Demonstration of Carbohydrates in Tissue Sections (PDF)

DEMONSTRATION OF CARBOHYDRATES IN TISSUE SECTIONS BY B. A. OWUSU 27/10/2017 OBRIGHTA 1  CARBOHYDRATES Carbohydrates are organic compounds containing carbon, hydrogen and oxygen (CHO) in specified proportion...

DEMONSTRATION OF CARBOHYDRATES IN TISSUE SECTIONS BY B. A. OWUSU 27/10/2017 OBRIGHTA 1  CARBOHYDRATES Carbohydrates are organic compounds containing carbon, hydrogen and oxygen (CHO) in specified proportions. In humans it occurs in a variety of forms depending on the functions allocated to it. Carbohydrates can be found in their simplest form monosaccharide or linked together as polysaccharides Associated with non-carbohydrate substances as in Glycoproteins, Glycolipids etc 27/10/2017 OBRIGHTA 2 CLASSIFICATION OF CARBOHYDRATES — Chitin — Starch — Cellulose — Glycogen — Mucins 27/10/2017 OBRIGHTA 3  CHITIN Chitin is a mucoprotein in the form similar to homopolyaminosaccharide and contains N- acetyl-D-glucosamine. A hyaline substance of wide distribution in non- human tissue, forming the exoskeleton of various insects. In human it is seen lining the walls of hydatid cysts of lung and liver due to infestation by the dog tapeworm (Echinococcus granulosus). 27/10/2017 OBRIGHTA 4  STARCH — Starch granules may appear free or contained within macrophages IN HUMANS — It is mostly found free in tissue as a result of contamination from surgical glove powder. — Very occasionally, starch granules are allowed to enter tissue during minor surgery, which may result in the formation of a granuloma — There are peritoneal granulomas caused by food starch residue from perforated bowels. — Starch granules can occur in pulmonary alveolar walls of drug addicts who have injected themselves with starch containing barbiturates. 27/10/2017 OBRIGHTA 5  CELLULOSE — Undigested food particles can be seen in sections from the gastrointestinal tract. This is most evident in sections of appendix and very occasionally intestinal fistulae. — Foreign body granulomas containing cellulose are possible especially in rural communities. 27/10/2017 OBRIGHTA 6  GLYCOGEN — It is a simple polysaccharide, which consists of branched or straight-chained D-glucose units. — Glycogen is found in their greatest numbers in liver skeletal muscle cardiac muscle — Sufficient quantities can be found in Hair follicles Endometrial glands Ectocervical epithelium — It can also be found in umbilical cord, mesothelial cells, neutrophils and megakaryocytes 27/10/2017 OBRIGHTA 7  TWO FORMS OF GLYCOGEN 6 H H H H STRAIGHT O O O 5 4 1 4 1 4 1 3 2 O O OH H BRANCHED O 6 1 O CH H H O O 1 4 1 O OH 27/10/2017 OBRIGHTA 8  GLYCOGEN UNDER E.M. — In humans glycogen can be viewed under electron microscope in two forms (alpha and beta) — Alpha forms: as clusters of particles (60-250 nm diameters) — Beta forms: as free particles of 20-40 nm scattered all over or next to the alpha forms in the same cell. — A third form (gamma): Observed in mouse liver tissue are non-particulate and found between the beta particles. (when the tissue is fixed in osmium tetra oxide in which potassium ferricyanide is incorporated). 27/10/2017 OBRIGHTA 9  MUCINS — Mucins have been referred to as ¨ Mucopolysaccharide ¨ Glycosaminoglycans ¨ Mucosubstances. ¨ Mucoconjugates 27/10/2017 OBRIGHTA 10  FUNCTIONS OF MUCINS — Most surfaces are coated with mucins having an obvious lubricating function but it has been suggested that this coating also forms a favorable medium for ionic and molecular diffusion. — Their presence increases cell-to-cell adhesion and serve as determinants for cell surface specificity. — In the bladder, the epithelial mucus may provide an anti-adhesive effect on bacteria allowing the bacteria to be washed away by urine. 