Special stains lecture 1 (1)

Special StainsSpecial StainsDr.Sara Seif el Din

Stains for collagenStains for muscleStains for elastic tissueStains for reticulin fibresStains for carbohydratesStains for amyloidStains for lipidStains for pigments & mineralsStains for nerve tissueStains for microorganismsStains for decalcified bone

Broadly classifying the special stains under following category…..

Demonstration of Collagen

o Massons trichrome staino Van Geisons stain

MASSON'S TRICHROME STAIN

PRINCIPLE: The term ‘trichrome stain’ is a general name for a

number of techniques for selectively demonstration of muscle, collagen fibers, fibrin, and erythrocytes.

The general rule in trichrome staining is that the less porous tissues are colored by the smallest dye molecule; whenever a dye of large molecular size is able to penetrate, it will always do so at the expense of the smaller molecule.

Human skin Results

Nuclei Blue- Black

Cytoplasm, keratin, Redmuscle fibers, intercellular fibers RBCs, Fibrin Collagen, mucous, Blue Cartilage, Amyloid,adult or mature bone

Van Gieson’s Stain (1889)

o Van Gieson’s mixture of picric acid and acid fuchsin

o The simplest method for the differential

staining of collagen.

Principle Picric acid is employed. It provides acidic

pH and acts as a counterstain for muscle and cell cytoplasm. It forms with dyes a complex which has affinity for collagen.

Results:

Collagen – deep red Muscle, – yellow cornified epithelium Nuclei – blue to black

Demonstration of muscle striations

- Haematoxylin and eosin and trichrome methods can demonstrate muscle striations.

- They may also be stained by using Heidenhain iron haematoxylin and Mallory’s phosphotungstic acid haematoxylin. Both these methods will give better definition of muscle striations than the trichromes.

Mallory's PTAH stain

Purple skeletal muscle striations in tumor with rhabdomyoblasts.

Demonstration of elastic tissue fibres

- Numerous techniques have been evolved for the demonstration of elastic tissue fibres, although few are in current use.

- Of these, the most popular are . Verhoeff , . Orcein, . Weigert’s resorcin-Fuchsin, . Aldehyde fuchsin.

Verhoff’s Elastic Stain

Verhoeff's Elastic stain is used to demonstrate pathologic conditions such as atrophy, breaks, thinning, loss etc. in elastic fibers.

•Principle

•The tissue is overstained with a soluble lake of hematoxylin-ferric chloride-iodine. Both ferric chloride and iodine serve as mordants, but they also have an oxidizing function that assists in converting hematoxylin to hematein.

• The mechanism of dye binding is probably by formation of hydrogen bonds, but the exact chemical groups reacting with the hematoxylin have not been identified.

• This method requires that the sections be overstained and then differentiated, so it is regressive.

Results : Elastic Fibres – Bluish

Black to Black Nuclei - Blue to Black Collagen - Red Other tissue elements –

Yellow

This section shows elastic cartilage stained with hematoxylin and potassium iodine to reveal elastic fibers (arrow), which are dark-stained linear structures embedded in the cartilage matrix.

Elastic fibres• Elastic fibres are thinner than collagen fibres

and are arranged in a branching pattern to form a three dimensional network. They give tissue the ability to cope with stretch and distension. Elastic fibres are interwoven with collagen fibres in order to limit distensibility and to prevent tearing.

• Elastic material is found in certain ligaments (elastic ligaments), some cartilage (called elastic cartilage) and in large arteries (elastic arteries). Instead, the elastin is laid down in fenestrated (having gaps or openings) sheets or lamellae arranged in concentric layers between layers of smooth muscle.

Methods of demonstartion

• aldehyde Fuchsin Elastic Stain

Movat’s Pentachrome Stain

Verhoeff’s Elastic Stain

Demonstration of reticulin fibres:

Reticulin fibres are demonstrated either by using dyes dyes as means of coloring agent or by metal impregnation methods.

techniques :1.Gordon and sweet’s method, 2. Gomori’s method,

Silver impregnation is the best method because it

provides good contrast enabling even the finest fibers to be resolved.

Wilder’s Reticulin Stain

o Used to demonstrate reticular fibers in tissue sections

o Differential diagnosis of carcinomas, sarcomas, and lymphosarcomas.

o Certain liver diseases where a change from normal reticular fiber pattern is seen.

Resulto Reticulin fiber – Blacko Collagen – Rose color o Other tissue elements

– Depending on counter stain used

• Gomori Stain for Reticular Fibers

Gordon & Sweets Stain for Reticular Fibers

Diagnostic applications

• Liver cirrhosis• Liver fibrosis

• Stains for carbohydrates ?• Mechanism of PAS technique ?• Differentiation of the mucin by

different stains ?• Clinical application of PAS stain?

