NOT by PAS.

* hyaluronidase sensitive (useful to differentiate mesothelioma

from adenocarcinoma.

4. Acid (complex or sulfated, epithelial) mucin.* found in the colonic epithelium and adenocarcinoma.

* PAS is usually weakly positive and they diastase digestion.

* Alcian blue is positive at pH1 and pH2.5 and positive forhigh iron diamine.

* Colloidal iron, mucicarmine and metchromatic stains are

also positive. They resist digestion with hyaluronidase.


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Type of Mucin (contd)

5. Acid (complex, connective tissue).* found in tissue stroma, cartilage and

bone e.g. chondroitin sulfate or keratinsulfate.

* there are PAS negative but they do stainselectively with Alcian blue at pH 0.5.


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STAINS FOR MUCIN

A. Colloidal iron – iron particles are stabilized in amonia and

glycerin and are attached to acid mucopolysaccharides. The

actual blue comes from a Prussian blue reaction.

B. Alcian blue – the pH of this stain can be adjusted to give more

specificity.

C. PAS (periodic acid-Schiff) – this stains glycogen as well as

mucins, but tissues can be pre-digested with diastase (oramylase) to remove glycogen. It stains neutral mucin,

basement membrane, most types of fungi and parasites.

D. Mucicarcmine – this is very specific for epithelial mucins. Themucin stain with the most specificity is mucicarcmine, but it is

very sensitive, so it is not really very useful. The stain that is

most sensitive is PAS.


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STAINS FOR BIOGENIC AMINES

Cell that produce polypeptide hormones, activeamines, or amine precursors (epinephrine,

noreepinephine) can be found individually

(Kulchistky cell of GI tract) or as a group (adrenal

medulla).

Staining patterns based upon the ability of the cells

to reduce ammniacal silver nitrate to metallic silver

(black deposit in tisuse section)


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STAINS FOR BIOGENIC AMINES

A] Chromaffin.

Types of stains for chromaffin include:

1. Modified Giemsa.

2. Schmorl’s.

3. Wiesel’s.

B] Argentaffin.

Types of stains for argentaffin include:

1. Diazo (diazonium salts).2. Fontana-Masson.

3. Schmorl’s.

4. Autofluorescence.C] Argyrophil (pre-reduction step necessary).

Types of stains for argyrophil include:

1. Grimelius (Bouin’s fixative preferred).

2. Pascual’s.

MELANIN STAINS


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MELANIN STAINS

• Melanin is normally found in the skin, eye and substantia nigra.It may also be found in tumors such as malignant melanomas.

Fontana-Masson reduce ammoniacal silver nitrate.

• Schorml’s method uses the reducing properties of melanin tostain granules blue-green.

• The most specific method of all is an enzyme histochemical

method called DOPA-exidase.• Bleaching techniques remove melanin in order to get a good

look at cellular morphology.

• Formaldehyde-induced fluorescence can be used to highlight biogenic amines and autofluorescence.

• The pseudomelanin pigment of melanosis coli is PAS positive

whereas true melanin is not.


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LIPOCHROME (LIPOFUSCIN)

PIGMENTS

• There are the breakdown products within cellsfrom oxidation of lipids and lipoproteins.

• Lipochrome can be stained by Sudan Black B,long Ziehl-Neelsen acid fast and schorml’s

methods. Lipochrome may also exhibit astrong orange autofluorescence in formalin-

fixed, unstained paraffin sections.


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IRON (HEMOSIDERIN)

• Hemosiderin (storage iron granules) may be present in areas of old hemorrhage or be

deposited in tissues with iron overload(hemosiderosis and hemochromatosis).

• Perl’s iron stain is the classic method for

demonstrating iron in tissues. The section is

treated with dilute hydrochloric acid to

release ferric ions from binding proteins.These ions then react with potassium

ferrocyanide to produce an insoluble bluecompound (the Prussian blue reaction).


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CALCIUM

• Only calcium that is bound to an anion (such as phosphates or carbonates). Calcium forms a

blue-black lake with hematoxylin to give a bluecolor on H & E stain, usually with sharp edges.

• Von Kossa stain is a silver reduction method

that demonstrates phosphatase and carbonates,

but there are usually present along with calcium.

This stain is most useful when large amounts are present, as in bone.


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URATES

• In tissue, urates are present as sodium urate.

They are soluble in aqueous solutions and

slightly soluble in weak alcoholic solutions.Therefore, tissues must be fixed in 95% or

absolute alcohol to prevent leaching ofurates.

• Methenamine silver stains urates black.

• Sodium urate crystals are also birefringent

on polarization.


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Slide 28.1


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EXOGENOUS PIGMENTS AND MINERALS

• These comes from industrial or environmental exposure by

inhalation, ingestion or contact. Sometimes, exposure comes

from work-related activities (miners). Sometimes they are planned, e.g. tattoo.

• Carbon appears as anthracotic pigment in the lungs. It can

be distinguished from melanin by doing a melanin bleach.Poorly fixed tissues may contain formalin-heme pigment,

which is black and finely granular, but this is widely

scattered in the tissues without regard to cellular detail.Formalin-heme pigment is also birefringent on polarization.


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Exogenous pigment (contd)

• Asbestos is a special type of long, thin silica crystal,

usually of the mineral group chrysotile. In tissue, these

crystals are highly irritative and highly fibrogenic. The

fibers become coated with a protein-iron-calcium matrix,

giving them a shish-kebab or dumbbell-shaped appearance.These are called “ferruginous bodies: because they are

stained blue using the iron stain (Prussian blue).



