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Carbs & Amyloid
Staining
| Question | Answer |
|---|---|
| 3 classifications of carbs | Monosaccharides, oligosaccharides, polysaccharides |
| What is the only monosaccharide found in the body? | Glycogen |
| Monosaccharides and oligosaccharides are_ | Soluble in aq. solution, difficult to demo |
| 4 groups of polysaccharides | Group I: neutral polysaccharides Group II: acid mucopolysaccharides Group III: glycoproteins Group IV: glycolipids |
| Group I includes_ | glycogen, starch, cellulose, chitin, PAS-positive |
| Group II includes_ | Hyaluronic acid, heparin, chondroitin sulfates A, B, & C, human aorta, bovine cornea, "connective tissue mucins," PAS-negative |
| Group III includes_ | ovimucoid, mucin, Paneth cell granules, sialomucins, sialogycoproteins, serum glycoproteins, "epithelial mucins," maybe PAS-positive |
| Group IV includes_ | Cerebrosides: fatty subs. w/ carb, phosphatides: non-carb lipids, PAS positive |
| PAS purpose | Demo polysaccharides, neutral mucosubstances, basement membranes |
| PAS facts | 10% NBF or Bouin; blood smear - methanol, kidney 1-2 μm, other 4-5μm, QC: kidney; demo glycogen - liver or cervix |
| PAS solutions | Periodic acid + Schiff Reagent + potassium metabisulfite + Harris w/ acetic acid or light green |
| PAS results | Positive reaction: bright rose - nuclei: blue if hematoxylin - background: light green if counterstained |
| Schiff reagent solution | sodium metabisulfite + HCL = sulfurous acid + basic fuchsin + activated charcoal + dH2O reacts w/ aldehydes to restore color structure |
| What substances give positive PAS reaction? | Glycogen, neutral mucosubstances, some epithelial sulfomucins & sialomucins, colloid material of thyroid, pars intermedia of pituitary, basement membranes, fungal walls |
| PAS w/ diastase digestion purpose | Demo glycogen |
| PAS w/ diastase digestion facts | 10% NBF, formalin alcohol, or absolute alcohol QC: 2 sections of liver or cervix w/ glycogen - label one "w/," one "w/o" |
| PAS w/ diastase digestion solutions | malt diastase solution + periodic acid + Schiff reagent + potassium metabisulfite + Harris w/ acetic acid |
| PAS w/ diastase digestion results | Glycogen on "w/o" section: bright rose red - glycogen on "w/" section: no stain |
| Malt Diastase solution | diastase of malt + phosphate buffer, pH 6 |
| Phosphate buffer, pH6 | NaCl + sodium phosphate + H2O |
| Mayer mucicarmine purpose | Demo "epithelial" mucin |
| Mayer mucicarmine facts | 10% NBF QC: small intestine, appendix, or colon |
| Mayer mucicarmine solutions | Mucicarmine solution + Weigert hematoxylin + Metanil yellow or light green + H2O |
| Mayer mucicarmine results | epithelial mucin, capsule of cryptococcus: deep rose to red - nuclei: black - other: blue or yellow |
| Mayer mucicarmine stock solution | carmine + alum lake + aluminum hydroxide + anhy. aluminum chloride + 50% ethanol + 25% ethanol |
| Metanil yellow solution | metanil yellow + acetic acid + H2O |
| Alcian blue, pH 2.5 purpose | demo acid mucopolysaccharides |
| Alcian blue, pH 2.5 facts | 10% NBF or Bouin QC: small intestine, appendix, or colon positive control |
| Alcian blue, pH 2.5 solutions | acetic acid + alcian blue + nuclear-fast red |
| Alcian blue, pH 2.5 results | acid mucopolysaccharides: dark blue - background/other: pink to red |
| Nuclear-fast red (Kernechtrot) solution | nuclear-fast red + aluminum sulfate + H2O |
| Alcian blue, pH 1.0 purpose | demo sulfated mucosubstances |
| Alcian blue, pH 1.0 facts | 10% NBF or Bouin QC: small intestine, appendix, or colon positive control |
| Alcian blue, pH 1.0 solutions | HCl + alcian blue, pH 1 + Nuclear-fast red |
| Alcian blue, pH 1.0 results | sulfated mucosubstances: pale blue - background: pink to red |
| Alcian blue w/ hyaluronidase purpose | Differentiate epithelial & connective tissue mucins, hyaluronidase digests out connective mucins |
| Alcian blue w/ hyaluronidase facts | 10% NBF QC: 2 sections of umbilical cord - one labeled "w," one "w/o," may use small intestine, appendix, or colon as 2nd control for epithelial mucins |
| Alcian blue w/ hyaluronidase solutions | hyaluronidase digestion soln. + acetic acid soln. + alcian blue + nuclear-fast red |
| Alcian blue w/ hyaluronidase results | w/o, acid mucopolysaccharides, sialomucins: deep blue - w/, mucosubstances w/ hyaluronic acid, chondroitin sulfates A & C: marked loss of staining |
| Buffer solution, pH 6.0 | potassium phosphate + sodium phosphate |
| Hyaluronidase digestion solution | testicular hyaluronidase + buffer soln. |
| Alcian blue-PAS-hematoxylin purpose | Differentiate between neutral & acidic mucosubstances |
| Alcian blue-PAS-hematoxylin facts | 10% NBF, Zenker, kidney 2-3 μm, other 4-5μm QC: kidney or mucin control depending on diagnostic tissue |
| Alcian blue-PAS-hematoxylin solutions | acetic acid soln. + alcian blue, pH 2.5 + periodic acid soln. + Schiff reagent + reducing rinse + Harris w/ acetic acid |
| Alcian blue-PAS-hematoxylin results | acid mucosubstances: blue - neutral polysaccharides: magenta - substances stained by PAS & alcian blue: purple |
| Reducing rinse solution | sodium metabisulfite + H2O prepare just before use |
| Müller-Mowry colloidal iron purpose | Demo carboxylated & sulfated, (acid) mucopolysaccharides & glycoproteins, less specific than alcian blue, Prussian blue reaction |
| Müller-Mowry colloidal iron facts | 10% NBF, Carnoy, alcoholic formalin; avoid chromate fixatives QC: small intestine, appendix, or colon |
| Müller-Mowry colloidal iron solutions | acetic acid soln. + Müller-Mowry colloidal iron + ferrocyanide-HCl + Nuclear-fast red |
| Müller-Mowry colloidal iron results | acid mucopolysaccharides & sialomucins: deep blue - nuclei: pink-red - cytoplasm: pink |
| Müller-Mowry colloidal iron working solution | ferric chloride + acetic acid + H2O |
| Ferrocyanide-hydrochloric acid solution | potassium ferrocyanide + HCl + H2O |
| What is amyloid? | Fibrillar protein, deposits in tissue under pathologic conditions, contains some acid mucopolysaccharides |
| What is amyloidosis? | Disease where deposits replace cellular elements causing progressive loss of function & eventual death |
| Primary amyloid | Occurs in absence of disease, primarily muscle, heart, skin, tongue |
| Secondary amyloid | Associated w/ disease, esp. inflammatory disease, e.g. RA & TB, primarily kidneys, liver, spleen, adrenal glands |
| Myeloma-associated amyloid | Associated w/ diseases of immunological system, primarily muscle, heart, skin, tongue |
| Tumor-associated amyloid | Associated w/ tumors, esp. those of amine precursor uptake & decarboxylation system |
| Alkaline Congo-red method facts | Demo amyloid, alcohol or Carnoy 8-10 μm (or no birefringence) QC: sections w/ amyloid (brain, kidney) stain intensity decr. over time |
| Alkaline Congo-red method solutions | Harris w/ acetic acid + alkaline salt soln. + Congo red working soln. |
| Alkaline Congo-red method results | amyloid: deep pink to red - elastic tissue: pale pink - nuclei: blue |
| Alkaline salt solution | 80% alcohol sat'd w/ NaCl + NaOH |
| Congo red working solution | Congo red + 80% alcohol sat'd w/ NaCl + NaOH |
| Stock 80% alcohol sat'd w/ NaCl | NaCl + 100% ethanol + H2O |
| Crystal violet purpose | Rapid screening of amyloid, not as specific as Congo red |
| Crystal violet facts | 10% NBF or alcohol 10-12 μm QC: section w/ amyloid polychromatic |
| Crystal violet solutions | 95% sat'd w/ CV + Apathy mounting medium, seal w/ nail polish |
| Crystal violet results | amyloid: purplish violet - other elements: blue |
| Modified Apathy mounting medium solution | gum arabic + cane sugar + NaCl + thymol + H2O |
| Thioflavin T Fluorescent method facts | Demo amyloid, less specific than Congo red 10% NBF 6-10 μm QC: section w/ amyloid |
| Thioflavin T Fluorescent method solutions | Mayer + thioflavine T + acetic acid soln. |
| Thioflavin T Fluorescent method results | amyloid: fluoresces yellow to yellow-green |
| How to test Schiff reagent quality? | Schiff reagent should be clear/light amber; to test quality add formaldehyde, reddish-purple - good, deep blue-purple - solution breaking down |
| Best carmine facts | Demo glycogen, less specific, alcohol, Bouin, Carnoy |
| Best carmine reagents | carmine + K2CO3 + KCl NH4OH + methanol + ethanol + hematoxylin |
| Best carmine results | glycogen: pink - nuclei: blue |
| Alcian blue is a: | Copper phthalocyanin dye |
| Prussian blue reaction: | Acidic potassium ferrocyanide, ferric iron reacts to form insoluble bright blue pigment |
| Congo red birefringence under polarized light should be: | Bright green, 8-10 µm, too thin - red, too thick - yellow |
| Turnball blue reaction: | Acidic potassium ferrocyanide, ferrous iron reacts to form insoluble bright blue pigment - ferrous ferricyanide |
| The colored product of the PAS reaction is formed by: | Restoration of the quinoid structure |
Created by:
CCF
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