SPECIAL STAINS FOR THE CARBOHYDRATES, PROTEIN, (1).pptx

Cyto histopathlogy special stains

1. SPECIAL STAINS FOR THE CARBOHYDRATES, PROTEIN, LIPID, NUCLEIC ACID AND PIGMENTS DR. DAMPTEY (BSC. MBCHB)

2. SCOPE

3. INTRODUCTION Other than routine haematoxylin and eosin staining, various special stains are now essential parts of routine lab work Applications of special stains include Demonstration of various cellular products for diagnosis Carbohydrates Proteins Lipids Pigments Demonstration of extracellular material for the identification of diseases such as amyloid Identification of microbial organisms Estimation of DNA and RNA content of the cell

4. CARBOHYDRATES Carbohydrates are compounds that contain polyhydroxy aldehydes or polyhydroxy ketones groups C n (H 2 O) m They can be classified depending on the number of subunits as monosaccharides , oligosaccharides and polysaccharides They are also further classified depending on their binding with protein and lipids material

5. Simple carbohydrates Monosaccharides : these are the simplest form of carbohydrates with the empirical formula (CH 2 O) n They are the building blocks of various other carbohydrates They contain an aldehyde or ketone group and the varying number of carbon atoms (5C-pentose, 6C-hexose etc ) The monosaccharides are water soluble and therefore difficult to demonstrate in the routine histology section Oligosaccharides : these are the polymers of monosaccharides that contain 2 – 10 monosaccharides units Polysaccharide : consists of multiple monosaccharides linked by covalent bonds eg . Glycogen, starch

6. Glycoconjugates / Proteoglycans These are primarily groups of extensively glycosylated proteins The proteoglycans have a central core protein that is covalently linked with polysaccharides The carbohydrate part of the proteoglycan is known as glycosaminoglycans Different types of glycosaminoglycans include Chondroitin sulphate : present in cartilage, ligament, bone Dermatan sulphate : skin Heparan sulphate : aorta Heparin: granules of mast cell Hyaluronic acid: synovial fluid

7. MUCINS Mucins are glycoproteins of high molecular weight and are composed of a polysaccharide chain and a protein component Mucin =80% carbohydrate ( hexosamine containing carbohydrate)+ 20% protein Mucins cover the epithelial cells and makes a physical barrier that protects the cells from any external injury. Tow types exist Secretory mucin : secreted in the respiratory tree, GIT and cervical part of the female genital tract Membrane-associated mucin : the mucin attached to the membrane of cells. Mucin may also be noted in non-epithelial tissues

8. Tissue specificity of MUC genes

9. The carbohydrate part of the mucin consists of 80% of the molecular weight of mucin . The polysaccharide part of the mucin may be neutral, weakly acidic or strongly acidic 1. Neutral mucin : noted in the surface of epithelial cells of the stomach, prostate and Brunner`s gland of the duodenum Staining: these mucins are positive for Periodic acid Schiff`s (PAS) stain and negative for Alcian blue stain 2. Acidic mucin : the polysaccharide chain is anionic sialomucin : contains sialic acid. Sialic acid is derived from the acetylation of neuramic acid. Theis is a weak acid mucin . Sialomucin is found in goblet cells, salivary glands etc Sulphomucin : contains a sulphate group, and it is stronger acidic. This type of mucin is seen in the mucus glands of the bronchus Staining: these mucins are positive for Alcian blue stain at low pH(2.8) and are negative for PAS stain

10. Strongly sulphated acid mucins Connective tissue mucin : chondroitin sulphate , keratin sulphate , heparin sulphate Bronchial glands Some fractions of goblet cells of the intestine Strongly sulphated mucins are PAS negative and Alcian blue positive at pH 0.5 Weakly sulphated acid mucins : they are epithelial mucin (Colonic goblet cells and Bronchial glands) Weakly sulphated acid mucins are Alcian blue positive at pH1 and negative for PAS Both neutral and acidic mucins are positive for mucicarmine

11. Significance of mucin demonstration Mucin secreting adenocarcinomas are positive for mucin stain. Whereas the lymphomas or other poorly differentiated carcinomas do not show intracellular mucin Intestinal metaplasia of the stomach show sulphomucin , which is prone to the development of malignancy Pleural mesotheliomas are positive for acid mucin

12. GLYCOGEN Glycogen is demonstrated by Periodic Acid Schiff`s (PAS) reaction PERIODIC ACID SCHIFF`S (PAS) STAIN It stain demonstrates neutral polysaccharides present in the basement membrane and the secretion of various glands in our body INDICATIONS TO DO PAS STAIN To demonstrate polysaccharides: glycogen, cellulose and starch. The basement membrane of glnds , glomeruli etc. the capsule of various fungi; Cryptococci, Histoplasma , blastomycosis etc Glycoproteins: neutral mucins ., also helpful for staining mucin for endocervical glands, intestinal glands, and bronchial glands

