4. mucin histochemistry_seminar

Histochemistry of mucin

1. HISTOCHEMISTRY OF MUCIN PRESENTER: Dr G SANTHIPRIYA MODERATOR: Dr DIVYASHREE B N 5/28/2018 Seminar

2. CONTENTS Introduction Properties of mucin Histochemistry of mucin Functional Classicifation Fixatives for mucin Methods of demonstration of mucin Expression of mucin in various tumors Summary of different types of mucin and their staining pattern 5/28/2018 Seminar

3. INTRODUCTION Are high molecular weight glycoproteins Synthesis - RER & golgi complex 5/28/2018 Seminar

4. INTRODUCTION 2 types Membrane bound MUC1 MUC4 Secreted MUC2 MUC5AC MUC5B MUC7 Unclassified : MUC3, MUC6, and MUC8 Partial c DNA technique- MUC 11,12,13,16, 17, 19, 20, 21, 22 5/28/2018 Seminar

5. MUC1 –breast, pancreas and ovaries MUC2 —large and small intestine MUC3 —jejunum MUC4, 5AC, 5b — transbronchial mucosa MUC6 —gastric mucosa MUC7 — submaxillary gland 5/28/2018 Seminar

6. Carbohydrates Simple Carbohydrates • Monosaccharides • Oligosaccharides • Polysaccharides Glycoconjugates Glycosaminoglycans Glycoproteins - mucins Glycolipids 5/28/2018 Seminar

7. Properties of mucin Stain strongly with basic dyes Metachromatic : They stain red to reddish blue with toluidine blue and thionine Precipitated by acetic acid (except gastric mucin ) Soluble in alkaline solution 5/28/2018 Seminar

8. Histochemistry of mucin Reactive groups Types of mucin 1, 2 glycol Neutral mucins 1, 2 glycol+ COOH Sialomucins 1, 2 glycol+ COOH + OSO 3 H Sulfo - sialomucins 5/28/2018 Seminar

9. Functional Classification 5/28/2018 Seminar

10. Types of mucosubstances location ACID MUCO SUBSTANCES Acid mucosubstances ( carboxylated / sialo )(non- sulfated) Enzyme labile Sub mandibular salivary glands, bronchial sub mucous glands and goblet cells of LI Enzyme resistant Mucosal glands of LI, submaxillary gland mucins , SI mucins Acid mucosubstances (sulfated and carboxylted ) Strongly sulfated Epithelial mucin (e.g. bronchial serous glands) Connective tissue mucin or proteoglycans (e.g. chondroitin sulfate, heparin/ heparin sulfate, keratin sulfate) Weakly sulfated Usually epithelial in origin, e.g. colonic goblet cells Sulfated sialo - mucosubstances Prostatic carcinoma, synovial sarcoma NEUTRAL MUCOSUBSTANCES Surface epithelium of stomach, Brunner’s glands of duodenum, prostate epithelium 5/28/2018 Seminar

11. CLASSIFICATION OF MUCINS 5/28/2018 Seminar

12. Fixatives for mucin Usually carbohdrates are water soluble and better results are obtained by use of fixatives such as formal- calcium, acidic fixatives, formaldehyde- alcohol and Carnoy’s fixatives Over fixation in formalin tends to reduce the strength of the PAS reaction in mucins owing to polymerization This can be reversed by treating hydrated sections with 0.2 NaOH for 10-15 minutes prior to staining 5/28/2018 Seminar

13. Demonstration There are 3 approaches to the histochemical study of carbohydrates 1. Carbohydrates with adjacent hydroxyl (-OH) groups( diols ): PAS 2. Electrostatic bonding: alcian blue, mucicaramine and metachromatic dyes 3. Affinity for lectins 5/28/2018 Seminar

14. PAS Technique Is the widely used and most useful technique for the demonstration of carbohydrates or glycoconjugates Fixatives: 10 % neutral buffered formalin or Bouin’s solution can be used Blood smears for PAS staining should be fixed with methyl alcohol for 10- 15 minutes 5/28/2018 Seminar

15. PAS- positive cells, tissues and other structures Glycogen Starch Mucin : sialomucin , neutral mucin Basement membranes Fungi (capsules): Candida albicans , Histoplasma capsulatum , Cryptococcus, Blastomucosis , Actinomycosis 5/28/2018 Seminar

