Method Description

Hemoglobin Electrophoresis:

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 hemolyzing solution is injected by aspiration. A high-voltage protein separation occurs with direct detection of the hemoglobin protein fractions 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: HbA2', C, A2/O-Arab, E, S, D, G-Philadelphia, F, A, Hope, Bart, J, N-Baltimore and H.(Louahabi A, Philippe M, Lali S, Wallemacq P, Maisin D. Evaluation of a new Sebia kit for analysis of hemoglobin fractions and variants on the Capillarys system. Clin Chem Lab Med. 2006;44[3]:340-345; instruction manual: CAPILLARYS Hemoglobin(E) using the CAPILLARYS 2 flex-piercing instrument. Sebia; 06/2014)

High-Performance Liquid Chromatography Hemoglobin Variant:

Hemolysate of whole blood is injected into an analysis stream passing through cation exchange column using high-performance liquid chromatography. 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 absorbance are displayed as a chromatogram of absorbance versus time.(Huismann TH, Scroeder WA, Brodie AN, Mayson SM, Jakway J. 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; Szuberski J, Oliveira JL, Hoyer JD: A comprehensive analysis of hemoglobin variants by high-performance liquid chromatography (HPLC). Int J Lab Hematol. 2012;34(6):594-604; instruction manual: Bio-Rad Variant II Beta-thalassemia Short Program Instructions for Use, L70203705. Bio-Rad Laboratories, Inc; 11/2011)

Unstable Hemoglobin:

Two different hemoglobin stability tests are performed: isopropanol and heat stability.

Unstable hemoglobins will precipitate in dilute solutions of isopropanol. Washed erythrocytes are hemolyzed and cleared by centrifugation. Isopropanol is added. The hemolysate is incubated at 37° C for 20 minutes and examined for turbidity. There is no turbidity with normal hemoglobins.(Fairbanks VF, Klee GG: Biochemical aspects of hematology. In: Burtis CA, Ashwood ER, eds. Tietz Textbook of Clinical Chemistry. 3rd ed. WB Saunders Company; 1999:1685-1687; Greene DN, Vaughn CP, Crews BO, Agarwal AM. Advances in the detection of hemoglobinopathies. Clinica Chimica Acta. 2015;439:50-57)

Unstable hemoglobins can also be precipitated by heating to 50° C. Washed erythrocytes are hemolyzed and cleared by centrifugation. The hemolysate is incubated at 50° C for 90 minutes and examined for turbidity. There is no turbidity with normal hemoglobins.

Osmotic Fragility:

Specimens for erythrocyte osmotic fragility tests are anticoagulated with EDTA. Osmotic lysis is performed using sodium chloride solution, 0.5 g/dL. An incubated fragility test is performed following 24-hour incubation at 37° C at the following sodium chloride 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° 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; King MJ, Zanella A. Hereditary red cell membrane disorders and laboratory diagnostic testing. Int J Lab Hematol. 2013;35:237-243)

Band 3/Eosin-5-Maleimide Binding Assay:

Eosin-5-maleimide (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 red blood cells is assessed and compared to normal-value patients.(King MJ, Behrens J, Rogers C, Flynn C, Greenwood D, Chambers K. Rapid flow cytometric test for the diagnosis of membrane cytoskeletal associated hemolytic anemia. Br J Haematol. 2000;111:924-933; King MJ, Zanella A. Hereditary red cell membrane disorders and laboratory diagnostic testing. Int J Lab Hematol. 2013;35:237-243)

Glucose-6-phosphate dehydrogenase:

Glucose-6-phosphate dehydrogenase 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 (NADPH) and the reaction is measured spectrophotometrically on an automated chemistry analyzer.(Beutler E: Red Cell Metabolism: A Manual of Biochemical Methods. 3rd ed. Grune and Stratton: 1984:68-71; van Solinge WW, van Wijk: Enzymes of the red blood cell. In: Rifai N, Horvath AR, Wittwer CT: eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier; 2018:chap 30)

Pyruvate Kinase:

Pyruvate kinase catalyzes the phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) by converting phosphoenolpyruvate to pyruvate. The amount of pyruvate formed is quantitated by adding lactate dehydrogenase and reduced nicotinamide adenine dinucleotide (NADH) and measuring the rate of decrease in absorbance spectrophotometrically at 340 nm as the NADH is oxidized to NAD(+) on an automated chemistry analyzer.(Beutler E: Red Cell Metabolism. A Manual of Biochemical Methods. 3rd ed. Grune and Stratton; 1984:68-71; van Solinge WW, van Wijk: Enzymes of the red blood cell. In: Rifai N, Horvath AR, Wittwer CT: eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier; 2018:chap 30)

Glucose Phosphate Isomerase:

Glucose phosphate isomerase interconverts glucose-6-phosphate (G6P) and fructose-6-P (F6P). In this assay, the F6P is then further converted to 6-phosphogluconate (6-PG) through the G6PD reaction resulting in the reduction of NADP(+) to NADPH. The reduction of NADP(+) is measured spectrophotometrically by the increase in absorbance at 340 nm on an automated chemistry analyzer.(Beutler E: Red Cell Metabolism: A Manual of Biochemical Methods. 3rd ed. Grune and Stratton; 1984:40-42; van Solinge WW, van Wijk. Enzymes of the red blood cell. In: Rifai N, Horvath AR, Wittwer CT: eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier; 2018:chap 30)

Hexokinase:

Hexokinase catalyzes the reaction of ATP and glucose to G6P and ADP. In this assay the formation of G6P is measured by linking its further oxidation to 6-PG to the reduction of NADP(+) through the G6PD reaction. The increase in absorbance which occurs as NADP(+) is reduced to NADPH is measured spectrophotometrically at 340 nm on an automated chemistry analyzer.(Beutler E: Red Cell Metabolism: A Manual of Biochemical Methods. 3rd ed. Grune and Stratton; 1984:38-40; van Solinge WW, van Wijk: Enzymes of the red blood cell. In: Rifai N, Horvath AR, Wittwer CT: eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier; 2018:chap 30)

Adenylate Kinase:

Adenylate kinase (myokinase) catalyzes the dismutation of ADP into adenosine-5'-monophosphate (AMP) and ATP. In this assay, the reverse reaction is measured by following the formation of ADP with pyruvate kinase (PK) and lactate dehydrogenase reactions resulting in NADH being oxidized to NAD(+). The decrease in absorbance that occurs as NADH is oxidized is measured spectrophotometrically at 340 nm by an automated chemistry analyzer.(Beutler E: Red Cell Metabolism: A Manual of Biochemical Methods. 3rd ed. Grune and Stratton; 1984:93-95; van Solinge WW, van Wijk: Enzymes of the red blood cell. In: Rifai N, Horvath AR, Wittwer CT: eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier; 2018:chap 30)

Phosphofructokinase:

Phosphofructokinase catalyzes the phosphorylation of F6P by ATP to fructose-1,6-diphosphate (FDP). FDP is then converted to dihydroxyacetone phosphate (DHAP) through subsequent aldolase and triosephosphate isomerase (TPI) catalyzed reactions. The rate of formation of DHAP is measured by linking its reduction to alpha-glycerophosphate by alpha-glycerophosphate dehydrogenase, which results in the oxidation of NADH to NAD(+). The decrease in absorbance at 340 nm is measured spectrophotometrically as the NADH is oxidized on an automated chemistry analyzer.(Beutler E: Red Cell Metabolism: A Manual of Biochemical Methods. 3rd ed. Grune and Stratton; 1984:68-71; van Solinge WW, van Wijk: Enzymes of the red blood cell. In: Rifai N, Horvath AR, Wittwer CT: eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier; 2018:chap 30)

Phosphoglycerate Kinase:

Phosphoglycerate kinase catalyzes the phosphorylation of ADP to ATP by conversion of 1,3-diphosphoglycerate (1,3-DPG) to 3-phosphoglyceric acid (3-PGA). In this assay, the reaction is driven in the reverse direction. The formation of 1,3-DPG is then measured through the glyceraldehyde phosphate dehydrogenase reaction as 1,3-DPG is converted to glyceraldehyde-3-phosphate (GAP) resulting in the oxidation of NADH to NAD(+). The decrease in absorbance that occurs as NADH is oxidized is measured spectrophotometrically at 340 nm on an automated chemistry analyzer.(Beutler E: Red Cell Metabolism: A Manual of Biochemical Methods. 3rd ed. Grune and Stratton; 1984:53-55; van Solinge WW, van Wijk: Enzymes of the red blood cell. In: Rifai N, Horvath AR, Wittwer CT: eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier; 2018:chap 30)

Triosephosphate Isomerase:

TPI interconverts GAP and DHAP. The rate of DHAP formation is measured by further converting it to alpha-glycerophosphate by alpha-glycerophosphate dehydrogenase which results in the oxidation of NADH to NAD(+). The oxidation of NADH is measured spectrophotometrically by the decrease in absorbance at 340 nm on an automated chemistry analyzer.(Beutler E: Red Cell Metabolism: A Manual of Biochemical Methods. 3rd ed. Grune and Stratton;1984; van Solinge WW, van Wijk: Enzymes of the red blood cell. In: Rifai N, Horvath AR, Wittwer CT: eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier; 2018:chap 30)

Glutathione:

Virtually all of the nonprotein sulfhydryl of red cells is in the form of reduced glutathione. 5,5'-dithiobis (2-nitrobenzoic acid) is a disulfide compound that is readily reduced by sulfhydryl compounds, forming a highly colored yellow anion. The absorbance of this resultant yellow substance is measured by 412 nm and compared to that of a known standard.(Beutler E: Red Cell Metabolism: A Manual of Biochemical Methods;  3rd ed. Grune and Stratton: 1984; Alisik M, Neselioglu S, Erel O. A colorimetric method to measure oxidized, reduced and total glutathione levels in erythrocytes, J Lab Med. 2019:43(5), 269-277. doi:10.1515/labmed-2019-0098)

Pyrimidine 5' Nucleotidase:

Pyrimidine nucleotides have a spectral absorption curve that is markedly different from that exhibited by (normally present) adenine nucleotides, eg, adenosine triphosphate. The former have a peak at about 270 nm; the latter at about 257 nm. Thus, pyrimidine 5' nucleotidase deficiency may be ascertained by demonstrating a very high spectral absorption maximum of 270 nm in erythrocyte extracts.(Beutler E: Red Cell Metabolism: A Manual of Biochemical Methods. 3rd ed. Grune and Stratton; 1984:100-102; van Solinge WW, van Wijk: Enzymes of the red blood cell. In: Rifai N, Horvath AR, Wittwer CT: eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier; 2018:chap 30)

Morphology Review:

A hematopathologist who is an expert in these disorders evaluates the slides and an interpretive report is issued.