Dr. Tarek atia

1. Histochemistry Dr. Tarek atia

2. Visualizing Chemicals and Enzymes in Tissue. • Enzyme histochemistry serves as a link between tissue biochemistry and morphology. • It is a sensitive dynamic technique that mirrors early metabolic changes in any pathological tissue lesion. • Histochemistry: Based on chemical reactions between cell components and stains. • The end products of the reaction are permanent, coloredprecipitatesthat can be viewed under the microscope.

3. Basic Principles of Histochemistry Histochemistry combines the methods of histology with those of chemistry or biochemistry, to reveal the biochemicalcomposition of tissues and cells beyond the acid-base distribution shown by standard staining methods (Hx&E), without disrupting the normal distribution of the chemicals.

4. application • Demarcate the chemical functional boundaries. • Identify, quantify, and localize: ……………………… • chemical substances • gene expression • biological structures; cell organelles • specific cell types

5. Limitations of the current methods (Disadvantages) • Cannot be used for real time in vivo analysis of any tissue (requires the removal and killing of the tissue). • Uses in human limited to biopsied tissues. • For looking at changes in tissue over time, each point in time requires a new tissue sample from a new animal. • Tissue preparation and histochemical analysis may alter specimen morphology or chemistry depending on the methods and materials used.

6. The Goal of Histochemistry 1- Presentation of Normal Chemical Distribution:The substance being analyzed must not diffuse away from its original site. 2- Presentation of Normal Chemical Composition:The procedure must not block or denature the reactive chemical groups being analyzed, or change normally non reactive groups into reactive groups.

7. 3- Specificity of the Reaction: The method should be highly specific for the substance or chemical groups being analyzed, to avoid false-positive results. 4- Detectability of the Reaction Product:The reaction product should be colored or electron scattering, so that it can be visualized easily with a light or electron microscope.

8. 5- Insolubility of the Reaction Product:The reaction product should be insoluble, so that it remains in close proximity to the substance it marks.

9. Some important biologic substances & classic methods for detecting them

10. 1- Ions • It is difficult to localize most ions accurately because of their small size and tendency to diffuse. • However, certain ions are normally immobilized by their association with tissue proteins. Examples:-

11. Iron stain )hemosiderin) • Hemosiderin (storage iron granules) may be present in areas of old hemorrhage or be deposited in tissues with iron overload. • Hemosiderosisis the term used if the iron does not interfere with organ function; but hemochromatosisrefers to a condition of iron overload associated with organ or tissue pathology.

12. Hemosidrosis vs Hemochromatosis • Hemochromatosis is the result of a mutation in the iron metabolism gene and body absorbs more iron than needed and it gets deposited in tissues and causes disease. • Hemosidrosisusually occurs in people who need repeated transfusions of red cells, e.g, sickle cell disease, or thalassemia, the excess iron in transfused red cells deposits in the tissues

13. Perl's iron stain is the classic method for demonstrating iron in tissues. The section is treated with dilute hydrochloric acid to release ferric ions from binding proteins. These ions then react with potassium ferrocyanide to produce an insoluble blue compound (Prussian blue reaction). Mercurial fixatives seem to do a better job of preserving iron in bone marrow than formalin.

14. Copper - Rhodanine Method • Purpose: To identify copper deposits, such as in Wilson's Disease (Autosomal recessive liver disease). • Principle: It has been suggested that the rhodanine demonstrates the protein to which the copper binds rather than the copper itself. • Results: Copper deposits: Bright red to orange Nuclei: Blue

15. Calcium-Von Kossa's Method • Purpose: Abnormal deposits of calcium may be found in any area of the body. • With the H&E stain, calcium appear deep blue-purple. • Principle: Tissue sections are treated with silver nitrate solution, the calcium is reduced by the strong light and replaced with silver deposits, visualized as metallic silver. • Results: Calcium salts: black Nuclei: red Cytoplasm: pink

16. Urate Crystals -Gomori's Methenamine Silver • Purpose: To demonstrate urate crystals deposit in tissues. • Principle: The urates take-up the silver, the silver is then reduced to its metallic state. • Results: Urates: black Background: green • NOTE: Large calcium deposits may also stain black.

17. Melanin- Fontana-Masson Silver Method • Purpose: To identify argentaffin granules and melanin. Melanin is a non-lipid, non-hematogenous pigment. • It is a brown-black pigment present normally in the hair, skin, retina, iris and certain parts of the CNS. Argentaffin granules are found in carcinoid tumors.

18. Principle: A positive argentaffin reaction means the cells take-up silver and then reduce it to a visible metallic state, without the aid of a reducing agent. • Results: Melanin, argentaffin cells: black Nuclei: red

19. enzyme histochemistry • The techniques of enzyme histochemistry, which relate structure and function, can be used to locate many enzymes, including acid phosphatase, dehydrogenases, and peroxidases. • Because fixation and clearing typically inactivate enzymes, frozen sections are commonly used. • The sections are incubated in solutions containing substrates for the enzymes of interest and reagents that yield insoluble colored or electron-dense precipitates at the sites of enzyme activity.

20. Acid Phosphatase:Owing to their characteristic content of acid phosphatase in lysosomes. • Dehydrogenases: Dehydrogenases can be localized by incubating tissue sections with an appropriate substrate and tetrazole. • Peroxidases: Peroxidases are most often demonstrated by incubating tissue with 3,3' diaminobenzidine (DAB) and hydrogen peroxide.