16. PAS- positive cells, tissues and other structures Others: thyroid colloid, adrenal lipofuscin Corpora amylacea of prostate Pancreatic zymogen granules α 1 -antitrypsin Macrophages in whipple’s disease Lipofuscin pigment Reticulin Russell bodies, megakaryocytic granules Hyaline globules in yolk- sac tumors Lymphoblast in acute lymphoblastic leukemia(block positivity) Intracytoplasmic crystals in alveolar soft part sarcoma 5/28/2018 Seminar

17. Principle of the PAS technique Periodic acid cleaves certain carbohydrates elements of tissue/ cells 5/28/2018 Seminar

18. Any substance which satisfied the following criteria will give positive PAS reaction 1. the substance must possess the 1, 2 glycol groupings or the equivalent amino or alky amino derivative or the oxidation product CHOH- CO. 1,2 glycol + periodic acid = aldehyde 2. The substance must not diffuse away from the tissue during fixation and must give an oxidation product which is not diffusible 3. The substance should be present in sufficient quantity to give detectable final color 5/28/2018 Seminar

19. Mechanism of PAS technique Basic fuchsin + Sulfurous acid + Aldehyde Deep(bright) red/ magenta color Colorless due to loss of quinoid structure 5/28/2018 Seminar

20. Demonstration of Schiff reagent Basic fuchsin - not a specific dye but a mixture of triarylmethane dyes such as pararosaniline , rosaniline and new fuchsin Method: aqueous solution of sulfurous acid The reaction of sodium metabisulfite (Na2S2O5) with a mineral acid, hydrochloric acid ( HCl ), or by the reaction of thionyl chloride (SOCl2) with water . 5/28/2018 Seminar

21. Preparation of Schiff Reagent 5/28/2018 Seminar

22. Method of staining with PAS 5/28/2018 Seminar

23. Results of PAS Component Color PAS Positive substances: Glycogen, neutral/ Sialomucins , various glycoproteins Magenta(purplish red) Nuclei Blue/ violet Other tissue constituens Yellow 5/28/2018 Seminar

24. 5/28/2018 Seminar

25. Enzyme digestion technique in carbohydrate identification Distinction of mucins from glycogen cannot be done by the PAS technique. The positive identification of some polysaccharides needs their selective removal following specific enzyme digestion( the amylase or diastase techniques for glycogen digestion) Addition of a glycogen digestion step helps in the correct identification of mucosubstances such as mucin or glycogen Glycogen is digested and not stained by PAS but it stains glycoproteins , glycolipids and glycomucins 5/28/2018 Seminar

26. The various enzymes used Alpha amylase Beta amylase Diastase: commonly used Saliva: not advised 5/28/2018 Seminar

27. PAS with Diastase Purpose: To demonstrate glycogen Principle: Diastase and α - amylase breaks down glycogen into smaller sugar units Results: positive PAS without diastate becomes negative PAS after enzyme digestion- indicates that the substance is glycogen 5/28/2018 Seminar

28. PAS PAS with Diastase 5/28/2018 Seminar

29. Alcian Blue Technique Cationic Water soluble conjugated dye molecule- central copper containing phthalocyanine ring AB 8GX –acid mucosubstances Mechanism- Electrostatic linkages with polyanionic molecules within tissues 5/28/2018 Seminar

30. Advantages Simplicity Specificity Strong coloration Insolubility of staining Permanent results 5/28/2018 Seminar

31. AB and pH A tissue component is more intensely stained if the AB dye is used at the pH at which the reacting groups are fully ionized At pH 1.0- only sulfated, GAGs and glycoproteins : cartilage, goblet cell mucins of LI and mucins of bronchial serous glands At pH 2.5- sulphated GAGs, glycoproteins , hyaluronic acid, components of CT and cartilage Acid epithelial mucins – sialo and sulfo mucins of LI are reactive at pH 2.5 5/28/2018 Seminar

32. AB with varying electrolyte concentration Molarity of magnesium chloride[CEC] Mgcl 4 ( in gms )/ 100 mL of AB solution Type of mucin that stains 0.06 M 1.2 all acid mucins 0.2-0.3 M 6.10 only sulfated mucins 0.5-0.6M 10.15 only strongly sulfated 0.7-0.8M 14.20 only heparin/ heparan sulfate and keratan sulfate >0.9 M 18.30 only keratan sulfate 5/28/2018 Seminar

