Histopatholgy staining by suchit kumar
- 1. SEMINAR PRESENTED on HISTOPATHOGY STAINING Moderated by:- Presented by:- Dr. Charu Batra Atreja Mr. Suchit Kumar Msc(mlt) Final year Department of pathology (MMIMSR) M.M. Deemed to be university
- 2. Introduction H&E stain is routine stain. โข - It is the first stain applied to the tissue sections . โข Gives diagnostic information in most cases.
- 3. Hematoxylin and Eosin stain ๏ Hematoxylin and Eosin (H & E) staining is the most common staining technique in histopathology. ๏ This uses a combination of two dyes, Hematoxylin and Eosin used for demonstration of nucleus and cytoplasmic inclusions in clinical specimens.
- 5. Hematoxylin and Eosin stain Principle :- ๏ Alum acts as mordant and hematoxylin containing alum stains the nucleus light blue. ๏ This turns red in presence of acid, as differentiation is achieved by treating the tissue with acid solution. ๏ Bluing step converts the initial soluble red color within the nucleus to an insoluble blue color. ๏ The counterstaining is done by using eosin which imparts pink color to the cytoplasm.
- 6. Hematoxylin and Eosin stain Reagents :- ๏ Harriโs Hematoxylin stain A = 1 gm hematoxylin in 10 ml ethanol B = 20 gm ammonium alum in hot distilled water Mix A & B, boil and add 0.5 gm of mercuric oxide and filter. ๏ Eosin solution Yellow eosin = 1 gm Distilled water = 80 ml Ethanol = 320 ml Glacial Acetic Acid = 2 drops ๏ 0.5% HCl ๏ Dilute ammonia water
- 7. Hematoxylin and Eosin stain Procedure :- ๏ Deparaffinize the section : flame the slide on burner and place in the xylene. Repeat the treatment. ๏ Hydration : Hydrate the tissue section by passing through decreasing concentration of alcohol baths and water. (100%, 90%, 80%, 70%) ๏ Stain in hematoxylin for 3-5 minutes ๏ Wash in running tap water until sections โblueโ for 5 minutes or less. ๏ Differentiate in 1% acid alcohol (1% HCl in 70% alcohol) for 5 minutes. ๏ Wash in running tap water until the sections are again blue by dipping in an alkaline solution (e.g.. ammonia water) followed by tap water wash. ๏ Stain in 1% Eosin Y for 10 minutes ๏ Wash in tap water for 1-5 minutes ๏ Dehydrate in increasing concentration of alcohols and clear in xylene ๏ Mount in mounting media ๏ Observe under microscope
- 8. Hematoxylin and Eosin stain Result and Interpretation:- ๏ Nuclei : blue, black ๏ Cytoplasm : Pink ๏ Muscle fibers : deep red ๏ RBCs : orange red ๏ Fibrin : pink
- 9. Special stains โข A special stain is a staining technique to highlight various individual tissue component once we have preliminary information from the H&E stain. โข Special stains are used to identify certain normal and abnormal substance present in the cells and tissue, which can not be identified on routine Hematoxylin & Eosin staining or are better appreciated on special stain.
- 10. Classification 1. Stains for carbohydrates ๏ Periodic Acid Schiff (PAS) stain ๏ Enzymatic digestion technique ๏ Alcian blue 2. Stains for amyloid ๏ Congo red stain ๏ Crystal/Methyl violet stain 3. Nucleic acid stains ๏ Feulgen stain ๏ Methyl green pyronin stain 4) Lipid stains ๏ Oil red o stain ๏ Sudan Black B stain 5. Stains for microorganisms โข ZiehlโNeelsen stain โข Gomori methenamine silver stain 6. Connective tissue stains โข Reticulin stain โข Massonโs Trichrome โข Van gieson stain 7. Stains for pigments and minerals โข Perlโs stain
- 11. Carbohydrate staining ๏Periodic Acid Schiff (PAS) stain ๏Enzymatic digestion technique ๏Alcian blue
- 12. Periodic Acid Schiff (PAS) stain Principle:- Substance containing vicinal glycol groups or their amino or alkylamino derivatives are oxidized by periodic acid into di-aldehydes which on reaction with Schiff โs reagent give insoluble purple- magenta compound. ๏ Control : Appendix Uses of PAS: โข PAS is used to demonstrate glycogen and neutral mucoprotein. โข In diagnosis of poorly differentiated adenocarcinoma of various tissue like stomach, pancreas, lung. โข In diagnosis of hepatocellular carcinoma.