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• Silica is present in many minerals and building materials.

Most forms are very inert and cannot be stained in tissue

but can be demonstrated by white birefringence on

polarization. It is most often present in lung, but can make

its way into lymph node.

• Street drugs for injection often are diluted with compounds

containing minerals such as silica or talc. Lymphoreticular

tissues and are also be found in the lung.

• Tattoo pigment is usually black and is inert and non-

polarizable. Red tattoo pigment often contains cinnabar

(which has mecury in it).• In general, minerals are best demonstrated or identified by

microincineration techniques or by scanning electron

microscopy with energy dispersive analysis.


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MICROORGANISMS

• Bacteria appear on H & E as blue rods or cocci regardless of

their gram reaction. Colonies appear as fuzzy blue clusters.

Tissue gram stains are all basically the same as that used in the

microbiology lab except that neutral red is used instead ofsafranin. Gram positive organisms usually stain well, but gram

negatives do not.


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Spirochetes are very difficult to stain. The best

method is the Warthnin-Starry. Steiner stain isvery good at identification of helicobacter

pylori. The Giemsa stain is also useful in

demonstrating donovan bodies and leishmania.

Intestinal spirochetosis occurs in AIDS

patients, and is readily identified without theaid of special stains, it is found as a fuzzy

basophilic band along the luminal aspect of

the mucosa, but avoid attachment to the goblet

cells.


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ACID FAST BACILLI (AFB) STAIN

• The AFB or Ziehl-Neelsen stain uses carbol-fuchsin to stain the lipid

walls, binding to the mycolic acid moieties of acid fast organisms

such as mycobacterium tuberculosis. Once the carbol-fuchsin attaches,

it resists acid alcohol decolorization, hence the term “acid fast bacilli”.

A modification of this stain is known as trhe Fite stain and has a

weaker acid for weakly acid fast bacilli such as mycobacterium leprae.The most sensitivie stain for mycobacteria is the Auramine-

Rhodamine stain.


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ACID FAST BACILLI (AFB) STAIN

• There are things other than mycobacteria that are acid fast.Cryptosporidium in a wet stool prep is AFB positive,

although these organisms are not AFB positive on a

surgical specimen that has been processed. The hookletsof cysticerci are AFB positive. Patients with lead

poisoning may slough off renal tubule epithelial cells, and

the cells containing lead are AFB positive.


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FUNGAL STAINS

Fungi stains blue with H & E, red with the PAS stain and

black with the Gomori methenamine silver stain.


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IMMUNOHISTOCHEMISTRY


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IMMUNOHISTOCHEMISTRY AS A

LABORATORY TEST

• The objective of immunohistochemistry isto use antibodies to identify antigens,

imparting a much greater specificity of the

stain to the tissue with which it reacts. Indoing so, immunohistology has transformed

surgical pathology from a highly subjectivediscipline into a much more objective

science.

ADVANTAGES OF IMMUNOHISTOCHEMISTRY


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• Identification of the microanatomic (cellular) location of the

antigen.

• Can identify the lineage of cell populations.

• Defining distinct populations of cells within the same lineage

• Defining functional differences.

• The technique has had success as a factor in selecting treatment

regimens.• Markers useful in this regard include tumor cell proliferation

markers, cell cycle regulators, oncogene and tumor suppressor

gene products, microvessel density determinations and occut

metastasis determination.

• To determine the presence of infectious agents.

• This technique preserves the histologic architecture and enablesthe pathologist to confirm that the positive cells are the cells in

uestion.

TECHNICAL CONSIDERATIONS


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• The quality of an immunohistochemical stain depends onthe integrity of an antibody-antigen interaction and

depends on the extent to which the relevant antigen has

been preserved during tissue fixation and processing.• The reagents and techniques employed need optimization

and thorough validation to ensure consistent, reliable and

clinically meaninful results.• Premanufactured kits.

• Automated staining.

• Positive and negative controls.

• Results and reporting.


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LIMITATATIONS OFIMMUNOHISTOCHEMISTRY

• Experience.

• Availability of antibodies.• Loss of antigenicity in stored cut paraffin

sections.• Antigen retrieval.

CURRENT APPLICATIONS OF IMMUNOHISTOCHEMISTRY


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Immunohistochemistry as a Diagnostic Tool:

Define origin.

1. Establish prognosis.

2. To determine treatment and response.

• Much more common is the diagnosis of tumor of uncertain origin.

Such situations include when:

1. The tumor is identified first in a metastatic site and the primary

site is not apparent.

2. The tumor is so poorly differentiated that no specific morphologic

appearance of the tumor is compatible with more than one pattern.

3. The histogenesis of a tumor is clear (e.g. adenocarcinoma) but the

primary site is in question.

Current applications (contd)


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• Test selection

• Keratin, vimentin, CD45, S-100

• Keratin-positive tumors

• Keratin-negative tumors

* Lymphomas

* Melanomas

* Sarcomas and Soft tissue tumors* Myogenic sarcomas

* Fibrohistiocytic tumors

* Neurogenic tumors* Normal and neoplastic vessels

* Anaplastic spindle cell tumors

* Neural and neuroendocrine tumors

* Glial fibrillary acid protein-positive tumors


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PROGNOSTIC MARKERS IN CANCER


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• Microinvasion and pseudoinvasion• Occult metastases

• Tumor cell proliferation

• Oncogenes growth factors and receptors

* Her-2/neu

• Tumor suppressor genes and gene products

* Rb

* p53

* Cyclin dependent kinase inhibitors

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Role of Histochemistry and Immunohistory in Diagnostic Patho