33. AB Method for acid groups(pH 2.5) Purpose: to demonstrate acid mucins , proteoglycans and mast cell granules Principle: when used in a 3% acetic acid solution(pH 2.5), alcian blue stains both acid mucins and proteoglycans The specificity of AB is partly due to its large molecular size and thus stains acidic substances of low density 5/28/2018 Seminar

34. Solutions and their components Quantity Alcian blue solution yielding a pH of 2.5 Alcian blue 8GX 1 gm 3% acetic acid solution 100 mL Nuclear fast red Aluminium sulfate 5 gm Distilled water 100 mL Nuclear fast red 0.1 gm Dissolve the alluminum sulfate in the water with heat. Add the nuclear fast red to water while still hot and filter 5/28/2018 Seminar

35. Method 5/28/2018 Seminar

36. Results Component Color Acid mucins Blue Proteoglycans and hyaluronic acid Blue Mast cell granules Blue Mucoid capsule of cryptococci Blue Nuclei Red 5/28/2018 Seminar

37. Alcian blue staining of acidic mucin 5/28/2018 Seminar

38. Alcian Blue method(pH 1) for sulfate groups Purpose: sulfate mucosubstances , GAGs and glycoproteins Principle: when used in a 0.1 N hydrochloric acid solution (pH 1.0), AB stains only sulfated acid mucosubstances (skin, cartilage, blood vessels, cornea and lung) And sulfate sialomucins - mucins of the colon 5/28/2018 Seminar

39. Components Quantity Alcian blue 8GX 1 gm 0.1 N hydrochloric acid 100 mL 5/28/2018 Seminar

40. Results Component Color Sulfated mucosubstances , GAGs and glycoproteins Blue Cartilage, goblet cell mucins of the LI, and the mucins of the bronchial serous glands Nuclei Red 5/28/2018 Seminar

41. Alcian blue pH 1.0 5/28/2018 Seminar

42. Combined alcian blue-PAS To differentiate neutral mucins from acidic mucins . Useful for studying inflammatory and metaplstic lesions of GIT Useful for recognizing Barrett’s esophagus Helps in differentiating intestinal metaplasia in stomach where goblet cells will appear to be differentiated from gastric mucosa Used as a broad means of detecting mucins 5/28/2018 Seminar

43. Solutions Alcian blue solution (in 3% acetic acid). Periodic acid solution Preparation of Schiff reagent Method 5/28/2018 Seminar

44. Results Alcian blue Acid mucins proteoglycans Blue PAS Glucogen , neutral mucin and various glucoproteins Deep red , magenta 5/28/2018 Seminar

45. Complete intestinal metaplasia (Periodic acid-Schiff [PAS] and Alcian blue). 5/28/2018 Seminar

46. Methods of demonstratin of mucin Traditional methods to demonstrate mucin are Metachromatic technique Mucicarmine Mucihematin 5/28/2018 Seminar

47. Southgate’s mucicarmine technique Stains mucins Specific for the mucins of epithelial origin Detection of the capsule of the fungus: cryptococcus neoformans 5/28/2018 Seminar

48. Principle: mucicarmine stain contains carmine bound in soultion to aluminum. Concentrated stock solution- just before use Aluminium in the stain acts as a mordant and carmine aluminum compound binds to the acid groups of the many mucins Deep pink or red color to the mucin 5/28/2018 Seminar

49. Stock stolution Constituent Quantity Carmine 1 gm Aluminum hydroxide 1 gm 50 % ethanol/ alcohol 100 mL Avoid reagents and 50% ethanol are mixed by shaking in a 500 mL flask. To this add 0.5 gm of anhydrous aluminum chloride. Boil it in a water bath for 2 and hald to 3 min. Agitate while boiling. Cool the flask under running tap water. Filter and store at 4oc. The stock solution is stable for several months Acidified ferric chloride stock solution Ferric chloride 2.48 gm Distilled water 97 mL Concentrated HCl 1 mL Alcoholic hematoxylin stock solution Hematoxylin 1 gm Ethanol (95%) 100 mL 5/28/2018 Seminar

50. Working solution Constituent Quantity Southgate’s mucicarmine working solutoin Stock solution 10 mL Dw 90 mL Weigerts iron hematoxyline working solution Alcoholic hematoxyline 50 mL Acidified ferric chloride solution 50 mL This solution should be mixed just before use Metanil yellow working solution Metani yellow 0.25 gm Dw 100 mL Glacial acetic acid 0.25 mL Mix and store in a brown bottle or a bottle completely wrapped with aluminum foil 5/28/2018 Seminar

