special stain

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1. SPECIAL STAINS IN PATHOLOGY Moderator : Dr. B P Bommanahalli Professor and Head

2. Contents : Stains for carbohydrates Stains for Mucin Stains for collagen and Elastic fibres Stains for amyloid Stains for lipid Stains for – Melanin, Calcium, Iron Stains for microorganisms

3. Introduction Stains : chemical substances used to achieve visible color contrast in the microscopic picture of a prepared tissue Staining : treating tissues or cells with a series of reagents so that it acquires a color ; no particles of dye are seen and the stained element is transparent. Dyes : Essentially Aromatic benzene ring compounds or derivatives that possess the twin properties of color and ability to bind to tissue.

4. A polyvalent metal ion which forms coordination complexes with certain dyes. A cts as an intermediary between dye and tissue , T hus increasing the affinity between them.

5. Ac c entua t o rs : Substances which increase the staining power of dye. They increase the intensity & selectivity of stain. e.g KOH in Lofflers methylene blue phenol in carbol fuschin & carbol thionin. Accelerators Accentuators used in metallic impregnation technique for the nervous system. e.g chloral hydrate Trappin g agen t s Agents which holds dye combination with tissue or bacteria . e.g tannic acid/iodine

6. Regressive Staining Tissue is initially overstained and then partially decolorized (differentiated) until the proper endpoint is reached. Sharper degree of differentiation is obtained The differentiation is controlled visually by microscopic examination. Faster and more convenient . Progressive Staining Tissue is stained for a predetermined time for adequate staining of the nuclei and leaves the background tissue relatively unstained. Once the dye is taken up by the tissues, it is not removed. The tissue is left in the dye solution until it retains the desired amount of coloration. The differentiation solely relies on the selective affinity of dyes for different tissue elements.

7. Removal or washing out of excess stain until the color is retained only by the tissue component that is to be studied. Done with acid alcohol or ethyl alcohol Exposure to air may oxidize and improve the process. Differentiation

8. Vital Staining Applied to living tissue Accomplished by injecting staining solution into some part of the body Mixing of stain with living cells Primarily used for research. Routine Staining Stains tissues with minimal differentiation except between nucleus and cytoplasm. Demonstrates general relationship among cells, tissues and organs. e.g Hematoxylin and Eosin stains Special Staining Demonstrates selective features of tissues : Particular cell products. Microscopic intracellular and intercellular structure. e.g PAS stain for mucopolysaccharide.

9. Periodic Acid-Schiff (PAS) stain Principle: Substance containing vicinal glycol groups oxidized by periodic acid into dialdehydes on reaction with schiff’s reagent insoluble purple- magenta compound. Dyes : 1%periodic acid Schiff’s reagent Control : Appendix Result : PAS +ve substance : Purple –magenta color Nuclei : Blue

10. PAS positive substances : Glycogen Neutral mucoprotein Glycoprotein Glycolipid Basement membrane All fungi Phosphorylated sugar Cerebrosides

11. PAS Diastase reaction : glycogen treated with diastase enzyme digest the glycogen and negative PAS reaction. to differentiate glycogen from other PAS positive elements such as mucin

12. Uses of PAS: D emo n s t r a t e gl y c o g e n a n d neut r al mucoprotein. In d i agno s is of poorl y d i f f e r e n t i a t ed ad e no c a r ci n o ma of various tissue like stomach, pancreas, lung. In diagnosis of hepatocellular carcinoma. Seminoma, Dysgerminoma RCC- Renal cell carcinoma: Clear cell type T o demo s t r a t e – i n t r a c y t op l a s m i c c r y st a l i n al v eolar soft part sarcoma T o demo s t r a t e baseme n t m e mb r ane e . g. adenoid c y s tic ca. To demostrate neutral mucin in gastrointestinal tract

13. To demostrate fungi in tissue sample Candidiasis Actinomycosis Hi s toplasm o sis Blastomycosis In diagnosis of ALL- block positivity and AML- diffuse cytoplasmic positivity in M5, M3 and cytoplasmic granular/block positivity in M6 and cytoplasmic granular and blebs positivity of blast in M7.