27/10/2017 OBRIGHTA 11  MUCIN GENES — MUC 1: Encodes for ‘episialin’ a membrane mucin strongly expressed in breast, pancreas and ovaries. — MUC 2: Strongly expressed in the large and small intestines — MUC 3: Strongly expressed in the jejunum — MUC 4, MUC 5A &C and MUC 5B: are isolated from trans-bronchial mucosa. — MUC 6: Expressed in gastric mucosa. — MUC 7: Is a small gene whose products are isolated from sub-maxillary gland. MUC genes are located in more than five different chromosomes 27/10/2017 with a cluster of them on chromosome 11. 12 OBRIGHTA  TYPES OF MUCINS Mucins can be divided into two groups ¨Acidic mucins Sulphated Carboxylated ¨ Neutral mucins 27/10/2017 OBRIGHTA 13  ACIDIC MUCINS (Sulphated) — Strongly sulphated mucins ¨ Connective tissue ¨ Epithelial — Weakly sulphated mucins — Atypical sulphated mucins 27/10/2017 OBRIGHTA 14  Strongly sulphated connective tissue mucins — These mucins react at low pH values. — They are usually PAS-negative under standard periodic oxidation. — Cells, which produce these highly sulphated substances, are: ¨ fibroblasts ¨ endothelial cells ¨ osteocytes ¨ chondrocytes ¨ mast cells. 27/10/2017 OBRIGHTA 15  Sub-types of Strongly sulphated connective tissue mucins -1 1. Chondroitin sulphate A (chondrotin-4-sulphate); Found in cartilage and contains D-galactosamine and D-glucuronic acid. It is sulphate-esterified at the C4 position on the galactosamine molecule. 2. Chondroitin sulphate B (dermatan sulphate); Found in aorta, heart valves and dermis of the skin. It is similar to type A but also contain 1-iduronic acid. 3. Chondroitin sulphate C (chondroitin-6-sulphate); The chemical composition is similar to type A but it is sulphate- esterified at C6 position on the galactosamine molecule. Found in cartilage, umbilical cord and dermis of the skin 27/10/2017 OBRIGHTA 16  COOH 6 H O H 5 4 H 1 H 3 2 OH SOH NH2 OH 27/10/2017 OBRIGHTA 17  Sub-types of Strongly sulphated connective tissue mucins -2 4. Heparin/heparan sulphate (heparitin sulphate) They contain: N-acetyl-D-glucosamine, N-sulphate-D-glucosamine D-glucoronic acid. The sulphate ester groups are found on: ¨ C6 of the glucosamine ¨ C2 of the glucoronic acid Differences: ¨ Heparin contains in addition: sulpho-amino group and iduronic acid in place of D-glucoronic acid. ¨ Heparin is found in mast cells while heparan is found in aorta and cardiac connective tissue. 27/10/2017 OBRIGHTA 18  COOH 6 H O OH 5 4 H 1 H 3 2 OH OH N-SH 27/10/2017 OBRIGHTA 19  Sub-types of Strongly sulphated connective tissue mucins -3 5. Keratan sulphate (keratosulphate); Consists of sulphate- esterified N-acetyl-D-glucosamine and galactose and no uronic acid. Found in cornea, pulposus of the intervertebral discs and in aging cartilage. 6. Hyaluronosulphate (chondroitin); Contains N-acetyl-D- glucosamine and D-glucoronic acid and found only in cornea 27/10/2017 OBRIGHTA 20  COOH 6 H O OH 5 4 H 1 H 3 2 SOH N-CH3 OH N- ACETYL- D- GLUCOSAMINE 27/10/2017 OBRIGHTA 21  Strongly sulphated epithelial mucins — These are similar in many ways to the strongly sulphate connective tissue types except that they originate from epithelial cells. — They are PAS-positive and have been demonstrated in goblet cells and bronchial serous glands 27/10/2017 OBRIGHTA 22  Weakly sulphated epithelial mucins — The weakly sulphated mucins are different from the strongly sulphated type in reacting at a slightly higher pH. — They are epithelial type and consist of polysaccharides with sulphate esters in which the sulphate esters are linked to various hexosamines. 27/10/2017 OBRIGHTA 23  ATYPICAL SULPAHTED MUCINS These are sulphated mucins, which stain alcian blue but not the traditional technique for sulphated mucins. 