- 13. Periodic Acid Schiff (PAS) stain Reagents: ๏ Preparation of staining solutions 1) Periodic Acid Solution : ๏ Periodic Acid : 1 gram ๏ Distilled water : 100 ml 2) Schiffโs Reagent : ๏ Fuchsin Basic : 1 gm ๏ Distilled water : 100 ml ๏ Sodium metabisulphite : 2 gm ๏ Conc. HCl : 2 ml ๏ Charcoal activated : 0.3 gm Procedure: 1) Fix films for 15 min. in methanol. 2) Rinse in running tap water and air dry. 3) Treat slides with 1% periodic acid for 10 min. 4) Rinse in running tap water for 10 min. and air dry. 5) Now treat with Schiffโs reagent for 30 min. 6) Rinse in running tap water and air dry. 7) Counter stain with aqueous haematoxylin for 5 min. then wash and air dry.
- 14. Periodic Acid Schiffโs โข Glycogen, neutral/sialo mucins โข Various glycoproteins Schiffโs โข NucleiCounterstain (Optional)
- 15. Enzymatic digestion technique ๏ Various enzymatic digestion techniques have been applied to increase or verify the specificity of carbohydrate staining. ๏ For example, the amylase or diastase techniques for glycogen digestion are commonly utilized in laboratories to enhance the specificity of the PAS technique .
- 16. Diastase digestion ๏ The PAS technique is unique among the methods described that it detects a varied number of mucosubstances, e.g. glycogen, mucins, and glycoproteins.
- 17. Diastase digestion Solutions :- ๏ Phosphate buffer Monobasic sodium phosphate 1.97 g Dibasic sodium phosphate 0.28 g . ๏ Distilled water - 1000 ml . ๏ This solution may be kept in the refrigerator for several months. ๏ Diastase solution Malt diastase -0.1 g . ๏ Phosphate buffer - 100 ml .
- 18. Diastase digestion Method :- 1. Dewax two serial sections in xylene and rehydrate through graded ethan0lโs to water. 2. Place one slide in the diastase solution for 1 hour at 37ยฐC. The other slide is an untreated control and may remain in water for 1 hour. 3. Wash both slides in running tap water for 5โ10 minutes. 4. Proceed with the PAS technique.
- 19. โข PAS Diastase reaction : Glycogen is diastase sensitive, hence section containing glycogen when pretreated with diastase, the enzyme will digest the glycogen and will give negative PAS reaction. โข The purpose of using PAS with diastase staining procedure is to differentiate glycogen from other PAS positive elements such as mucin that may be present in the tissue sample. Gastric signet ring cell carcinoma on PASstain
- 20. PAS positive substances โข Glycogen โข Neutral mucoprotein โข Glycoprotein โข Glycolipid โข Basement membrane โข All fungi โข Phosphorylated sugar โข Cerebrosides
- 21. ๏ Presence of glycogen will be evidenced by loss of staining after enzyme treatment when compared to the untreated sections. PAS StainPAS with Diastase
- 22. Alcian blue stain Principle :- 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. Purpose: Alcian blue may be used in combination with the PAS staining procedure, so that both acid and neutral mucins can be demonstrated in the same tissue sample.
- 23. Alcian blue stain ๏ Control: Small intestine, appendix, large intestine. ๏ Fixative: 10% NBF, Bouinโs, or Hollande's. Solutions ๏ Alcian blue solution 1. Alcian blue 8GX - 1 g 2. 3% acetic acid solution - 100 ml 3. Nuclear fast red Aluminum sulfate - 5 g 4. Distilled water 100 ml 5. Nuclear fast red 0.1 g
- 24. Alcian blue โข Acid mucins (sulfomucins and sialomucins) โข Proteoglycans and hyaluronic acid Alcian blue โข Nuclei Nuclear fast red
- 25. Combined Alcian blue-PAS Differentiate neutral mucins from acidic mucins within a tissue section
- 26. 2. Stains for amyloid Amyloid :- A protein that is deposited in the liver, kidneys, spleen, or other tissues in certain disease. This condition of deposition of amyloid in tissues is known as Amyloidosis. ๏ Congo red stain ๏ Crystal/methyl violet stain
- 27. Congo red stain Introduction :- ๏ Congo red histological staining technique is the gold standard technique for the diagnosis of amyloidosis. Principle :- ๏ Congo red dye forms non-polar hydrogen bonds with amyloid and red to apple green birefringence occurs when viewed by polarized light due to alignment of dye molecules on the lineraly arranged amyloid fibrils. ๏ The high pH enhances the non-polar hydrogen bonding of Congo red and amyloid.
