Special stains in histopathology

1. SPECIAL STAINS DR. EKTA JAJODIA

2. H&E stain is routine stain. - It is the preliminary or the first stain applied to the tissue sections - Gives diagnostic information in most cases. A special stain is a staining technique to highlight various individual tissue component once we have preliminary information from the H&E stain

3. Classification 1. Stains for carbohydrates 2. Stains for amyloid 3. Nucleic acid stains Lipid stains Stains for microorganisms Connective tissue stains Stains for pigments and minerals

4. CARBOHYDRATES SIMPLE CARBOHYDRATES (molecules composed purely of carbohydrates) Monosaccharides ( glucose,mannose,galactose ) Oligosaccharides ( sucrose,maltose ) Polysaccharides ( glycogen,starch ) GLYCOCONJUGATES (molecules composed of carbohydrates and other molecules such as protein and lipid) Proteoglycans Mucins Others glycoproteins

5. 90-95% of their molecular weight is due to the carbohydrate component The carbohydrate component is known as glycosaminoglycans (GAG) A GAG is composed of repeating disaccharide units , each made up of 2 different monosaccharides Each disaccharide is composed of a carboxylated uronic acid ( glucuronic or iduronic acid) and a hexosamine such as N- acetylglucosamine or N- acetylgalactosamine TYPES OF GYCOSAMINOGLYCANS Chondroitin sulfate Dermatan sulfate Keratan sulfate Heparin sulfate Heparin Hyaluronic acid PROTEOGLYCANS

6. MUCINS Neutral mucins : surface epithelia of gastric mucosa brunner’s glands prostatic epithelia Sialomucins : bronchial submucous glands goblet cells salivary glands 3. Sulfomucins : bronchial mucous glands Sialomucins and sulfomucins are acidic mucins

7. FIXATIVES Tissue placed in fixative promptly after removal – capable of autolytic reaction If immediate fixation not possible, tissue refrigerated until adequate fixation possible Recommended fixatives : Rossman’s fluid Alcoholic formalin with picric acid Glutaraldehyde fixatives avoided free aldehyde groups are capable of undergoing schiff reaction – so increased background staining

8. HEPATOCYTES SHOWING STREAMING ARTIFACT Fixation should be carried out at 4 ̊ C to minimize streaming artifact

9. Periodic acid schiff (PAS) technique Alcian blue method Combined alcian blue- PAS techhnique Mucicarmine technique Colloidal iron technique Metachromatic staining Iodine staining for glycogen Enzymatic digestion technique Diastase digestion, Sialidase digestion, Hyaluronidase digestion CARBOHYDRATE STAINING

10. PERIODIC ACID SCHIFF METHOD 1 st histochemical use was by McManus for demonstration of mucin Reagents – 1. periodic acid 2. schiff reagent 0.5-1% solution of periodic acid (oxidant) used for 5-10 minutes oxidation of hydroxyl group within the formation of two free aldehyde groups free aldehydes react with schiff reagent carbohydrate bright red magenta end product RESULT – glycogen and mucins : magenta nuclei : blue

11. CANDIDA IN PAS STAIN ALPHA ANTITRYPSIN IN PAS STAIN

12. Other oxidants like potassium permanganate/ chromic acid not used – further oxidise aldehyde groups to carboxylic groups – not reactive to schiff reagent MILD PAS TECHNIQUE – 0.01% periodic acid used for shorter period for N-acetyl sialic acid containing mucins as the hydroxyl groups are highly susceptible to periodic acid oxidation

13. PAS REACTIVE CELLS AND TISSUE COMPONENTS GLYCOGEN STARCH MUCIN BASEMENT MEMBRANE ALPHA ANTI TRYPSIN RETICULIN FUNGI(CAPSULES) PANCREATIC ZYMOGEN GRANULES THYROID COLLOID CORPORA AMYLACEA RUSSELL BODIES

14. Diagnosis of several medical conditions: Glycogen storage disorder Staining macrophages in Whipple's disease Mucins in adenocarcinoma of large intestine Demonstration of fungi Seminoma,rhabdomyosarcoma,ewing’s sarcoma contain glycogen