14. Gastric signet ring cell carcinoma on PAS stain

15. Candida seen on PAS stain

16. Presence of glycogen will be evidenced by loss of staining after enzyme treatment when compared to the untreated sections. PAS Stain PAS with Diastase

17. Mucin T wo major categories- epithelial and stromal. Epithelial mucin (membrane bound or secreted) composed of central protein core and sialic acid sulfated or nonsufated. Stromal mucin known as glycosaminoglycans contain hyaluronic acid may be sulfated or non sulfated . They are better known as myxoid substance. Their functions are lubrication, chemical barrier and cell signaling.

18. The types of mucin are as follows : Neutral - Found in glands of stomach and in prostate . stain with PAS but not with Alcian blue, colloidal iron and mucicarmine. Acid (simple, or non-sulfated ) – typical mucins of epithelial cells containing sialic acid. They stain with PAS, Alcian blue at pH 2.5, colloidal iron. E.g . salivary glands. Acid (simple, sulfated -mesenchymal ) – These contain hyaluronic acid and are found in stroma. do not stain with PAS , stain with Alcian blue at pH 2.5, colloidal iron

19. Acid (complex, epithelial ) – These are found in adenocarcinomas of colon, breast,ovary , mucoepidermoid carcinoma . PAS is usually positive . Alcian blue is positive at pH 1 , Acid (complex, connective tissue ) – Found in tissue stroma, cartilage, and bone substances such as chondroitin sulfate or keratan sulfate. They are PAS negative stain selectively with Alcian blue at pH 0.5.

20. Stains for mucin Alcian blue PAS Mayer mucicarmine Hale colloidal iron stain

21. Alcian blue PRINCIPLE Alcian blue is a copper phthalcyanin dye C ontains positively charged groups capable of salt linkage with polyanions of the acid mucins By varying the pH of the solution of Alcian blue more information can be gained concerning the types of acid mucin present . It does not stain neutral mucin .

22. Dyes : 1%Alcian blue in 3%acetic acid Results : Acid MPS – deep blue Nuclei – faint blue PAS-Alcian blue is best ‘ pan-mucin ’ stain combination , it demostrates mucin both neutral, slightly acidic and highly acidic.

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

24. Colonic mucosa showing sialomucin that have acid and neutral mucopolysaccharide stain purple on alcian blue-PAS.

25. USES T o differentiate gastric metaplasia into complete and incomplete . Complete metaplasia is small intestinal type , shows well defined brush border and well formed goblet cells. I ncomlete metaplasia is colonic type and shows multiple irregular mucin droplets of varying size in the cytopla s m and absence of brush border. D iagnosis of pleomorphic adenoma, mucoepidermoid carcinoma, in stroma of chordoma at sacrococcygeal region. Excessive amounts of non-sulfated acidic mucosubstances are seen in mesotheliomas .

26. Mixed salivary gland. Acid mucopolysaccharides in mucous cells are stained blue (Alcian blue at pH 2.5).

27. Chordoma on alcian blue stain at ph 2.5

28. MUCICARMINE STAIN PRINCIPLE :- Positively charged carmine complex bonds with the negatively charged acid mucin . Strongly sulphated mucins are variable in their reaction , neutral mucin do not stain at all , acidic mucin stain strongly.

29. REAGENTS: Southgate's Mucicarmine Solution: Carmine, alum lake Aluminum hydroxide 50% alcohol -1.0 gm -1.0 gm -100.0 ml Aluminum chloride, anhydrous- 0.5 gm Mayer's Hematoxylin:

30. Uses: Mucicarmine is useful to identify acidic mucin. It is used to identify adenocarcinoma particularly of gastrointestinal tract. It also stains the capsule of fungus- cryptococcus neoformans lung & nervous tissue of immunocompromised patient.