13. 3. Glycolipid: cerebrosides and gangliosides . Glucocerebrosides and galactocerebrosides are accumulated in Gaucher`s and Krabbe`s dx resp. gangliosides are accumulated in rare lysosomal storage dx 4. Pigments : certain pigments such as lipofuscin and pigments of Dubin Johnson syndrome are demonstrated by PAS 5. Plasma cells : Russel bodies of plasma cells are stained by PAS

14. PRINCIPLE The hydroxyl group (OH) of the carbohydrate molecule is oxidized to aldehyde (CHO) group by periodic acid These aldehyde grps react with Schiff`s reagent to form a magenta coloured compound

15. Components of solution Solution 1: Periodic acid (1%) Periodic acid :1g Distilled water 100ml Solution 2: Schiff`s reagent Basic fuchsin 1g Distilled water 200ml Potassium metabisulphite 2g 1 M hydrochloric acid (HCL) 20ml Activated charcoal 2g

16. Preparation Dissolve basic fuchsin (1g) in 200ml of boiling distilled water Cool the solution Add 1N hydrochloric acid and mix well Add potassium Metabisulphite (2g) Add activated charcoal (2g) Keep the solution in the dark

17. Steps of staining Deparaffinise Pass through graded lower concentration of alcohol and section/smear to bring in water Oxidise with periodic acid (1%) for 5-10mins Clean with water Schiff`s reagent for 20-30min Clean in running tap water for 5min Counterstain with haematoxylin Wash in tap water for bluing Absolute alcohol Clear in xylene

18. Result Glycogen and glycoprotein: magenta colour Materials that are positive for PAS reaction: glycogen, starch, mucin , reticulin , basement membrane, capsule of fungi Testing the Schiff`s reagent: add drops of Schiff`s reagent will quickly change the colour formation to pink

19. ALCIAN BLUE Alcian blue stains acid mucin (in acidic pH 2.5), such as sialomucin and sulphomucin It stains mucin of salivary glands, prostate, and large intestine Alcian blue also stains proteoglycans of the cartilaginous material

20. Indications Intestinal metaplastic cells in Barrett`s oesophagus and stomach biopsy: the intestinal metaplastic cells contain acid mucin , and these cells in Barrett`s oesophagus are better demonstrated by Alcian blue Mucinous adenocarcinoma of the ovary Pleural mesothelial cell: the pleural mesothelial cells contain hyaluronic acid that is Alcian blue positive and sensitive to hyaluronidase enzyme. Adenocarcinoma cells are Alcian blue positive and resistant to hyaluronidase enzyme Myxoma : mucin secreting tumours such as myxomas are positive for Alcian blue stain Others: mucinous materialmucinous material in myxedema, discoid lupus erythematous lesion etc are also demonstrated by Alcian blue stain

21. Basic principle Alcian blue is a group of water soluble polyvalent basic dyes. The dye is made of a copper-containing phthalocyanine ring with a copper atom in its centre . The Phthalocyanine ring is also attached with four isothiouronium groups that are positively charged. This positively charged Alcian blue dye complex has an attraction with anionic sites of the mucin . Copper imparts the blue colour of the dye- mucin complex

22. Solution Alcian blue solution Alcian blue, 8g 1% aqueous solution: 1g Acetic acid (3%) solution: 100ml Neutral red solution Neutral fast red: 1g Aluminium sulphate : 5g Deionized water: 100ml

23. Steps Dissolve Aluminium sulphate in deionized water and heat Mix neutral fast red in hot water Filter

24. Method of staining Depraffinise Rehydration of the section/smear by graded alcohol Rinse in deionized water Keep the smear in Alcian blue for 30mins Rinse in running water: 5min Counterstain with Neutral fast red: 10min 95% ethyl alcohol Absolute alcohol Xylene mount

25. Result Acid mucin ( sialomucin , sulphomucin ), proteoglycans, and hyaluronic acid will take blue colour

26. COMBINED PAS-ALCIAN BLUE STAINING INDICATIONS Combined use of Alcian blue and PAS in the same sections helps to demonstrate both acidic and neutral mucin in the same section This is frequently applied in gastrointestinal biopsy sections Solution: Alcian blue, periodic acid and Schiff`s reagent

27. Method of staining Deparaffinise Rehydration of the section/smear by graded alcohol Rinse in deionized water Keep the smear in Alcian blue for 30mins Wash in tap water followed by deionized water Periodic acid (1%) for 5 to 10min Clean in running tap water for 5min Schiff`s reagent for 20-30min Clean in running tap water for 5min Counterstain with haematoxylin Wash in tap water for bluing Absolute alcohol Clear in xylene mounting

28. Result Glycogen; magenta colour Acid mucin : Blue colour

29. MUCICARMINE STAIN