15. ALCIAN BLUE Alcian blue 8 GX – recommended Comprised of copper containing pthalocyanine ring linked to 4 isothiouronium groups – strong bases - account for cationic nature of the dye Sulfate and carboxylate groups of proteoglycans ionised at pH 2.5 and carry a negative charge Sialo - and sulfo mucins also reactive at pH 2.5 So, they stain with alcian blue at pH 2.5 Neutral mucins are not reactive with alcian blue

16. REAGENTS : 1. Alcian blue 2. Aluminium sulfate 3. Nuclear fast red RESULTS sulfomucin,sialomucin Proteoglycans Hyaluronic acid Nucleus  red Blue

17. GOBLET CELLS BY ALCIAN BLUE

18. COMBINED ALCIAN BLUE- PAS TECHNIQUE PRINCIPLE Demonstrate presence of mucin Differentiate acid mucin from neutral mucin 1st stain all acid mucin with alcian blue (blue) Those acid mucin which are PAS + ve will not be stained on PAS reaction  only neutral mucin will be stained(magenta)

19. ALCIAN BLUE WITH VARYING ELECTROLYTE CONCENTRATIONS This technique is based upon phenomenon known as CRITICAL ELECTROLYTE CONCENTRATION (CEC) CEC is defined as point at which amount of electrolyte such as MgCl2 is sufficient to prevent staining from AB Competition between cations of salt and dye occurs for polyanionic sites within tissue Different acidic carbohydrates have different CEC value So can differentiate acidic mucins and proteoglycans

20. MUCICARMINE Active dye molecule is aluminium – carminic acid complex known as CARMINE carminic acid produced from dried bodies of female Coccus Cacti insects Carmine complex has a positive charge and so attracts polyanions such as sialomucins and sulfomucins Useful for identification of adenocarcinoma ( especially of GIT) Capsule of fungus – cryptococcus neoformans is also detected

21. REAGENTS : Southgate’s mucicarmine solution Alcoholic hematoxylin Acidified ferric chloride solution Weigert’s iron hematoxylin solution Metanil yellow solution RESULTS : Acidic mucins – deep rose to red Nuclei – black Other tissue elements – light yellow

22. COCCUS CACTI CRYPTOCOCCUS STAINED BY MUCICARMINE

23. GOBLET CELLS BY MUCICARMINE

24. NUCLEIC ACIDS 2 nucleic acids are : 1. DNA ( In the nucleus) 2. RNA (In the cytoplasm) They consist of : Sugar ( Deoxyribose / Ribose), Phosphate and Nitrogenous base Demonstration of Nucleic acids depends upon either : Reaction of the dyes with the phosphate groups , or , Production of aldehydes from the sugar ( deoxyribose ) No histochemical methods are available to demonstrate the nitrogenous base

25. Feulgen technique ( demonstrate sugar) Methyl green pyronin technique (demonstrate phosphate) Acridine orange (by fluorescent method) Gallocyanin -chrome alum method Demonstrates both DNA and RNA The last staining method do not separate the 2 nucleic acids (stains both DNA and RNA blue) and suitable extraction technique must be used DNA IS DEMONSTRATED BY

26. EXTRACTION TECHNIQUES DIGESTION METHODS : Pure deoxyribonuclease will digest DNA and pure ribonuclease will digest RNA CHEMICAL METHODS : a) By perchloric acid : To remove RNA – 10% perchloric acid at 4̊ C overnight b) Trichloroacetic acid c) Hydrochloric acid

27. FEULGEN STAIN SOLUTIONS USED ARE : 1M HCL acid - used for acid hydrolysis to break the purine-deoxyribose bond and yield an aldehyde . - Done at 60̊ C (HCL should be preheated to 60 ̊ C ) - Time (minutes) depends upon the fixative used - For carnoy’s and formalin – 8 minutes used B) Schiff reagent - The aldehydes are then demonstrated by schiff’s reagent C) Bisulfite solution RESULT DNA : red-purple CYTOPLASM : green

28. DNA BY FEULGEN STAIN

29. METHYL GREEN PYRONIN METHOD Reagents : Methyl green - impure dye contains methyl violet – removed by washing with chloroform - pure methyl green specific for DNA - NH2 of dye reacts with phosphate of DNA Pyronin - binds to any negatively charged tissue constituent - apart from RNA, binds to acid mucins and cartilage RESULTS – DNA : green-blue RNA : red

30. NUCLEIC ACIDS BY METHYL GREEN PYRONIN

31. Most suitable technique for identifying DNA is In-situ hybridization Bouin’s fixative is not suitable as it causes over hydrolysis of the nucleic acid during fixation RNA cannot be demonstrated by feulgen stain because ribose- purine bond is unaffected by hydrolysis/ 1 M HCL Control method for the standard feulgen technique is – Naphthoic acid hydrazide (NAH) method – DNA is acid hydrolysed by 1M HCL. Aldehydes are coupled with naphthoic acid and then again coupled with diazonium salt, fast blue B. results are identical to true feulgen reaction Blue thionin-feulgen reaction – used for studying cancer cell nuclear morphology and ploidy . Here DNA is stained blue and cytoplasm remains unstained POINTS TO REMEMBER

32. LIPIDS SIMPLE LIPIDS - FATS - OILS - WAXES COMPOUND LIPIDS - c/o fatty acids, alcohol and one more group such as phosphorus or nitrogen DERIVED LIPIDS Derived from simple or compound lipids by hydrolysis cholesterol Bile acids

33. Lipids with melting point below staining temperature can be stained with fat stains So only lipids which are liquid at staining temp. are stained. Those in solid or crystalline state remains unaffected Melting point of a lipid is inversely related to its fatty acid chain length Simple lipid is best demonstrated with fresh frozen sections Best fixative – Formal calcium (2% calcium acetate + 10% formalin)

34. 1 st Sudan dye was Sudan 3 Most sensitive of all fat dyes is – Sudan black B Sudans must be dissolved in organic solvents to penetrate fats Some organic solvents used are – 70% ethanol Isopropanol Propylene glycol Triethyl phosphate SUDAN BLACK B

35. Sudan black b has 2 fractions – 1 st stains neutral fats blue-black 2 nd stains phospholipids gray This gray reaction can be enhanced as a bronze dichroism if section is viewed in polarised light It fails to stain crystalline cholesterol, lecithin and free fatty acids Bromine pre treatment converts crystalline cholesterol to oily derivatives and hence permeable to Sudan dye

36. AMYLOID Extracellular , amorphous , eosinophilic material Composed of protein in an antiparallel  -pleated sheet configuration In H&E stain , can be confused with hyaline and fibrinoid substances Earliest special stain used for amyloid was Iodine by Virchow

37. CONGO RED STAIN Acidic dye and composed of 2 identical halves Each half has a phenyl ring bound to a naphthalene moiety by a diazo group 2 phenyl groups bound by a diphenyl bond - gives a linear dye molecule It stains amyloid by hydrogen bonding and other tissue components by electrochemical bonds Electrochemical staining of other tissues can be suppressed by – using alkaline-alcoholic solvents using competitive inhibition by salt solution

38. 2 factors are important to the congo red- amyloid reaction Linearity of the dye molecule -pleated sheet configuration of the amyloid If the spatial configuration of either is altered, the reaction fails

39. Fixation – Not critical Solution- 0.5 % Congo red in 50% alcohol 0.2% Potassium Hydroxide in 80% alcohol Results- Amyloid - red Nuclei - Blue TECHNIQUE

40. AMYLOID BY CONGO RED

41. ALKALINE CONGO-RED TECHNIQUE High concentration of NaCl is used Background electrochemical staining is reduced hydrogen bonding of congo -red to amyloid is enhanced

42. POLARIZING MICROSCOPY AND CONGO-RED Under polarized light, congo red stained amyloid exhibits apple-green birefringence Most reliable diagnostic test for amyloid currently

43. POINTS TO REMEMBER Thickness of section is critical – 8-10 micro meter is ideal Too thin section – show faint red color Too thick section – show yellow birefringent Other structures giving apple-green birefringence : 1. neurofibrillary tangles of alzheimer’s 2. intracellular inclusions seen in adrenal cortical cells 3. cellulose and chitin 4. dense collagen Thioflavin T – flourescent method

44. To differentiate AA and AL amyloid : Section pretreatment with trypsin or potassium permanganate done AA amyloid lose their affinity for congo -red but AL amyloid is resistant

45. METHYL /CRYSTAL VIOLET METHOD Methyl violet contains a mixture of tetra- , penta - , and hexa - methyl pararosaniline Amyloid stained due to selective affinity for one of the colored fractions Hence, polychromasia seen Ammonium oxalate accentuates polychromatic effect RESULT : AMYLOID, MUCIN , HYALINE – red-purple BACKGROUND - blue