31. Col o ni c Mu c o s a sh o wing sial o msucin -- - magenta RESULTS: Mucin - magenta Nuclei - black Other tissue elements- yellow

32. Cryptococcus neoformans in lung of AIDS patient capsule stains red

33. HALE’S COLLOIDAL IRON STAIN Positive staining with hale’s colloidal iron stain is considered diagnostic feature of chromophobe renal cell carcionoma and has been used as discriminatory feature to differentiate it from other renal tumour. Result Acid mucopolysaccharides: blue Nuclei: red

34. Classic chromophobe RCC classic chromophobe RCC on hale’s colloidal iron stain Eosinophilic chromophobe Eosinophilic chromophobe on hale’s colloidal iron stain e.oncocytoma f. oncocytoma diffusely negative cytoplasmic reaction with hale‘s colloidal iron stain

35. RETICULIN STAIN-GOMORI’S METHOD Reticulin stain demonstrate both reticular fibers and basement membrane material. Reticular fibers consist of very thin fiber of type III collagen Basement membrane are largely composed of type IV collagen and laminin.

36. Princi p le : Reticulin fibers have little natural affinity for silver solutions . On treatment with potassium permangenate it produce sensitised sites on fibers where silver deposition can be initiated . A reducing agent formalin causes deposition of silver in the form of metal.

37. Dyes used: Silver nitrate 10% NaOH 10% KMnO4 1% aqu. oxalic acid 5% aqu Iron alum 2.5% Formalin 10% Control: Cirrhosis of liver Result : Reticular fiber – Black Nuclei- Gray Other elements- According to counter stain used

38. Reticulin fiber- Black Nuclei -Black

39. Uses of reticulin stain Diagnosis of liver cirrhosis. To distinguish epithelial neoplasms from non- epithelial neoplasms. Foci of carcinoma have reticulin around tumour nest but not in between tumour cell, whereas in most sarcomas and large cell lymphoma reticulin separates single cells. To differentiate between in-situ and invasive carcinoma

40. Trichrome stain This stain is mainly used to evaluate the type and amount of extracellular material like- collagen, fibrin, muscle and elastic fiber . Various technique includes: Masson trichrome stain Van gieson stain Mallory, Phosphotungstic or phosphomolybdic acid stain Verhoeff-Van Gieson(VVG) stain

41. The general rule 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.

42. Masson trichome stain This method is used for detection of collagen fibers in the tissues The collagen fibers will be stained blue and T he nuclei - black.

43. Masson trichrome stain Principle: 3 dyes are used which selectively stain muscle, collagen fibers, fibrin and erythrocytes . Acid fuchine stain all the connective tissue , PMA ( phosphomolybdic acid) competes with fuchine and gain access to collagen displacing fuchine . If reaction stopped at appropriate time, collagen will be free to be stained by Methyl Blue.

44. Reaent used – Weigerts iron hematoxylin solution Phosphomolybdic- phosphotungstic acid solution Biebrich scarlet acid fucshin solution Aniline blue solution 1% acetic acid solution Result - Nuclei : Black Collagen : Blue Cytoplasm, Muscle, RBC : Red Positive control : skin, lung, stomach, intestine

45. Result Collagen- Blue Cytoplasm, RBCs- Red Membranoproliferative glomerulonephritis on masson trichome stain

46. Uses It is used to differentiate between collagen and smooth muscle in tumour. To identify increased collagen deposition in condition like cirrhosis, keloid, benign prostatic hyperplasia, membranoproliferative glomerulonephritis etc.

47. VAN GIESON STAIN Principle combined solution of picric acid & acid fuchsin used small molecules of picric acid penetrate all the tissue rapidly , only firmly retained in the close textured red blood cells and muscle . The larger molecules of ponceau S displaces picric acid molecule from collagen fibres, which has larger pores and allow larger molecules to enter. It is used for detection of collagen. Result --

48. R es u l t s: Nuclei : Blue / Black Collagen - Red Cytoplasm , muscle , fibrin, RBCs : Y e l l o w

49. Phosphotungstic acid- Hematoxylene test (PTAH) test: Principle There is much more phosphotungstic acid in the solution th a n hematein . The phosphotungstic acid binds all the available hematein to form blue lake pigment . This lake stains muscle cross striations, fibrin, nuclei, and other tissue elements blue . The rest of phosphotungstic acid stains the red-brown components, such as collagen.

