Test Id : HAEV1

This is a consultative evaluation in which the case will be evaluated, the appropriate tests performed at an additional charge, and the results interpreted. If a peripheral blood smear is provided, the morphologic features will be incorporated into the interpretation.

Red blood cell enzymes will always be performed. Capillary electrophoresis, cation exchange high-performance liquid chromatography, and hemoglobin stability studies will always be performed. Reflex testing required to positively identify a hemoglobin abnormality may be added as the case requires. Osmotic fragility (OF) and eosin-5-maleimide binding band 3 flow cytometry will be performed on all cases. A normal shipping control for OF is necessary to exclude false-positive results due to preanalytic artifact.

The protein and molecular test results will be reported separately, which may result in incomplete data until all testing has been finalized.

One or more of the following molecular tests may be reflexed on this test:

-ATHAL / Alpha-Globin Gene Analysis, Varies

-WASQR / Alpha Globin Gene Sequencing, Blood

-WBSQR / Beta-Globin Gene Sequencing, Blood

-WBDDR / Beta-Globin Cluster Locus Deletion/Duplication, Blood

-WGSQR / Gamma-Globin Full Gene Sequencing, Varies

An additional comprehensive consultative interpretation that summarizes all results will be provided after all tests are completed to incorporate results into an overall evaluation.

For more information see Hereditary Hemolytic Anemia Evaluation Testing Algorithm

Preliminary screening tests, such as complete blood cell count with peripheral smear and direct Coombs test with a negative result, should be run before ordering this evaluation.

Cold agglutinin disorders and autoimmune disorders should be excluded prior to testing. This evaluation is not suitable for acquired causes of hemolysis.

Specimens must arrive within 72 hours of collection.

At minimum, include recent transfusion information and most recent complete blood cell count results.

Metabolic Hematology Patient Information (T810) is strongly recommended. Testing may proceed without this information, however if the information requested is received, any pertinent reported clinical features and data will drive the focus of the evaluation and be considered in the interpretation.

The laboratory has extensive experience in hemoglobin variant identification and many cases can be confidently classified without molecular testing. However, molecular confirmation is always available, subject to sufficient sample quantity (eg, multiplex ligation-dependent probe amplification testing requires at least 2 mL of specimen in addition to protein testing requirements). If no molecular testing or specific molecular tests are desired, utilize the appropriate check boxes on the form. If the form or other communication is not received, the reviewing hematopathologist will select appropriate tests to sufficiently explain the protein findings, which may or may not include molecular testing.

The following specimens are required for testing:

2 Whole blood EDTA specimens

2 Whole blood ACD specimens

1 EDTA control specimen

2 Well-made peripheral blood smears (Wright stained or fixed in absolute methanol)

Patient:

Specimen Type: Whole blood

Container/Tube: Lavender top (EDTA) and yellow top (ACD)

Specimen Volume:

EDTA: Two 4-mL vials

ACD: Two 6-mL vials

Collection Instructions:

1. Immediately refrigerate specimens after collection.

2. Send whole blood specimens in original tubes. Do not aliquot.

3. Rubber band patient specimen and control vial together.

Specimen Type: Slides

Container/Tube: Blood smears

Specimen Volume: 2 Peripheral blood smears

1. Prepare 2 peripheral blood smears from 1 of the EDTA tubes collected from the patient

2. Either stain the smear with Wright stain or fix the smear with absolute methanol prior to shipping.

Normal Shipping Control:

Specimen Type: Whole blood

Container/Tube: Lavender top (EDTA)

Specimen Volume: 4 mL

Collection Instructions:

1. Collect a control specimen from a normal (healthy), unrelated, nonsmoking person at the same time as the patient.

2. Clearly hand write normal control on the outermost label.

3. Immediately refrigerate specimen after collection.

4. Send specimen in original tube. Do not aliquot.

5. Rubber band patient specimen and control vial together.

• Informed Consent for Genetic Testing
• Metabolic Hematology Patient Information
• Benign Hematology Evaluation Comparison
• Informed Consent for Genetic Testing (Spanish)
• Hereditary Hemolytic Anemia Evaluation Testing Algorithm

1. New York Clients-Informed consent is required. Document on the request form or electronic order that a copy is on file. The following documents are available:

-Informed Consent for Genetic Testing (T576)

-Informed Consent for Genetic Testing-Spanish (T826)

2. Metabolic Hematology Patient Information (T810)

3. If not ordering electronically, complete, print, and send a Benign Hematology Test Request (T755) with the specimen.

EDTA Blood: 3 mL

ACD Blood: 5 mL

Evaluation of lifelong or inherited hemolytic anemias, including red blood cell membrane disorders, unstable or abnormal hemoglobin variants, and red blood cell enzyme disorders

This evaluation is not suitable for acquired causes of hemolysis.

This is a consultative evaluation in which the case will be evaluated, the appropriate tests performed at an additional charge, and the results interpreted. If a peripheral blood smear is provided, the morphologic features will be incorporated into the interpretation.

Red blood cell enzymes will always be performed. