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Hemolytic Anemia Evaluation:
A hematopathologist who is an expert in these disorders evaluates the case, appropriate tests are performed and an interpretive report is issued.
Hemolysate of whole blood is injected into an analysis stream passing through a cartridge containing diethylaminoethyl-resin using HPLC. A preprogrammed gradient controls the elution buffer mixture that also passes through the analytical cartridge. The ionic strength of the elution buffer is raised by increasing the percentage of a second buffer. As the ionic strength of the buffer increases the more strongly retained hemoglobins elute from the cartridge. Absorbance changes are detected by a dual-wavelength filter photometer. Changes in absorbances are displayed as a chromatogram of absorbances versus time.(Huismann TH, Scroeder WA, Brodie AN, et al: Microchromotography of hemoglobins. III. A simplified procedure for the determination of hemoglobin A2. J Lab Clin Med 1975;86:700-702; Ou CN, Buffone GJ, Reimer GL, Alpert AJ: High-performance liquid chromatography of human hemoglobins on a new cation exchanger. J Chromatogr 1983;266:197-205)
The CAPILLARYS System is an automated system that uses capillary electrophoresis to separate charged molecules by their electrophoretic mobility in an alkaline buffer. Separation occurs according to the electrolyte pH and electro-osmotic flow. A sample dilution with hemolysing solution is injected by aspiration. A high voltage protein separation occurs and direct detection of the hemoglobin protein fractions is at 415 nm, which is specific to hemoglobins. The resulting electrophoregram peaks are evaluated for pattern abnormalities and are quantified as a percentage of the total hemoglobin present. Examples of position of commonly found hemoglobin fractions are, from cathode to anode: Hb A2', C, A2/O-Arab, E, S, D, G-Philadelphia, F, A, Hope, Bart, J, N-Baltimore and H.(Louahabi A, Philippe M, et al: Evaluation of a new Sebia kit for analysis of hemoglobin fractions and variants on the Capillarys system. Clin Chem Lab Med 2006;44:340-345)
Stability-unstable hemoglobins precipitate in dilute solutions of isopropanol. Washed erythrocytes are hemolyzed and cleared by centrifugation. Isopropanol is added. The hemolysate is incubated at 37 degrees C for 20 minutes and examined for turbidity. There is no turbidity with normal hemoglobins.(Fairbanks VF, Klee GG: Biochemical aspects of hematology. In Tietz Textbook of Clinical Chemistry. Third edition. Edited by CA Burtis, ER Ashwood. Philadelphia, WB Saunders Company, 1999, pp 1685-1687)
Specimens for erythrocyte osmotic fragility tests are anticoagulated with EDTA. Osmotic lysis is performed using sodium chloride (NaCl) solution, 0.50 g/dL. An incubated fragility test is performed following 24-hour incubation at 37 degrees C at the following NaCl concentrations: 0.60, 0.65, and 0.75 g/dL. Results are reported and interpreted.(Larson CJ, Scheidt R, Fairbanks VF: The osmotic fragility test for hereditary spherocytosis: use of EDTA-anticoagulated blood stored at 4 degrees C for up to 96 hours. Am Soc Clin Pathol Meeting Abstract, 1988; Larson CJ, Scheidt R, Fairbanks VF: The osmotic fragility test for hereditary spherocytosis: objective criteria for test interpretation. Am Soc Clin Pathol Meeting Abstract, 1988)
Glucose-6-Phosphate Dehydrogenase (G6PD):
G6PD in a hemolysate catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconate. Concomitantly, nicotinamide adenine dinucleotide phosphate (NADP) is changed to its reduced form (nicotinamide adenine dinucleotide phosphate-oxidase [NADPH])), a reaction measured spectrophotometrically.(Beutler E: Red Cell Metabolism: A Manual of Biochemical Methods. Third edition. New York, Grune and Stratton, 1984, pp 68-71)
Eosin-5-malemide (EMA) is a fluorescent dye that binds to Lys-430 of the extracellular loop of the band 3 protein. Using a 1-color flow cytometry method (number of events plotted against fluorescence), the fluorescent intensity of EMA-stained RBC, is assessed and compared to normal-value patients.(King MJ, Behrens J, Rogers C, et al: Rapid flow cytometric test for the diagnosis of membrane cytoskeletal associated hemolytic anemia. Br J Haematol 2000;111:924-933)
A red cell hemolysate is incubated with adenosine diphosphate and phosphoenolpyruvate. The amount of pyruvate formed is quantitated by adding lactic dehydrogenase and reduced nicotinamide adenine di-nucleotide and measuring the rate of decrease in absorbance at 340 nm.(Beutler E: Red Cell Metabolism: A Manual of Biochemical Methods. Third edition. New York, Grune and Stratton, 1984, pp 68-71)
Glucose Phosphate Isomerase:
Washed erythrocytes are hemolyzed and the hemolysate is mixed with glucose, adenosine triphosphate (ATP), glucose-6-phosphate dehydrogenase, and nicotinamide adenine dinucleotide phosphate (NADP). The reduction of NADP is measured spectrophotometrically and is proportional to the enzymatic conversion of ATP and glucose to glucose-6-phosphate.(Beutler E: Red Cell Metabolism: A Manual of Biochemical Methods. Third edition. New York, Grune and Stratton, 1984, pp 40-42)
Hexokinase (in the presence of magnesium) catalyzes the reaction of ATP and glucose to G-6-P and ADP. In this assay the formation of glucose-6-phosphate (G-6-P) is measured by linking its further oxidation to 6-phosphogluconate (6-PG) to the reduction of NADP through the glucose-6-phosphate dehydrogenase (G-6-PD) reaction. The increase in absorbance which occurs as NADP+ is reduced is measured at 340 nm.(Beutler E: Red cell metabolism: A Manual of Biochemical Methods. Third edition. Grune and Stratton, New York, 1984, pp 38-40)
A hematopathologist who is an expert in these disorders evaluates the slides and an interpretive report is issued.
Monday through Friday; Varies