50. USES Traditionally used for demonstration of muscle striations, glial cells and fibrin. This technique has been largely replaced by immunohistochemistry techniques RESULTS Muscle, cross striations : Blue - black Fibrin and neuroglia – Deep blue Connective tissue: Pale orange-pink to brownish red Bone and cartilage- yellowish to brownish red

51. Skeletal muscle striation on mallory’s PTAH stain

52. Gliosis on PTAH stain

53. Verhoeff-Van Gieson(VVG) stain : This method is used for identifying elastic fiber in tissue Result – Elastic fiber : Blue -black to black Nuclei: Blue to black Collagen: Red other tissue elements: Yellow

54. Vessel wall on Verhoeff van gieson stain

55. This section shows elastic cartilage and elastic fibers (arrow), which are dark-stained linear structures embedded in the cartilage matrix.

56. Fibrin Fibrin is formed by polymerization of smaller soluble fibrinogen. Found in tissue damage and acute inflammation, fibrinoid necrosis in vessel wall. Stained by : Mallory PTAH MSB Stain

57. MSB (Maritus, Scarlet, Blue)Stain for fibrin : This is trichrome method for selective demostration of fibrin . Dyes Used : Maritus Yellow Crystal Scarlet Methyl Blue, PTA. Results : Fibrin : Red RBC’S : Yellow Muscle : Red Collagen : Blue

58. Fibrin stains red, collagen stains blue, muscle stains red, nuclei stains blue/black, red cells stain yellow

59. Stain s for Amyloid Amyloids are insoluble protein . They arise from inappropriately folded protein and polypeptides present naturally in the body . Stains used to demostrate amyloid: Congo red Crystal/methyl violet PAS Thioflavin T & S

60. Congo red stain Principle : Amyloids are homogeneous and eosinophillic, proteinaceous deposits, extracellular When stained with the congo red stain the amyloid will show birefringence an apple green color, under the polarizing microscope.

61. Reagent: Solution a - 0.5% Congo red in 50% alcohol Solution b - 0.2% potassium hydroxide in 80% alcohol RESULTS: Amyloid, elastic fibers, eosinophilic granules, --- red to pink Nuclei – blue

62. It is used to demostrate amyloid deposits in R enal amyloidosis- in patients on dialysis for long time Medulary carcinoma of thyroid, Vessel wall in case of Alzheimer’s disease Cardiac arrhythmias, etc.

63. 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.

64. Crystal/Methyl violet for amyloid Crystal violet or methyl violet stain are used for metachromatic amyloid staining . They stain amyloid as purple-red in blue background. Reagents used Crystal/methyl violet 95% alcohol 1% aqueous ammonium oxalate 0.2% acetic acid

65. Amylo i d – purple Background - blue

66. Stains for Lipid Oil Red O Sudan Black B

67. Oil red o stain 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. Result Lipid – Red Nuclei- Blue

68. REAGENTS : 60 % isopropanol Alum hematoxylin: Glycerin Jelly mounting medium Oil Red O working solution: To make oil red O stock solution Oil red O - 0.5gm Isopropanol 100 ml Working solution should be made fresh each time.