21. Special Staining Dr. Tarek Atia

22. Carbohydrates • Complex carbohydrates, i.e, oligosaccharides and polysaccharides, can be localized by many histochemical techniques. • In addition, some carbohydrates are immunogenic owing to their large size or their presence as covalently linked components (proteoglycans, glycoproteins, glycolipids); these can be analyzed by immuno-histochemical methods.

23. 1-Periodic Acid Schiff's ( PAS ) • Purpose: Glycogen is present in liver, kidney, skeletal and cardiac muscle. • The PAS stain is used to demonstrate neutral polysaccharides that are present in basement membranes, and mucus substances secreted from the epithelia of various organs. • A routine stain for: liver and kidney biopsies.

24. Principle: The PAS stain is a histochemical reaction in that the periodic acid oxidizes the carbon to carbon bond forming aldehydes, which react to the fuchsin-sulfurous acid to form the magenta color. • Results: Glycogen: magenta (Red)

25. 2- Alcian Blue pH 2.5 for Acid Mucopolysaccharides PURPOSE: Alcian blue stains acid mucus substances ormucopolysaccharides. • Excessive amounts of non-sulfated acidic mucus substances are seen in mesotheliomas, certain amounts occur normally in the wall of blood vessels but increase in early lesions of atherosclerosis.

26. 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. • Alcian blue stains both sulfated and carboxylatedacid mucopolysaccharides and sulfated and carboxylated glycoproteins. • It is believed to form salt linkages with the acid groups of acid mucopolysaccharides. • RESULTS: Acid mucins/mucus substances: blue

27. 3- Alcian Blue-PAS (PAb) • Purpose: To differentiate between neutral and acidic mucus substances. • Routine stain for G.I. biopsies. • Results: Acid mucus substances: blue Neutral polysaccharides: Red

28. Acid mucus substances: blue Neutral polysaccharides: magenta red

29. 4- Mucicarmine Stain - Mucin PURPOSE: To stain acid mucopolysaccharides (mucin), which is a secretion produced by a variety of epithelial cells and connective tissue cells. • Excess mucin is secreted by epithelial cells in certain inflammations and in certain intestinal carcinomas. • The mucicarmine technique is also useful in determining the site of a primary tumor in that finding mucin positive tumor cells.

30. Principle: aluminum is believed to form a chelation complex with the carmine, changing the molecule to a positive charge allowing it to bind with the acid substrates of low density such as mucins. • Results: Mucin: deep rose Nuclei: black Other tissue elements: yellow Mucin: deep rose Nuclei: black Other tissue elements: yellow

31. Nucleic Acids • The nucleic acids, DNA and RNA, can be localized by specific and non specific methods. • DNA is found mainly in nuclei, and its amount is much the same in every cell. • RNA is found both in nuclei and in cytoplasm, and its amount varies widely, depending on a cell's functional state. • Feulgen's reaction: determine the amounts of DNA. • Methyl Green Pyronin Stain todetermine DNA and RNA • Neoplastic and the rapidly growing cells contain more RNA in the cytoplasm Basophilic Cytoplasm

32. 1- Methyl Green Pyronin Stain • PURPOSE: The nuclear chromatin in the nucleus is composed of nucleoproteins. • The two types of nucleic acids are deoxyribonucleic acid (DNA), found in the nucleus, and ribonucleic acid (RNA), found in the nucleolus and cytoplasm. • DNAfunctions in cell heredity, and synthesis of RNA. • RNAfunctions principally in protein synthesis.

33. Principle: • This stain can demonstrate RNA and DNA. • DNAstains with methyl green. • RNA is colored red with pyronin. • RESULTS: DNA: blue-green to green RNA: pink to red

34. 2- Feulgen stain • A selective cytochemical reaction for DNA in which sections or cells are first hydrolyzed with hydrochloric acid to produce apurinic acid and then are stained with Schiff reagent to produce Red stained nuclei. • Generally the concentration of DNA in nucleoli and mitochondria is too low to permit detection by this stain

35. Basic dyes (Hematoxylin). • Both DNA and RNA stained nonspecificallywith basic dyes. Because of the strong affinity of RNA for such dyes, its distribution in cells and tissues may be studied by subtraction. • In this procedure, one of 2 adjacent sections is treated with ribonuclease (RNase) to remove RNA; then both are stained with basic dyes (eg, hematoxylin, toluidine blue, methylene blue). • Basophilic structures present in the untreated section (eg. ribosomes) but absent in the RNase-treated section contain RNA.

36. Lipids • Several methods used to show normal lipid distribution and disease-related lipid accumulation (eg, fatty change in the liver). • Fat occurring in an abnormal place, such as fatty emboli, and tumors arising from fat cells (liposarcomas) can be differentiated from other types of tumors. • Lipids are usually dissolved by organic fixatives or clearing agents, leaving gaps in the tissue, but they are preserved in frozen sections.

37. Oil Red O • Purpose: To demonstrate fat or lipids in fresh frozen sections. • Principle: Staining with oil-soluble dyes is based on the greater solubility of the dye in the lipoid substances than in the usual hydro-alcoholic dye solvents. • Results: Fat: red Nuclei: blue

38. Sudan Black • Purpose: For the demonstration of fat using fresh frozen sections(cryostat sections). • Principle: Sudan Black is slightly basic dye and will combine with acidic groups in compound lipids, thus staining phospholipids also. • Results: Fat: blue-black Nuclei: red