27/10/2017 OBRIGHTA 24 CARBOXYLATED MUCINS — SIALOMUCINS — SULPHATED SIALOMUCINS — HYALURONIC ACID 27/10/2017 OBRIGHTA 25  Carboxylated mucins (The sialomucins) — Sialomucins contain derivatives of neuraminic acid (N- acetylated neuraminic acid (sialic acid) — Sialomucin are epithelial and formed of a terminal sialic acid molecule on oligosaccharide side chains of a polypeptide. — There are two types of sialomucins a. Enzyme labile sialomucins b. Enzyme resistant sialomucins 27/10/2017 OBRIGHTA 26  The enzyme labile sialomucins — The enzyme labile sialomucins are digested by sialidase enzyme. — They have a wide distribution occurring either alone or mixed with other mucins. — Found in bronchial submucus glands, submandibular salivary glands and goblet cells of the small intestines. 27/10/2017 OBRIGHTA 27  The enzyme resistant sialomucins — Similar to the one described above but differ in two significant respects. — That they are resistant to denaturation with sialidase enzyme and are PAS-negative. — It consists of N-acetyl-O-acetyl neuraminic acids in which there is a masking O-acetyl group on the sialic acid molecule. 27/10/2017 OBRIGHTA 28  Sulphated sialomucins — There is a debate as to whether this group of mucins really exists or occurs in situations where sulphomucins are mixed with sialomucins. — They have reactions similar to those of weakly sulphated mucins and these are prevented when sialidase digestion is carried out before demonstration 27/10/2017 OBRIGHTA 29  Hyaluronic acid -1 — Is a widely occurring connective tissue mucin in fibroblasts. — It contains N-acetyl-D-glucosamine and D- glucoronic acid. — They have similar properties as sialic containing mucins especially reactions at the same pH. — The difference is in their reactions with enzymes hyluronidase and sialidase. 27/10/2017 OBRIGHTA 30  Hyaluronic acid -2 — An important component of synovial fluid, where its concentration is directly proportional to the viscosity of the fluid. — Hyaluronic acid is found in umbilical cord, early placenta, cardiac connective tissue and dermis of the skin. — It is found as a minor fraction of mucin in aorta, bone and cartilage 27/10/2017 OBRIGHTA 31  NEUTRAL MUCINS. — They have no acidic reactive group and consist of various hexosamine associated with free hexose groups — Epithelial in type and found in abundance in: Brunner’s glands, gastric linings, alimentary and respiratory tract, goblet cells as well as prostatic glands. 27/10/2017 OBRIGHTA 32  SUMMARY OF LOCATION OF THE DIFF. CHO TYPE SITUATION Cellulose Abnormally in skin and gastrointestinal tract Chitin Hydatid cysts of liver, brain and lung Chondroitin sulphate A Hyaline cartilage Chondroitin sulphate B Skin and heart valves Chondroitin sulphate C Umbilical cord and skin Glycogen Liver, voluntary muscle and hair follicles Heparin/heparan sulphate Mast cells/aorta, respectively Hyaluronic acid Umbilical cord, skin early placenta and synovium 27/10/2017 OBRIGHTA 33  SUMMARY OF LOCATION OF THE DIFF. CHO -2 TYPE SITUATION Keratan sulphate Hyaline cartilage and intervertebral discs Neutral mucins Stomach, prostrate and Brunner’s glands Sialomucin enzyme-labile Submandibular salivary glands Sialomucin enzyme-resistant Large intestine Starch Some skin and peritoneal granulomata Strongly sulphated mucins Bronchial sub mucous glands epithelial Sulphated sialomucin Prostatic carcinoma Weakly sulphated mucins Goblet cells and large intestine 27/10/2017 OBRIGHTA 34  Lecture 2 27/10/2017 OBRIGHTA 35  PREPARATION OF TISSUE FOR CHO DEMONSTRATION —Starch, Chitin and Cellulose: For these CHO the type of fixative used is not important. As a matter of fact, they do not really need fixation at all in order to be demonstrated 27/10/2017 OBRIGHTA 36  PREPARATION OF TISSUE FOR CHO DEMONSTRATION (GLYCOGEN) — Glycogen require fixation. — Fixation should be prompt to avoid an initial sharp loss of glycogen. This should be done at 4oC to avoid or minimize streaming artifacts. — Two schools of thought about the effects of fixatives on glycogen. 