69. Fat emboli seen as red dot within capillaries of lung on Oil red O stain

70. Fatty liver -stain Oil red O

71. U s es Oil red O stain is used for staining neutral triglycerides, lipids and lipoprotein. Tumors arising from fat cells (liposarcomas) can be differentiated from other types of tumors. Fat occurring in an abnormal place, such as fat emboli Lipid storage disorder like nieman-pick disease To demonstrate fat or lipids fatty liver. burkitt’s lymphoma and

72. SUDAN BLACK B PRINCIPLE : Sudan Black B is a dye that is insoluble in water but dissolves in fat. Therefore this dye will accumulate in fat globules within cells. It is slightly basic dye and will combine with acidic groups in compound lipids, thus staining phospholipids also. Result Fat- Blue / Black Nuclei- Red

73. REAGENTS : 85% Propylene Glycol: Propylene glycol Distilled water Hematoxylin: -85.0 ml -15.0 ml Sudan Black B/Propylene: Sudan Black B Propylene glycol -0.7 gm -100.0 ml

74. Uses Sudan black is used for staining neutral triglycerides, lipids, lipoprotein and phospholipid. M yeloblast vs lymphoblast .

75. Myeloblast on Sudan black stain

76. MASSON FONTANA METHOD- FOR MELANIN It is a brown-black pigment nonlipid , non hematogenous pigment . present normally in the hair, skin, retina, iris and certain parts of the CNS. PRINCIPLE: Melanin is insoluble in organic solvents but soluble in 1M sodium hydroxide . It is slowly bleached by strong oxidising agents. The solutions of ammoniacal silver nitrate are reduced by melanin to black metallic silver this is the basis of Masson Fontana method for demostrating melanin.

77. Melanin pigment of skin showing RESULTS: Me l ani n , a r g e n t a f fin granules - Black Nuclei -red

78. Melanin pigment in cells of malignant melanoma, Fontana-Masson stain.

79. US E S : To identify melanin and argentaffin granules. Argentaffin granules are found in carcinoid tumors . In diagnosis of malignant melanoma

80. VON KOSSA METHOD FOR CALCIUM PRINCIPLE: Tissue sections are treated with silver nitrate solution , S ilver deposit s by replacing the the calcium and then it is reduced by the strong light and visualized as metallic silver. USE: Abnormal deposits of calcium With the H&E stain, calcium appear deep blue-purple. On von kossa method it appear black .

81. Coronary artery showing calcified atheromatous plaque RESULTS : Calci u m salt s -b l a c k Nuclei -red Cy t op l asm -p i nk

82. PERL’S- PRUSSIAN BLUE PRINCIPLE : Dilute mineral acid hydrolysis releases ferric iron from protein bound tissue deposits I n the presence of ferrocyanide ions , It is precipitated as highly coloured and highly water soluble complex , P otassium ferric ferrocyanide- prussian blue. Ferrous ion do not produce a coloured reaction. Tissue deposits containing ferric iron are invariably hemosiderin

83. US E : To demonstrate ferric iron in tissue sections. hemochromatosis - with deposits found in the liver and pancreas hemosiderosis - with deposits in the liver, spleen, and lymph nodes. To a s ess the bone marrow iron content CONTROL : Postmortem lung – high number of heart failure cells that contain hemosiderin

84. R E A GEN T S: 2% Potassium Ferrocyanide: Potassium ferrocyanide Distilled water Neutral-fast Red: - 2.0 gm - 100.0 ml 1gm - 100 m l 1ml Neutral red Distilled water Glacial acetic acid 2% Hydrochloric Acid: Conc. Hydrochloric acid, Distilled water - 2.0 ml - 100.0 ml

85. Hemosiderin in liver RESULTS: I r o n ( hemos i der i n ) -b l ue Nuclei -red Background - pink

86. Staining for Microorganism Ziehl- Neelsen (ZN) stain Wade fite faraco stain Gomori Methenamine (hexamine) silver stain Warthin-Starry stain Giemsa stain Gram staining

87. Ziehl-Neelsen (ZN) Stain for Mycobacterium Mycobacteria are difficult to demonstrate by the Gram technique because they possess a h ydrophobic capsule . Phenolic acid or heat may be used to reduce the surface tension and increase the porosity.