STAINS FOR CARBOHYDRATE

o Periodic Acid Schiff Staino Alcian Blue o Mucicarmine Stain – Southgate’s

 PERIODIC ACID SCHIFF (PAS )FOR THE DEMONSTRATION OF GLYCOGEN 

• McManus, 1946)

• Glycogen is a simple intracytoplasmic polysaccharide found in greatest amount in liver, cardiac and skeletal muscle, with significant quantities also present in hair follicles, endometrial glands, vaginal and cervical epithelium, umbilical cord, neutrophil leucocytes and megakaryocytes.

• PRINCIPLE 

• Tissue containing glycol groups or their amino or alkylamino derivatives are oxidized by periodic acid to form dialdehydes, which combine with Schiff's reagent to form an insoluble magenta compound.

• The chromphoric groups of basic fuchsin in Schiff's reagent are broken by sulphuration to form a colorless solution. In the presence of free aldehyde groups an insoluble colored compound similar to the original dye is formed.

Results:

PAS positive substances - magenta Nuclei - blue

PERIODIC ACID SCHIFF (PAS) WITH DIASTASE FOR THE ENZYME EXTRACTION OF GLYCOGEN

PRINCIPLE Glycogen is digested with certain forms of amylase. Commercially available diastase, which á amylase or salivary amylase from saliva can be used to digest glycogen in tissue sections.

Purpose : - Glycogen is present in mucosa, skin, liver,

parathyroid gland and skeletal & cardiac muscle. - The PAS stain is used for demonstration of

basement membrane, mucosubstances secreted by the epithelia of various organs, fungi, etc.

• RESULTS Presence of glycogen will be evidenced by loss of staining after enzyme treatment when compared to the untreated sections.

PAS StainPAS with Diastase

Glycogen in Ewing Sarcoma

Note

o The most important PAS positive carbohydrates in tissues are polysaccharides (glycogen),Neutral mucopolysaccharides, mucoproteins, glyco proteins, and glycolipids.

o Acid mucopolyssacharides are only weakly positive or negative.

o The PAS reaction can be used to demonstrate many other normal and pathological tissue constituents, the most important of which are amyloid, basement membrane, cartilage, cerebrosides, epithelial mucins, fungi, hyaline membrane of neonatal lung, lipochrome pigments, mucoid cells, mucoid granules, zymogen granules, starch, thyroid colloid etc.

PAS stain for fungi

o The fungal cell wall contains polysaccharides that are oxidized by the periodic acid to aldehydes.

o The aldehydes react with Schiff's reagent to stain the fungi rose pink.

Candida Hyphae in chronic hyperplastic Candidosis

PAS-positive materials can simply be recognized by their shape (morphology) ,eg.Fungal hyphae

Clinical Application of PAS Stain

o Salivary Gland Tumors

o Soft tissue tumors Alveolar Soft-Part Sarcoma (Diastase

resistant crystals)

o Fungal Infection Candidiasis Actinomycosis

ALCIAN BLUE Ph2.5 – ACID MUCOPOLYSACCHARIDES Alcian blue is a group of polyvalent basic dyes that are water soluble. The blue color is due to the presence of copper in the molecule.

Acid mucin - strongly sulphated connective tissue mucins react at low pH values with suitable cationic dyes and are usually PAS-negative.

Submandibular salivary gland mucous cell showing sulfomucin Brown black, sialomucin blue, -Iron alcian blue technique

RESULTS: Acid mucins /mucosubstances - blue

Nuclei (using Nuclear fast red) -reddish pink

Background - Very pale red or colourless

Colonic mucosa showing sialomucin that have acid and neutral mucopolysaccharide stain purple

The goblet cells of the gastrointestinal tract are filled with abundant acid mucin and stain pale blue with this Alcian blue stain.

MUCICARMINE STAIN – SOUTHGATE’S – MUCIN

PRINCIPLE :-

Mucicarmine has large molecular size which allows the dye to penetrate and bind to acidic substrates of low density, i.e. mucins. Neutral mucins and some strongly acidic sulphated mucins do not show appreciable staining.

Colonic Mucosa showing sialomsucin stain -- - magenta

RESULTS:Mucin - deep rose

Nuclei - black

Other tissue elements- yellow

Mucoepidermoid carcinoma, mucin positive

Demonstration of amyloid

o Congo redo Thioflavin To Crystal Violet

Congo-Red - Putchler’s Modification – Amyloid

Principle : Amyloid is homogeneous and eosinophilic, the

deposits are extracellular and may become sufficiently large enough to cause damage to surrounding tissues. When stained with the congo red stain the amyloid, with aid of polarizing lenses, will birefringe an apple green color, under the microscope.