51. Method 5/28/2018 Seminar

52. Results Component color Acidic epithelial mucins pink/red Nuclei blue/black Other tissue components light yellow 5/28/2018 Seminar

53. Mucicarmine staining of colon tumor 5/28/2018 Seminar

54. Colloidal iron Anionic mucosubstances can also be stained by cationic colloids containing iron Principle: ferric cations in colloidal ferric oxide solution attract the negatively charged carboxyl and sulfate groups of acid mucins and proteoglycans Tissue bound ferric ions subsequently are visualized by treatment with potassium ferrocynide to form bright blue deposits of ferric ferrocyanide or prussian blue 5/28/2018 Seminar

55. Component Color Proteoglycans , hyaluronic acid, and acidic mucins Bright blue Collagen Red Muscle and cytoplasm yellow 5/28/2018 Seminar

56. Colloidal iron stain Mucinous spindle cell carcinoma of kidney 5/28/2018 Seminar

57. Combined colloidal iron- PAS Sections are incubated with a colloidal iron solution and stained with potassium ferrocyanide before oxidation by periodic acid Acid mucins , proteoglycans and hyaluronic acid – bright blue with colloidal iron/ potassium ferrocyanide reaction Neutral mucins and glycogen are colored red/ magenta by the schiff reaction 5/28/2018 Seminar

58. High iron diamie Useful for the detection the highly acidic sulfomucins Selctive for carbohydrates carrying a high negative charge density due to ionized sulfate groups Does not demonstrate hyaluronic acid and sialomucins 5/28/2018 Seminar

59. Combined high iron diamine and alcian blue technique Differentiation of sulfomucins from sialomucins in tissue section Sulfomucins and proteoglycans stain brown to black Sialomucins and hyaluronic acid- blue Useful for the demonstrate of the distribition of sialomucins and sulfomucins in the epithelia of the intestines 5/28/2018 Seminar

60. Metachromatic methods Methylene blue, azure A and toluidine blue are small planar cationic dyes which typically stain tissues blue. Metachromatically these dyes stain tissue components purple-red. The use of such dyes to identify charged mucins and proteoglycans . The highly anionic proteoglycans with alternating sulfate and carboxylate groups meet these criteria and produce metachromatic stains with dyes 5/28/2018 Seminar

61. Expression of mucin in various tumors 5/28/2018 Seminar

62. Type of tumor Glycogen(PAS + ve ) Acid mucin (strongly sulfated) Neutral mucin CARCINOMA GIT - + + Ovary +/- +/- +/- Kidney(RCC) - - -/+ Endometrium -/+ + + Mucoepidermoid - + + Colloid carcinoma - + + SARCOMA Liposarcoma - + - Rhabdomyosarcoma + + - Osteogenic sarcoma - + - Ewing’s sarcoma + - - Smooth muscle tumors +/- + - Neurogenic tumors - + - Seminoma + - - 5/28/2018 Seminar

63. Summary Type of carbohydrate PAS PAS-D Ab 2.5 Ab 1.0 Ab 2.5/ PAS Muc Meta Polysaccharides Glycogen + - - - +(M) - - Connective tissue glycoconjugates Proteoglycans - - + + +(B) v/- + Hyaluronic acid - - + - +(B) v/- + Mucins Neutral + + - - +(M) - - Sialomucin (enzyme labile ) + + + - +(M) + + Sialomucin (enzyme resistant) - - + - +(B) + + Sulfomucin v/- v/- + + +(BB) + + 5/28/2018 Seminar

64. Referances Totty B A.Mucins.In:Gamble M,Bancroft J D.Theory and practice of histological techniques.5 th ed.Philadelphia.Elsevier;2002:163-200 . Nayak R. Mucins . In: Nayak R. Histopathology technique and its management. Jaypee;2018: 176-99. Vilar J P, Hill R L. The Structure and Assembly of Secreted Mucins . Jbc ; vol. 274, no. 45, 1999: 31751–4. Ringel J, Lohr M. The MUC gene family: Their role in early detection of pancreatic cancer. Mol Cancer. 2003; 2-9. Jass J.R. Mucin staining. J Clin Pathol . 1996;49:787-90. Owen D.A. The stomach. In:Mills S.E,editor . Sternberg’s Diagnostic surgical pathology.4th ed.Philadelphia;2006:1321-73. 5/28/2018 Seminar