88. PRINCIPLE Mycobacterias (tubercle bacilli) have a lipid-rich cell wall C apable of taking up strong phenol dye solutions (eg. carbol fuchsin solution ) D ye is retained upon subsequent differentiation in acid or alcohol . They are said to be acid fast bacilli. Results Mycobacteria - Red Background - pale Blue

89. M e thod Bring the section to water level. Flood sections with carbol-fuchsin and heat to steaming by passing the flame of spirit swab underneath the slides on metal rack till, vapour just being formed. Wash the slides in distilled water. Shake off excess liquid. Decolourise the slides with 20% H2SO4 Wash well in tap water, till no more red colour runs off the surface when the slides is dipped in water Wash thoroughly with water.

90. Counter stain in methylene blue solution, 30 seconds. Blot and differentiate by alternate dehydration and rehydration until the background is a delicate pale blue. Examine microscopically screening at high power and confirming all suspicious organism with an oil immersion lens.

91. Mycobacteria seen as red rods

92. Acid fast bacteria seen on intestinal biopsy

93. Wade fite faraco stain This stain is used for staining leprocy bacilli in tissue sample. Leprocy bacilli are much less acid and alcohol fast than tuberculous bacilli . 10 % sulphuric acid is used as decolouriser in place of acid-alcohol solution . The section are also deparaffinised using ground nut oil and xylene mixture to protect more delicate waxy coat of lepra bacilli.

94. PROCEDURE Warm section and deparaffinised using mixture of two part xylene and one part of ground nut oil for 10 minutes. Blot dry and wash in water untill section is uniformly wetted. Stain with carbol fuschin for 20-30 minutes. Wash in tap water and blot dry. Decolorize in 10% sulphuric acid till no more red colour appear while dipping in water . Wash in tap water and counter stain with 0.2% methylene blue for 5-10 seconds. Blot dry and do not mount. Smears should be seen in oil immersion lens.

95. Leprocy and other mycobacteria- red Background- blue

96. Warthin-Starry method for spirochetes (Warthin & Starry 1920) PRINCIPLE Organisms are demonstrated by silver impregnation technique – based on the ability of certain organisms to bind to silver ions. It can also be used to demostrate Helicobacter pylori in gastric mucosa Leptospira organism in renal biosy Cat scratch disease – Bartonella Sp .

97. Result : Organisms - black Ba c kg r oun d - g ol d en yellow C a t s c r a t ch d is e a s e b a c i lli i n ly mph n o de b i op sy

98. H. Pylori on gastric mucosa on Warthin starry stain

99. Gomori methenamine silver stain GMS staining is a silver staining technique for demonstrating fungi in tissue sections. based on staining the polysaccharides in fungal cell walls , can be used to demostrate the basement membrane. PRINCIPLE This method depends upon the reduction of the silver by the aldehyde groups produced after oxidation of fungal wall components with chromic acid.

100. Pneumocystis carinii R esult: Fungi, pneumocystis carinii, melanin - black Mucin and glycogen- - grey-black RBCs - yellow Background - pale green

101. GMS stain for Cryptococcus neoformans

102. Basement membrane on GMS stain

103. Gram method for Bacteria Gram staining differentiate bacteria into 2 classes depending on their cell wall structure and composition Gram positive and Gram negative . PRINCIPLE Crystal Violet stains the nucleic acids of the bacteria (and background tissue ) after treatment with iodine, the sections are differentiated in acetone counterstained with basic fuchsin . The tissue background and Gram- negative bacteria lose their blue staining and are subsequently stained with counter stain basic fuchsin .

104. Gram-positive bacteria resist the decolourisation and retain the crystal violet/iodine blue staining. Result : Gram-positive organisms - blue Gram – negative organisms - red .

105. Gram Positive Gram Negative

106. Cutaneous anthrax – Gram positive Bacilli stained blue on skin biopsy

107. References Horobin R W , How histological stains work in : Suvarna S K, Layton C, Bancroft J D, Bancroft’s Theory and practice of histological techniques, 7 th ed , Elsevier, p.157-72. Introduction to staining and principles of staining, in : Shariff S, Kaler A K, Principles & interpretation of laboratory practices in surgical pathology by shameem shariff , 1 st ed , Jaypee , p.79-95

108. Thank you