1. Elimination of water 2. The accepted theory is that fixatives fix proteins associated with glycogen to form crosslinkages. 27/10/2017 OBRIGHTA 37  FIXATION OF GLYCOGEN 6 H H H H STRAIGHT O O 5 O 4 1 4 1 4 1 3 2 O O OH H BRANCHED O 6 1 CH O H H O O 1 4 1 O OH 27/10/2017 OBRIGHTA 38 GLYCOGEN FIXATION — Fixatives: Alcohol, picric acid and aqueous formalin Fixatives are generally favored for glycogen demonstration. — Avoid: Glacial acetic acid fixatives (Susa and Zenker solutions). — However, in calcified bone, use 5% trichloroacetic acid for glycogen 27/10/2017 OBRIGHTA 39  Summary(glycogen) To obtain best results in glycogen demonstration — Fix early asap after death or removal. — Use alcoholic, picric acid or formalin fixatives — Fix at low temperature (4oC) — Fix for at least 1-2 days 27/10/2017 OBRIGHTA 40  PREPARATION OF TISSUE FOR CHO DEMONSTRATION (mucin) — There is no need for prompt fixation and for most purposes 10% formalin is acceptable. — Alcohol fixation may be required in the demonstration of abnormal CHO (in certain mucopolysaccharidoses). — When hyaluronic acid is not mixed with other mucins you can use 10% formol-alcohol or formol- sublimate instead of 10% formalin. — In calcified bone, use chelating agents for mucins. 27/10/2017 OBRIGHTA 41  DEMONSTRATION OF GLYCOGEN — The traditional iodine methods of demonstrating CHO are still relevant but not important in pathology since the stain is both not permanent and non-specific. — In H&E, Glycogen will not stain Haematoxylin but stains weakly if at all with eosin. — In practice, cells containing abundant glycogen stains weakly. 27/10/2017 OBRIGHTA 42  Periodic acid-Schiff reaction (PAS) — The PAS reaction is a very useful indicator for CHO in tissue sections. — The principle: periodic acid oxidizes glycosyl units resulting in the cleavage of bonds between C-C links resulting di-aldehydes. — These aldehydes reacts with fuchsin-sulphurious acid, which combines with a basic pararosaniline to form a magenta-coloured compound (alkyl sulphonate in nature) 27/10/2017 OBRIGHTA 43  CH2OH CH2OH H O H O HIO4 O O F(SO2H) 2 CH2OH O F 27/10/2017 OBRIGHTA 44  PAS POSITIVE STAINING OF GOBLET CELLS 27/10/2017 OBRIGHTA 45 PAS POSITIVE STAINING OF GOBLET CELLS 27/10/2017 OBRIGHTA 46 Alternative oxidants — Chromic acid — Potassium per manganate — Lead tetra-acetate — Sodium bismuthate 27/10/2017 OBRIGHTA 47  Best’s Carmine method(1) — Carmine is extracted from cochineal beetle with water — Contains 56% carminic acid and proteins together with some small amounts of aluminum and calcium. — Although the carminic acid is almost certainly the essential staining ingredient, it is not often used in place of carmine. 27/10/2017 OBRIGHTA 48  Best’s Carmine (2) — Staining is accomplished when the OH groups in the glycogen form hydrogen bonds with the H atoms of carminic acids in carmine. — A useful stain for demonstrating glycogen but its stock solutions slowly deteriorate when stored. — Best’s carmine also stains fibrin and neutral mucins. — Glycogen stains deep red, while some mucins and fibrin stain weakly red. 27/10/2017 OBRIGHTA 49  Hexamine silver technique — Gomori 1946, modified by Grocott 1955 — The principle: When glycogen is oxidized with chromic acid, aldehydes are formed. These aldehydes then reduce hexamine-silver nitrate mixture to a black compound. 27/10/2017 OBRIGHTA 50  NOTES ON HEXAMINE SILVER TECHNIQUE — Any oxidant can be used but remember that some mucins will be stained as well. — Sections should be examined periodically during incubation in hexamine-silver nitrate solution. — Hexamine –silver solution is heated to 56oC before sections are incubated in it. — Avoid heating the hexamine –silver solution for too long. — Remember that the stock solution can deteriorate and therefore false negative results may occur. 