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Carbs & Amyloid

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 Fixative: 10% NBF or Bouin; blood smear - methanol, Sections: paraffin kidney 1-2 μm, other 4-5μm, QC: kidney; demo glycogen - liver or cervix
PAS solutions Periodic acid >> ox. tissue to aldehydes Schiff Reagent potassium metabisulfite >> remove excess Schiff Harris hematoxylin w/ acetic acid or light green
PAS results Positive reaction - bright rose nuclei - blue if hematoxylin counterstain background - light green if counterstained
Schiff reagent solution sodium metabisulfite + HCL = sulfurous acid >> red. fuchsin 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 Fixative: 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 hematoxyin 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 monobasic water
Mayer mucicarmine purpose Demo "epithelial" mucin
Mayer mucicarmine facts Fixative: 10% NBF QC: small intestine, appendix, or colon
Mayer mucicarmine solutions Mucicarmine solution Weigert hematoxylin Metanil yellow or light green water
Mayer mucicarmine results epithelial mucin, capsule of cryptococcus - deep rose to red nuclei - black other elements - blue or yellow
Mayer mucicarmine stock solution carmine, alum lake aluminum hydroxide & anhy. aluminum chloride >> form chelation product 50% ethanol 25% ethanol
Metanil yellow solution metanil yellow acetic acid water
Alcian blue, pH 2.5 purpose demo acid mucopolysaccharides
Alcian blue, pH 2.5 facts Fixative: 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 (Kernechtrot) aluminum sulfate water
Alcian blue, pH 1.0 purpose demo sulfated mucosubstances
Alcian blue, pH 1.0 facts Fixative: 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 Fixative: 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 solution acetic acid solution 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, monobasic sodium phosphate, dibasic
Hyaluronidase digestion solution testicular hyaluronidase buffer solution
Alcian blue-PAS-hematoxylin purpose Differentiate between neutral & acidic mucosubstances
Alcian blue-PAS-hematoxylin facts Fixative: 10% NBF, Zenker Sections: paraffin kidney 2-3 μm, other 4-5μm QC: kidney or mucin control depending on diagnostic tissue to be stained
Alcian blue-PAS-hematoxylin solutions acetic acid solution alcian blue, pH 2.5 periodic acid solution Schiff reagent reducing rinse Harris hematoxylin 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 water prepare just before use
Müller-Mowry colloidal iron purpose Demo carboxylated & sulfated mucopolysaccharides & glycoproteins, less specific than alcian blue, Prussian blue reaction
Müller-Mowry colloidal iron facts Fixative: 10% NBF, Carnoy, alcoholic formalin; avoid chromate fixatives QC: small intestine, appendix, or colon
Müller-Mowry colloidal iron solutions acetic acid solution Müller-Mowry colloidal iron ferrocyanide-hydrochloric acid 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 water
Ferrocyanide-hydrochloric acid solution potassium ferrocyanide HCl water
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
What are the 4 groups of amyloid? Primary amyloid, secondary amyloid, myeloma-associated amyloid, tumor-associated amyloid
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 purpose Demo amyloid
Alkaline Congo-red method facts Fixative: alcohol or Carnoy Sections: paraffin 8-10 μm (or no birefringence) QC: sections w/ amyloid (brain, kidney) stain intensity decr. over time
Alkaline Congo-red method solutions Harris hematoxylin w/ acetic acid alkaline salt solution Congo red working solution
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 water
Crystal violet purpose Rapid screening of amyloid, not as specific as Congo red
Crystal violet facts Fixative: 10% NBF or alcohol Sections: paraffin 10-12 μm QC: section w/ amyloid polychromatic
Crystal violet solutions crystal violet 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 water
Crystal violet working solution 95% sat'd w/ crystal violet HCl water
Thioflavin T Fluorescent method purpose Demo amyloid, not as specific as Congo red
Thioflavin T Fluorescent method facts Fixative: 10% NBF Sections: paraffin 6-10 μm QC: section w/ amyloid
Thioflavin T Fluorescent method solutions Mayer hematoxylin thioflavine T acetic acid solution
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 purpose Demo glycogen, less specific
Best carmine facts Fixative: alcohol, Bouin, Carnoy
Best carmine reagents carmine >> dye potassium carbonate & potassium chloride >> buffers NH4OH 28% >> incr. pH methanol >> solvent ethanol & methanol >> differentiators hematoxylin >> nuclear stain
Best carmine results glycogen - pink nuclei - blue
Alcian blue is a: Copper phthalocyanin dye
Prussian blue reaction: Sections are treated w/ acidic potassium ferrocyanide & any ferric iron present reacts to form insoluble bright blue pigment - Prussian blue
Congo red birefringence under polarized light should be: Bright green, if birefringence is pale yellow sections may be too thin
Turnball blue reaction: Sections are treated w/ acidic potassium ferrocyanide & any ferrous iron present reacts to form insoluble bright blue pigment - Turnball blue (ferrous ferricyanide)
Created by: CCF