Neutral mucopolysacharides – They contain hexoses (instead of hexuronic acid) as their second carbohydrate compound.
Chitin and capsules of some organisms like fungi and pneumococcus
What are the examples of Glycoproteins
These constitute the mucins secreted by GI epithelial cells, gonadotrophic and thyroid stimulating hormones, serum mucoproteins and agglutinogens
acid epithelial mucins secreted by GI tract epithelial cells are sialomucins, sulfomucins and sulfated sialoglycoproteins
Tracheobronchial tree – Sulfated mucin predominate on surface
Deeper mixed mucous glands – Sialomucins
what are Glycolipids
Cerebrosides (chiefly Phrenosin and kerasin) and abnormal kerasin found in Gauchers disease are PAS positive
What are the best fixatives for mucins and mucopolysaccharides
For mucins
Formol- calcium
Formol- alcohol
Acidic fixatives
Carnoys fluid
Mercurial fixatives – Zenkers, Bouins and Brazil
For acid mucopolysaccharides
Lead subacetate
Lead – formalin fixative
Prolonged formalin fixation reduces the strength of PAS why? how can this be reversed
Reduced strength of PAS reaction to mucins on prolonged fixation is due to polymerization.
This can be reversed by treating hydrated sections with 0.2 NaOH for 10 to 15 minutes prior to staining
What are the three dyes of Basic Fuchsin
Rosanilin
Pararosanilin
Magenta II
What is the Schiff reaction
H.Schiff (1866) showed that aldehydes restore the magenta colour to Fuchsin which has been decolorized (leucofuchsin) by sulfur dioxide
Leucofuchsin is colorless because its chromophoric double bond has been destroyed. Reoxidation slowly by exposure to light and air will restore double bond and the colour
what is the principle of PAS reaction
Periodic acid oxidizes glycogen, mucoprotein and other high molecular to aldehyde
Aldehydes which reacts with colourless Schiff reagent producing bright red or pink color
What is Feulgen Schiff reaction
Feulgen discovered that gentle acid hydrolysis of fixed tissues exposed the deoxypentose of nuclei in a potentially aldehydic form but still bound to nuclei acid chain by phosphate links
later on exposure to Schiffs reagent produces reddish purple colour in DNA containing structures
Ribonucleic acid (RNA) which contains ribose instead of deoxyribose of DNA, does not give Feulgen reaction because the glycol group of its ribose is substituted by phosphate
What are PAS positive substances
Glycogen
Neutral polysaccharides (capsules of bacteria and fungi)
Glycoproteins
mucin secretions of intestinal tract and glands
secretions of tracheobronchial tree
Gonadotrophic hormone
Thyroid stimulating hormone
Thyroglobulins
Serum mucoproteins
Hyaline proteinaceous renal casts
Russel bodies in plasma cells
cytoplasm of megakaryocytes
Fractions of serum albumin and globulin
Basement membrane
Reticulin and collagen
Sialoglycoproteins of ovarian pseudomucinous cystadenomas of ovary.
Glycolipids
Gangliosides in gray matter of brain, epithelioid cells and globoid cells in Krabbe’s Leukodystrophy and in Tay Sachs disease
cerebrosides in white matter
Kerasin in Gauchers disease
Non carbohydrate containing substances
Sphingomyelin (Neimann – Pick’s disease)
Pigments
Ceroid
Lipofuscin
Pigment in melanosis coli
Miscellaneous substances
Mast cells (less sulfated forms of heparin are PAS positive)
Plasmalogens
These are labile acetal phospholipids which are formed in tissues fixed only for a short time in formalin or has been mercury – fixed
What are PAS negative mucopolysaccharides
Acid mucopolysaccharides : Hyaluronic acid, Hyaluron sulfate, and chondroitic sulfate
Reactive groups are prevented from attack by HIO4 because of the affect of acid groups.
What is blocking of PAS reaction
On PAS stain, the red color of carbohydrate substance is due to presence of 1:2 glycol groups which are oxidized to stable dialdehydes
Blocking of PAS reaction is done by acetylation which blocks both 1,2-glycol and hydroxyamino group
This is used to differentiate whether false reaction is caused by preformed aldehyde groups. To demonstrate this false reaction section is treated by Schiff reagent alone without periodic acid pretreatment, which will be recovered by aldehyde group
What are different methods of blocking PAS reaction
Acetylation – Placing hydrated sections for 1 to 24 hrs at 250 C or 1/2 to 6 hrs at 580 C in a mixture of 16 ml acetic anhydride and 24 ml anhydrous pyridine
Benzoylation
Boration
Bromation
How is the blocking of PAS reaction reversed (Deacetylation)
This process is reversed by saponification
For this sections may be treated with 0.5% KOH in 70% alcohol for 30 minutes
During this procedure sections may detach hence 0.1% of chrome alum 1% of gelatin is used as adhesive
What is the technique of blocking preformed aldehyde groups in tissue sections
Treat sections with 10% aniline in acetic acid for 30 minutes at room temperature
wash in distilled water
Treat sections with Schiff reagent and continue as with routine PAS stain
What is PAS – diastase reaction
Sections are treated with 1% of malt diastase for 30 minutes at room temperature
Diastase digests the Glycogen in sections and later PAS staining will be negative
This is used to differentiate the glycogen form other PAS positive substances like mucins
What are the reagents required for PAS stain
Periodic acid 1%
Schiff’s reagent
Mayers Haematoxylin
What is the method for preparing Schiff’s reagent
Different methods available are
Lillie’s procedure
Itikawa and Ogura method
De Tomasi – Coleman method
Barger and Delamater method
Lillie’s procedure
Dissolve 1g basic fuscin and 1.19g Sodium metabisulfite in 100ml of 0.15N HCl.
Shake the solution at intervals for 2hrs
It should be clear and yellow to light brown
Add 0.5g of activated charcoal and shake 1 to 2 minutes
Filter into brown stock bottle and store at 0 to 40 C
How do we test the potency of Schiff’s reagent
Schiff’s reagent may be tested by adding a few drops to 3 to 5ml of 40% formaldehyde on a watch glass
Active reagent will give reddish purple colour rapidly.
If there is delayed development of colour it indicates the Schiff’s reagent is losing potency
What is the procedure for PAS stain
Deparaffinize and hydrate to water.
Oxidize in 0.5% periodic acid solution for 5 minutes
Rinse in distilled water
Place in Schiff reagent for 15 minutes (Sections become light pink color during this step).
Wash in lukewarm tap water for 5 minutes (Immediately sections turn dark pink color).
Counterstain in Mayer’s hematoxylin for 1 minute.
Wash in tap water for 5 minutes.
Dehydrate and coverslip using a synthetic mounting medium.
Interpretation
PAS positive material – Magenta or Purplish red colour
Background – blue
Diagnostic importance of PAS stain
It is used to demonstrate glycogen and neutral mucoproteins. It is used to diagnose –
Poorly differentiated adenocarcinoma of various tissues like stomach, pancreas, lung
Hepatocellular carcinoma
Renal cell carcinoma
Seminoma
Dysgerminoma
Demonstrate intracytoplasmic crystals in alveolar soft part of sarcoma
demonstrate basement membrane
Demonstrate neutral mucin in Gastrointestinal tract
Demonstrate fungi in tissues
Diagnosis of block positivity in AML and AML diffuse cytoplasmic positivity in M5, M6, M9
References
Cullings. Histotechniques. In: Lynch Medical Laboratory technology by Mathew J. Lynch, Stanely S. Raphael. Saunders publication 1983