27/10/2017 OBRIGHTA 51  The use of enzymes in glycogen demonstration — Alpha amylase — Beta amylase — Diastase — Saliva — Pectinase 27/10/2017 OBRIGHTA 52  Alpha amylase — Extracted from hog pancreas and certain organisms such as Bacillus subtilis and Aspergillus oryzae. — They are capable of breaking both branched and straight chains by splitting α-1:4 and α- 1:6 glucosidic linkages. — This releases glucose and maltose, which are soluble in the solvent used for the enzyme and can therefore be washed off the section. 27/10/2017 OBRIGHTA 53  Beta amylase — This can be obtained from barley or sweet potato. — It is an enzyme that can only digest straight- chained glycogen, which it does by splitting alternate α-1:4 glucosidic linkages. — A pure beta amylase releases only maltose. 27/10/2017 OBRIGHTA 54  Diastase: — This is the commonest glycogen-digesting enzyme — It is extracted from malt and contains both α and β amylase. — It is easy to use, stable and cheap. — Diastase is used at 1% aqueous concentration 27/10/2017 OBRIGHTA 55  Saliva: — Saliva is obtained from human — Saliva contains ptyalin (salivary amylase) and breaks both α-1:4 and α-1:6 linkages — In practice this is the most available and highly effective source of enzyme for digesting glycogen in tissue sections. 27/10/2017 OBRIGHTA 56  Pectinase: — This enzyme works well against abnormal glycogens found in some glycogenosis (glycogen storage disease). — These glycogens are usually not easily digested by the conventional enzymes 27/10/2017 OBRIGHTA 57  NOTES ON THE USE OF ENZYMES — Note that starch is also digested by these enzymes but at a slower rate. — Fixed tissues resist digestion and will require longer time for digestion — The type of fixative used can affect enzyme digestion (gluteraldehyde and OsO4) — Some workers prefer to buffer the enzyme. — Diastase should be prepared daily. — Celloidinisation can inhibit enzyme activity and should be avoided. 27/10/2017 OBRIGHTA 58  LECTURE 2B 27/10/2017 OBRIGHTA 59 DEMONSTRATION OF MUCINS -1 — H&E: In haematoxylin and eosin stains, mucins are generally weakly stained by eosin. — When Ehrlich’s haematoxylin is used, those of connective tissue origin turn to be stained blue. 27/10/2017 OBRIGHTA 60  MUCINS -2 — PAS — Mucins containing reactive (vicinal OH groups) hexose components are PAS-positive. Examples include; Neutral mucins, N-acetyl sialomucins epithelial tissue acid mucins. mucoproteins in basement membrane, thyroid colloids pituitary basophil cells cerebrosides and gangliosides. PAS-negative Sialomucins containing O-acetyl group (enzyme resistant) — Connective tissue mucins (alpha 1:4 linkages of those containing uronic acid can be positive if oxidation is extended (upto 24 hrs). 27/10/2017 OBRIGHTA 61  MUCINS -3 — Phenylhydrazine-PAS (neutral mucins) — Phenylhydrazine condenses the aldehydes formed from neutral mucin by periodic acid as against those of acid mucins. This blocks the Schiff’s reactivity to the former. — In this way it is possible to differentiate neutral mucins from acid mucins. 27/10/2017 OBRIGHTA 62  ALCIAN BLUE — Alcian blue is the most popular dye for the general demonstration of acid mucins and used in 3% acetic acid at pH 2.5 — Specificity; It has a high specificity for acid mucins (and calcium salts) — Strong coloration. — Insolubility of staining; Once the sections are stained, the dye-tissue complex is insoluble. — Permanence of results; Once the sections are stained, they seem to retain the dye for many years. This has nothing to do with the effect of the mountant. 