Cont’n of Phosphatases Non-Enzymatic Techniques Leukocyte Alkaline Phosphatase PAS-Schiff reagent Sudan Black B Toluidine Blue Reagents: Fixative: formalin 40mL Methanol 360mL Store in freezer. Tris buffer 0.2M pH9.1 Trizma base 48.44mg Distilled H2O 200mL Adjust pH to 9.1 w/ 1N HCl Substrate soL’n: Dissolve 0.6g of naphthol-AS- BI phosphate in 10mL N,N- dimethylformamide. Add 0.2M Tris buffer to 2L. Store in refrigerator. -colorless soL’n capable ofreacting w/ aldehyde groups in glycogen, mucoproteins and other high molecular weight carbohydrates. for the demonstration of certain phospholipids and lipoproteins Mechanism of action is uncertain in that it may be: 1. Selec tive adso rption 2. A chemical reaction 3. Or a combinati on SBB reaction is similar to the myeloperoxidase reaction. Advantage of SBB over the myeloperoxidase stain: 1. Its stability to heat and storage 2. more sensitive to primitiv e myeloid cells 3. the reagent are not car cinoge nic Disadvantages: 1. Requi re 1-2 hours st ainin g time 2. its speci fici ty 3. increased background staining on bone marrow specimen secondary to the fatty nature ofbone marrow itselfa dye that can bind w/ acid mucopolysaccharides in blood cells to form metachromatic complexes Ex. granules of both basophils&mast cells are stromgly metachromatic-stain reddish violet Usefulness: Recognition of mast cell diseases and acute or chronic basophilic leukemias. Fixative: formalin-10mL Absolute methanol- 90mL Keep capped&refrigerated. 1% Periodic-kept refrigerated, should be made fresh every 7 days Schiff’s rgt: commercially prepared. Keep refrigerated. Colorless, if it turns pink—it must be discarded Harris hematoxylin Ammonia water 3-5 drops conc. NH4OH in 50mL distilled H2O IncubationMixture: Working soL’n: 50mg of fast blue BBn dissolve in 50mL ofsubstrate, prepared fresh&filter before use. Procedure: Cold fixatives (4°-10°C)-30 secs Working soL’n-20 minutes Nuclear fast red- counterstain-20mins Controls: Slides taken from pregnant women in their last trimester Interpretation: Count 50 segmented neutrophils. Score staining reaction from 0- 4 + 0=no granules 1 + =very few granules 2 + =moderate # of granules scattered throughout the cell 3 + =numerous granules starting to coalesce 4 + =cytoplasm packed w/ granules Glycogen is predominant in leukocytes. + color-bright fuschia-pink Immuno Cytochemical Techniques Immunochemistry is defined as the identification of the immunologic phenotype of a given cell population throught the use of specific monoclonal or polyclonal antibodies against selected cell antigens. Specimens used: Cell Suspension Paraffin or cryostat section Smears Imprints Cytospin prep A slide-based technique is advantageous because: 1. it allows for counting of designated cell populations 2. permanent slide can be filed for future review =both practical and cost effective =no expensive equipment is required = specimen may be as much as 1 week old Application of this technique in hema lab has been primarily for the identification of the cell types involved in acute or chronic leukemias. Enzyme Immunocytochemistry (Immunoperoxidase) Peroxidase-antiperoxidase (PAP) Plant enzyme used—horseradish peroxidase Immunoalkaline phosphatase PAP technique uses 3 rgts: 1. primary antibody that is specific for the antigen in question 2. secondary AB that will bind to the Fc p ortion of the 1 AB&carries the PAP complex as a tag 3. substrate used to make the perox idase reaction visible Advantages: 1. easil y obtai nabl e&stab le 2. endogenous peroxidase is easily blocked 3. variety of chromogens are available to react w/ peroxidas e to form a colored end product inexpensive Advantages: lack of cross reactivity w/ the pseudoperoxidase oferythrocytes/granulocytes Method-depend on the kit commercially available Control-buffy coat preparation made from peripheral blood from healthy individual Ferric Iron-Prussian Blue Reaction Presence of iron granules in otherwise mature, nonnucleated red cells (siderocytes) may be seen in iron overload or poor iron utilization syndromes Reagents: Fixative:absolute methyl alcohol Interpretation: Bone marrow iron storage is usuallyreported as absent, decreased, increased or normal Other principal use of the iron stain in hematology is the evaluation of red cell iron utilization. Troubleshooting: Presence of excessive amounts ofextracellular material that stains + for iron can be caused by contaminated reagents. [-] staining in a + control may be secondary to improper pH of the Kferrocyanide.