- 28. Congo red โข Amyloid, elastic fibers, eosinophil granules Congo red Hematoxylin โขNuclei
- 29. Crystal/Methyl violet for amyloid โข Crystal violet or methyl violet stain are used for metachromatic amyloid staining. โข They stain amyloid as purple-red in blue background. Reagents used โข Crystal/methyl violet โข 95% alcohol โข 1% aqueous ammonium oxalate โข 0.2% acetic acid
- 30. Crystal/Methyl violet stain Result :- Amyloid - Purple Background - Blue
- 31. 3. Nucleic acid stains ๏ Feulgen stain ๏ Methyl green pyronin stain
- 32. Feulgen stain Introduction :- ๏ Feulgen stain is a staining technique discovered by Robert Feulgen and used in histology to identify chromosomal material or DNA in cell specimens. ๏ It depends on acid hydrolysis of DNA, therefore fixating agents using strong acids should be avoided.
- 33. Feulgen stain RESULT :- DNA : Red-Purple Cytoplasm : Green
- 34. Methyl green pyronin stain ๏ Methyl green- pyronin (MGP) is a classical histological staining technique using two basic dyes for the demonstration and differentiation of DNA and RNA. ๏ Reagents : ๏ 1. Methyl green - impure dye contains methyl violet โ removed by washing with chloroform - pure methyl green specific for DNA - NH2 of dye reacts with phosphate of DNA ๏ 2. Pyronin - binds to any negatively charged tissue constituent - apart from RNA, binds to acid mucins and cartilage .
- 35. Methyl green pyronin stain
- 36. Lipid stain ๏ Oil red o stain ๏ Sudan Black B stain
- 37. Oil red ostain 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 โ Red Nuclei- Blue
- 38. Oil red o stain ๏ Reagents : ๏ 60% isopropanol ๏ Alum hematoxylin: ๏ Glycerin Jelly mounting medium ๏ Oil Red O working solution: ๏ o To make oil red O stock solution ๏ Oil red O - 0.5gm ๏ Isopropanol 100 ml
- 39. Fatty liver -stain Oil redO
- 40. Oil Red O stain Oil Red O Hematoxylin โขFat โขNuclei
- 41. Fatemboli seen asred dot within capillaries of lung onOil red Ostain
- 42. Sudan black B is a lipophilic dye that stains intra cellular phospholipids and other lipids . โข 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. โข It is slightly basic dye and will combine with acidic groups in compound lipids, thus staining phospholipids also. Sudan Black B stain
- 43. Sudan Black B stain Reagents:- ๏ Fixative:- 40% formaldehyde vapours ๏ Stain:- 0.3% SBB in absolute alcohol ๏ Phenolic buffer:- 16gm crystalline phenol in 30ml of absolute ethanol and final volume up to 100ml with buffer. ๏ Working stain solution:- add 40ml phenolic buffer to 60ml SBB solution. ๏ Counter stain :- Leishman stain
- 44. Sudan Black B stain Procedure :- 1) Fix air dried smear in in formalin vapours for 5- 10 min. 2) Then air wash for 15 min. 3) Now stain with working solution of SBB stain solution for 1 hour. 4) Now give 3 washings of ethanol for 30 sec each. 5) Wash with water and air dry. 6) Now counter stain with Leishman stain.
- 45. Results:- The reaction product is black and granular. All nuclei are blue.
- 46. Stain for micro-organisms ๏ ZiehlโNeelsen stain ๏ Gomori methenamine silver stain
- 47. ZiehlโNeelsen stain Principle :- ๏ This procedure is used to stain mycobacterium tuberculosis and mycobacterium leprae. ๏ These bacteria are also called acid fast bacilli. ๏ They stain with carbol fuschin, which is a red dye. They retain the dye when treated with acid, which is because of the presence of mycolic acid in their cell wall.
- 48. ZiehlโNeelsen stain Reagents :- ๏ Carbol fuchsin Basic fuchsin - 0.5 g Absolute alcohol - 5 ml 5% aqueous phenol -100 ml ๏ Mix well and filter before use. ๏ Acid alcohol Hydrochloric acid - 10 ml 70% alcohol - 1000 ml โขMethylene blue solution Methylene blue - 1.4 g 95% alcohol - 100 ml โข Methylene blue solution (working) Methylene blue - 10 ml โขTap water - 90 ml
- 49. ZiehlโNeelsen stain Procedure :- ๏ Fix the smear of the specimen over the glass slide, either by heating or alcohol fixation. ๏ Pour carbol fuschin over smear and heat gently until fumes appear. Do not overheat and allow it to stand for 5 minutes, then wash it off with water. ๏ Pour 20% sulphuric acid, wait for one minute and keep on repeating this step until the slide appears light pink in color. Wash off with water. ๏ Pour methylene blue, wait for two minutes, again wash with water ๏ Allow it to air dry and examine under oil immersion lens.