27/10/2017 OBRIGHTA 63  Other alcian dyes — Alcian blue 8 GX. (also known currently as Ingrain blue 1). — Alcian yellow. — Alcian green 2GX (staining an emerald green colour) — Alcian green 3BX (staining a blue–green colour) 27/10/2017 OBRIGHTA 64  Varying the pH of alcian blue for the different acid mucins - 1 — The tissue component is more intensely stained if the dye is used at the pH at which the reacting groups are fully ionized. — To this effect advantage can be taken of this to use alcian blue solutions of varying pH to separate and identify the different acid mucins. 27/10/2017 OBRIGHTA 65  Varying the pH of alcian blue for the different acid mucins - 2 — Strongly sulphated mucins; These react most consistently at low pH levels. Above pH 1.0 their staining becomes uncertain. — Weakly sulphated mucins; Stain well at pH 2.5 down to 1.0 and below. — Hyaluronic acid and N-acetyl sialomucins; pH ranges between 3.2 down to 1.7 — N-acetyl-o-acetyl sialomucins; As compared to N- acetyl sialomucins these will stain down to pH 1.5 — Combined alcian blue-alcian yellow; A lower pH alcian blue followed by a higher pH alcian yellow 27/10/2017 OBRIGHTA 66  Alcian blue- PAS technique — Acid mucins and neutral mucins are clearly separated by this method. — By first staining all acid mucins with alcian blue, those acid mucins, which are also PAS positive, would have been stained. — So in the subsequent PAS stain only the neutral mucins will be stained. 27/10/2017 OBRIGHTA 67  Deamination alcian blue-PAS (1) — Some proteins associated with the carbohydrate are either so much or bound to the carbohydrate in a way that will mask the carbohydrate moiety. — In certain types, sialic acid may be present but masked by a protein component. In such instances the mucoprotein will show PAS positive but Alcian blue negative 27/10/2017 OBRIGHTA 68  Deamination alcian blue-PAS (2) — Deamination is carried out to displace the protein (amino group) and expose the masked alcianophilic component. — Subsequent combined Alcian blue-PAS will show a change from PAS positive to Alcian blue staining. — The principle is that when the tissue is treated with Sodium nitrite and acetic acid, nitrous acid is formed leading to the displacement of amino groups by nitrogen. 27/10/2017 OBRIGHTA 69  Dialysed iron-Prussian blue technique for acid mucins — The principle of this technique is that at a low pH colloidal iron will be absorbed into tissue polyanions. — The absorbed iron is subsequently visualized by conversion to ferric ferrocyanide (Prussian blue) based on conventional Perl’s reaction. — The technique can be combined with PAS to differentiate between acid and neutral mucins. 27/10/2017 OBRIGHTA 70  Acridine orange technique for fluorescent staining of acid mucins — Acridine orange is a cationic fluorochrome and can be used for the demonstration of acidic tissue moieties including acid mucins. — It is also possible to vary the pH for the differentiation of the various acid mucins as done in alcian blue technique. — The disadvantages are that the staining remains for only 2 hours after mounting and requires the use of fluorescent microscope 27/10/2017 OBRIGHTA 71  Aldehyde fuchsin For sulphated mucins — A reliable means for the differentiation between sulphated mucins and carboxylated mucins. Aldehydes fuchsin stains sulphur-containing moieties in tissues when they are pre-oxidised with iodine or potassium permanganate. — In PAS, sulphurated basic fuchsin combines with aldehydes while aldehyde fuchsin technique employs basic fuchsin plus aldehydes to demonstrate sulfur- containing compounds. 27/10/2017 OBRIGHTA 72 PAS: Sulphur + basic fuchsin aldehyde = pink Aldehyde fuchsion: Aldehyde + basic fuchsin Sulphur = pink 27/10/2017 OBRIGHTA 73  Mild methylation-alcian blue — When sections are treated with methanol for 4 hours at 37oC, carboxylated mucins will be blocked. By subsequent staining with Alcian blue sialomucins and hyaluronic acid will be expressed by loss of staining. — Connective tissue sulphated mucins will also be blocked leaving epithelial sulphated mucins. — It is therefore wiser to use this technique only when differentiating between carboxylated mucins and epithelial sulphated mucins. 