46. STAINS FOR MICROORGANISMS

47. Gram staining of Bacteria ( MODIFIED BROWN-BRENN METHOD) Reagents : Crystal violet stain Gram’s iodine solution Ethyl alcohol – acetone solution( decolorizer ) Acetone- xylene solution Basic Fuchsin Picric acid, 0.1% in acetone RESULTS : GRAM POSITIVE BACTERIA – blue GRAM NEGATIVE BACTERIA – red NUCLEI – red OTHER TISSUE ELEMENTS - yellow

48. Dry picric acid is explosive – recommended to purchase picric acid – acetone solution pre made Do not allow sections to dry at any point in the staining process – decolorization will be difficult and uneven

49. Acid Fast Staining for Bacteria Mycobacteria cannot be demonstrated by gram’s stain – possess a capsule containing long chain fatty acid ( mycolic acid) that makes them hydrophobic Can be stained by a strong stain like carbol fuchsin Fatty capsule resist the removal of stain by acid-alcohol solution (acid and alcohol fastness) Mycobacteria are PAS positive due to carbohydrate content of their cell wall

50. Ziehl Neelson (ZN) stain Reagents Carbol fuchsin solution 1% acid alcohol 0.1%Methylene blue solution RESULTS Acid fast bacilli bright red Other tissue Pale blue Caseous material very pale grayish blue

51. Blue counterstain may be patchy if extensive caseation is present Avoid over counterstaining – scant organism can easily be obscured Decalcification using strong acids may destroy acid-fastness - so formic acid recommended

52. MODIFIED FITE TECHNIQUE REAGENTS : Carbol fuchsin solution 5% sulphuric acid in 25% alcohol Methylene blue solution RESULTS: M.leprae – bright red nuclei and other tissue elements – pale blue

53. Fite stain Modified ZN Uses mixture of xylol & liquid paraffin prior to stain Does not use Incubation in ZN carbolfuchsin at 37 ºc for 45 min Incubation In preheated ZN carbol fuchsin at 56 ºc for 60min Decolorize with 5% H2SO4 1% acid alcohol. Acid :20% H2SO4 Demonstrates M leprae M tuberculosis AFB : Fite vs modified ZN

54. Warthin Starry Method for Spirochetes REAGENTS : Acetate buffer pH-3.6 1% silver nitrate RESULTS : SPIROCHETES – black BACKGROUND – golden -yellow

55. SPIROCHETES BY WARTHIN STARRY

56. FUNGAL STAINS Fungal cell walls are rich in polysaccharides which can be converted by oxidation to dialdehydes Dialdehydes are then detected by silver solution

57. Gomori methenamine silver nitrate(GMS) technique Reagents 4% chromic acid 1% sodium bisulfite 5% sodium Thiosulfate 0.21% Silver nitrate(stock) Gold chloride 0.1% aqueous solution Light green solution

58. Results Fungi , Pneumocystis , melanin - Black Mucin & Glycogen - dark grey Background - Pale green Hyphae & yeast form - sharply delineated in black against green background

59. CRYPTOCOCCUS BY GMS STAIN

60. MISCELLANEOUS STAINS Cresyl violet acetate method for helicobacter pylori Macchiavello’s stain for rickettsia and viral inclusions Lendrum’s phloxine – tartrazine stain for viral inclusions Giemsa stain for parasites

61. CONNECTIVE TISSUES Provide a matrix that connects and binds the cells and organs and ultimately gives support to the body. Parent cell is embryonic mesenchyme

62. COLLAGEN FIBRES 1. Masson ‘s trichrome technique 2. Van Gieson’s stain 3. Mallory’s Phosphotungstic Acid Hematoxylin 4. MSB Technique 5. PAS 6. Heidenhain’s Azan stain 7. lillie’s allochrome method 8. Luxol fast blue G

63. FACTORS AFFECTING TRICHROME STAINING 1 . TISSUE PERMEABILITY AND DYE MOLECULAR SIZE When protein component of a tissue exposed to a fixative – insoluble protein network formed Structure of the protein network directly related to the staining reactions Erythrocyte protein – dense network with small pores Muscle cells – larger pores Collagen – least dense network and quite porous