- 50. ZiehlโNeelsen stain Result :- ๏ Acid fast bacilli - pink. ๏ The background appears blue.
- 51. Verhoeff-VanGieson(VVG)stain โข VVG is a two-part combination stain that enables differentiation of some connective tissue components in a tissue which are not easily distinguished by H&E staining: Verhoeff stain component: an iron- hematoxylin stain that is specific for elastic fibers.
- 52. VAN GIESON STAIN Principle :- โข When using combined solution of picric acid and fuchsin, the small molecules of picric acid penetrate all the tissue rapidly, but are only firmly retained in the close textured red blood cells and muscle โข The larger molecules of ponceau S displaces picric acid molecule from collagen fibres, which has larger pores and allow larger molecules to enter. โขIt is used for detection of collagen. Result-- Nuclei : Blue / Black Collagen :Yellow Cytoplasm, muscle, fibrin, RBCs : Red Yellow
- 53. van Gieson โข Nuclei Hematoxylin โข Cytoplasm, muscle, and erythrocytes Acid Fuchsin โข Collagen Picric acid
- 54. Gomori methenamine silver stain for fungi ๏ GMS staining is a silver staining technique for demonstrating fungi in tissue sections. ๏ It is primarily based on staining the polysaccharides in fungal cell walls, and 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.
- 55. Pneumocystis carinii Result: Fungi, pneumocystis carinii, melanin - black Mucin and glycogen- - grey-black RBCs - yellow Background - pale green
- 56. Stain for Connective tissue ๏ Reticulin stain ๏ Massonโs Trichrome
- 57. Reticulin stain Introduction :- ๏ In pathology, the reticulin stain, is a popular staining method in histology. ๏ It is used to visualize reticular fiber and used extensively in liver histopathology. Principle :- Reticulin fibres have little natural affinity for silver solutions so, they must be treated with potassium permanganate to produce sensitized sites on the fibres where silver deposition can be initiated.
- 58. Reticulin stain ๏ 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 โ Black Nuclei- Gray
- 60. Massonโs trichome stain Introduction :- ๏ This method is used for detection of collagen fibers in the tissues such as skin, stomach, intestine and lung. Principle: ๏ As per the name 3 dyes are used which selectively stain muscle, collagen fibers, fibrin and erythrocytes. ๏ As general rule in trichrome stain less porous tissues are stained by small dye molecule. ๏ Acid fuchsine stain all the connective tissue, PMA ( phosphomolybdic acid) competes with fuchsine and gain access to collagen displacing fuchsine. If reaction stopped at appropriate time, collagen will be free to be stained by Methyl Blue.
- 61. Masson Trichrome โข Nuclei Hematoxylin โข Cytoplasm, muscle, and erythrocytes Acid Fuchsin โข Collagen Methyl blue/ Light green SF
- 62. Stain for pigment and minerals ๏Perlโs stain
- 63. Perlโs stain Introduction :- ๏ Hemosiderin is present in tissues as intracellular pigment. ๏ It contains iron in the form of ferric hydroxide that is bond to a protein framework. Principle:- ๏ The reaction occurs with the treatment of tissue sections with acid ferrocyanide solution. ๏ Any ferric ion (Fe3+) in the tissue combines with ferrocyanide and results in the formation of a bright blue pigment called โPrussian blueโ or ferric ferrocyanide.
- 64. Perlโs stain Fixation :- ๏ Avoid the use of acid fixatives. Chromates will also interfere with the preservation of iron. Staining solution :- ๏ 1% aqueous potassium ferrocyanide = 20 ml ๏ 2% aqueous hydrochloric acid = 20 ml ๏ Mix both Procedure:- ๏ Deparaffinize and bring the sections to water. ๏ Treat the sections with freshly prepared acid ferrocyanide solution for 10-30 minutes. ๏ Wash well in distilled water. ๏ Lightly stain the nuclei with 0.5% aqueous neutral red or 0.1% nuclear fast red. ๏ Wash rapidly in distilled water. ๏ Dehydrate, clear and mount.
- 65. Perlsโ Prussian blue reaction โข Ferric ironPerlโs โข NucleiNeutral red
- 66. Thank you