27/10/2017 OBRIGHTA 74  High iron diamine technique — This method demonstrates sulphated mucins with high specificity. — Principle; when diamine salt is oxidized by ferric chloride it forms a black cationic chromogen which bonds with sulphate ester groups. — Counter staining with Alcian blue, which by exclusion will only stain carboxylated mucins, makes a clear colour distinction between the two groups (mucins from bronchial sub-mucus glands do not react). 27/10/2017 OBRIGHTA 75 SIALIC ACID-CONTAINING CARBOXYLATED MUCINS — Mild PAS technique — By utilizing a weak solution of periodic acid (0.01%) instead of the usual percentage, a normal PAS technique will selectively demonstrate N- acetyl sialomucins. — This is based on the findings that the side chain of sialic acid is more readily oxidized than others. 27/10/2017 OBRIGHTA 76  SIALOMUCINS — Sialidase digestion: Sialidase digestion is important for differentiating enzyme labile and enzyme resistant sialomucins. Used in combination with the Alcian blue-PAS technique. — Deacetylation-sialidase digestion for sialidase- resistant sialomucins: Alkaline aqueous alcohol is used to deacetylate the O-acetylation, which is responsible for the inhibition. After which the sialomucins becomes enzyme labile and can be digested by sialidase enzyme 27/10/2017 OBRIGHTA 77  URONIC ACID CONTAINING CARBOXYLATED MUCINS — The only member of this group is hyaluronic acid which has the same Alcianophilia as sialomucins. It can therefore be digested by hyaluronidase enzyme in the same way that sialidase is used for sialomucins 27/10/2017 OBRIGHTA 78  Demonstration of CHO in dx — The two most important diagnostic entities in CHO demonstration are glycogen and mucins. — Glycogen may be demonstrated in a number of lesions and is diagnostically significant in certain tumors. — Some glycogenosis may exhibit excess amounts of glycogen in tissues and this is paralleled by marked deposition of glycosaminoglycans in various tissues in mucopolysaccharidoses. 27/10/2017 OBRIGHTA 79  Demonstration of CHO in dx — The usual diagnostic requirement is to demonstrate the presence or absence of carbohydrates. However, there are circumstances where the precise identification may be of value. — Secondary tumors may show a marked variation from the primary neoplasm as seen by an H&E stained section, the component CHO are invariably constant in amount and types. 27/10/2017 OBRIGHTA 80  Glycogenosis TYPE EPONYM ENZYME DEFFICIENCY I Von Gierke Glucose-6-phosphatase II Pompe Acid maltase III Forbes -Cori Debrancher enzyme IV Andersen Brancher enzyme V McArdle-Schmid- Myophosphorelase pearson VI Hers Hepathophosphorelase VII - Phosphoglutamase VIII - Reduced phosphorelase IX - Markedly reduced phosphorelase X - Cyclic AMP-dependant kinase 27/10/2017 OBRIGHTA 81  Glycogen and mucin deposits of diagnostic significance CARBOHYDRATE PATHOLOGY Glycogen Carcinomas of bladder, kidney, liver, ovary (adeno), pancreas, lung (adeno). Also, Ewing’s sarcoma, seminoma, mesothelioma (some), juvenile rhabdomyosarcoma. Hyaluronic acid Follicular mucinosis, pre-tibial myxoedema, maxoid liposarcoma and some mesothelioma Neutral mucins Carcinoma of the stomach (diffuse type) Sialomucins (N-acetyl form) ‘Transitional’ epithelium colon/rectum and carcinoma of the breast Sialomucins (O-acetyl form) Some carcinoma of colon/rectum Sulfomucins Intestinal metaplasia of stomach (Type IIB, redesignated type III) 27/10/2017 OBRIGHTA 82 THANK YOU, ANY QUESTION??? 27/10/2017 OBRIGHTA 83