64. 2 . Heat : Increases rate of staining and penetration 3 . pH : Low pH ( 1.5 – 3) 4 . Nuclear stain for trichrome Iron hematoxylin preferred More resistant to acidity of dye solutions Alum hematoxylins are decolorized Can use Celestin blue- alum hematoxylin sequence

65. EFFECT OF FIXATION 10% NBF will not yield optimal results Treatment of formaldehyde fixed tissue with picric acid /mercuric chloride solution enhances intensity and radiance of trichrome Recommended fixatives are: Bouin’s Zenker’s , Formal-mercury Zinc formalin

66. Demonstrate collagen and muscle in normal tissue Differentiate collagen and Muscle in tumors Identify an increase in collagenous tissue Indicate fibrotic change in cirrhosis of liver Indicate fibrotic change in pyelonephritis Distinguish tumors that have arisen from muscle cells and fibroblasts Masson ‘s trichrome technique

67. REAGENTS Weigert’s iron hematoxylin 2. Acid fuchsin 3. Glacial acetic acid 4. Phosphomolybdic acid 5. Methyl blue RESULT Nuclei – Blue/ Black Cytoplasm, muscle , RBC → Red Collagen → Blue

68. Trichrome stain showing slight mesangial prominance

69. Van Gieson Technique REAGENT : Weigert’s iron hematoxylin Saturated Picric acid solution Acid fuchsin RESULTS : Collagen – bright red Nuclei – Blue/Black Cytoplasm, muscle, RBC , elastin , reticulin -yellow

70. PIGMENTS AND MINERALS ENDOGENOUS PIGMENTS hematogenous non- hematogenous EXOGENOUS PIGMENTS asbestos silica lead carbon ARTIFACT PIGMENTS formalin malaria mercury schistosome

71. Hemosiderin Breakdown product of haemoglobin composed of ferric iron and protein Seen as yellow-brown granules 3 methods for demonstration : 1.Perl’s prussian blue reaction – for ferric ion 2. Lillie’s method – for ferrous iron 3. Hukill and putt’s method – for both ferric and ferrous iron

72. PERL’S STAIN Principle : unmasking of ferric iron in hydroxide form by dilute HCl PRUSSIAN BLUE REACTION – Ferric Hydroxide + potassium ferrocyanide = Ferric ferrocyanide (insoluble blue compound) Results Ferric iron –Blue Nuclei – Red Reagents 2% aq. Potassium ferrocyanide 2% HCl Counterstain with 1% neutral red or saffranin

73. Best positive control – postmortem lung tissue that contains a reasonable amount of iron positive macrophages (heart failure cells) In Hb and myoglobin , iron bound tightly within protein complex – cannot be demonstrated by traditional technique Treatment with hydrogen peroxide releases iron - stained with perl’s stain

74. Modified Fouchet’s technique: bile pigment Demonstrates liver bile pigment Most common routine method Reagents a) Fouchet ‘s reagent : 25% aq trichloracetic acid 10% aq ferric chloride b)Van Gieson stain :acid Fuchsin + saturated aq picric acid RESULT Bile pigment : emerald to blue green Muscle :yellow Collagen :red

75. BILE PIGMENTS BY MODIFIED FOUCHET’S TECHNIQUE

76. MELANIN Normally occur as light brown to black granules in substantia nigra,hair , skin and eye Found Pathologically throughout the body :benign nevus,malignant melanoma

77. MELANIN DEMONSTRATED BY : Reducing methods : a) Masson fontana silver technique b) Schmorl’s ferric- ferricyanide reduction test Enzyme methods – DOPA reaction Solubility and bleaching characteristics Fluorescent method Immunohistochemistry

78. MASSON FONTANA STAIN ARGENTAFFIN REACTION – reduction of ammoniacal silver solution to form metallic silver without the use of extraneous reducer. Masson’s method( using fontana’s silver solution) rely on melanin’s argentaffin property Melanins are blackened by acid silver nitrate solution RESULT : Melanin – black Nuclei - red

79. Schmorl’s ferric- ferricyanide reduction test Schmorl reaction – Melanin reduce ferricyanide to ferrocyanide with production of prussian blue in the presence of ferric salts RESULT : Melanin – dark blue Nuclei - red

80. Special stains enhance detection & localization of individual tissue component But should not be substituted for routine H&E CONCLUSION