Purpose : To demonstrate amyloid deposits in tissue

sections

RESULTS:

Amyloid -red to pink

Nuclei -blue

Puchtler and Sweat Congo Red Method

Polarised Light showing“apple green”birefringence of amyloid deposits

Positive red staining is present around the large central artery and a smaller vessel to its upper right. The right panel shows the green birefringence that is diagnostic of amyloid when the Congo red stain is viewed with polarized light. All amyloids have a fibrillar ultrastructure that gives this reaction.

Stains for demonstration of lipid and principle?

Stains for Lipid - Oil Red O - Sudan Black B

PRINCIPLE :

Staining with oil-soluble dyes is based on the greater

solubility of the dye in the lipid substances than in

the usual hydroalcoholic dye solvents.

sebaceous gland stained with oil red O stain

Cytoplasm – red

Nuclei - blue

Oil red O stain of fat emboli in lung.

RESULTS:

Fat blue -black Nuclei -red

VON KOSSA METHOD FOR CALCIUM

PRINCIPLE: Tissue sections are treated with silver

nitrate solution, the calcium is reduced by the strong light and replaced with silver deposits, visualized as metallic silver.

PURPOSE: Abnormal deposits of calcium may be found

in any area of the body. With the H&E stain, calcium appear deep blue-purple.

Coronary artery showing calcified atheromatous plaque

RESULTS : Calcium salts -black

Nuclei -red Cytoplasm -pink

PERL’S- PRUSSIAN BLUE REACTION

PRINCIPLE : The reaction occurs with the treatment of

sections in acid solutions of ferrocyanides. Any ferric ion in the tissue combines with the ferrocyanide and results in the formation of a bright blue pigment called 'Prussian blue" or ferric ferrocyanide.

Hemosiderin, liver, iron stain.

RESULTS: Iron (hemosiderin) -blue Nuclei -red Background - pink

Staining for Microorganism

- Gram staining- Ziehl- Neelsen (ZN) stain - Warthin-Starry for spirochetes- Gomori Methenamine (hexamine) silver for fungi- Giemsa stain for parasite

gram stain of the abscess shows thin, gram positive rods in chains.

Result

Gram positive organism

- blue-black

Gram negative organism - red

Mycobacterium tuberculosis in lung, Ziehl-Neelsen acid fast stain.

Giemsa stain

A stain for hemopoietic tissue and hemoprotozoa consisting of a stock glycerol methanol solution of eosinates of Azure B and methylene blue with some excess of the basic dyes.

o Giemsa stain is used for the histopathological diagnosis of Malaria and other parasites. It is named after Gustav Giemsa, an early malariologist.

o Giemsa stain is also a differential stain. It can be used to study the adherence of pathogenic bacteria to human cells.

o It differentially stains human and bacterial cells pink and purple respectively. It can be used for histopathological diagnosis of malaria and some other spirochete and protozoan blood parasites.

Designed to differentiate blood cells and to stain intracellular parasites in red blood cells and plasma,

e.g. Plasmodium falciparum (malaria parasite).

Toxoplasma gondii

Stains for smears• Blood smears• Mucosal scrapes : buccal ,tongue• Mucinous sample : sputum• Fine needle aspiration

Hematologic stains

• Romanowsky stainsGiemsa stain Leishman stainsWright’s stains

Romanowsky stains

It will stain both nucleus and cytoplasm.

These histology stains are used for blood smear and bone marrow.

Examples of Romanowsky histology stains include Wright's stain, Giemsa stain and Jenner's stain.

These histology stains are based on a combination of eosin and methylene blue. .

PAPANICOLAOU STAIN• Papnicolaou formula

• Harris’s hematoxylinHematoxylin - 5gEthanol - 50mlPotassium alum - 100gDistilled water (5 C) – 1000ml0 0̊Mercuric oxide – 2-5gGlacial acetic acid - 40ml

• Orange G 6Orange G (10% aqueous) – 50mlAlcohol – 950mlPhosphotungstic acid – 0-15g

EA 500.04 M light green SF -10ml0.3 M eosin Y – 20mlPhosphotungstic acid - 2gAlcohol -750mlMethanol -250mlGlacial aceticmacid -20mlFilter all stains before use.

• Results:Nuclei are stained blue while cytoplasm displays varying shades

of pink, orange, yellow and green

• Cytologic image of a scraping of the buccal mucosa. Intermediate squamous cell with sex chromatin body (Barr body) (arrow) lying against the inner nuclear membrane (Papanicolaou stain).

- Cytology smear showing clusters of keratinizing squamous carcinoma indicating metastasis in the lymph node.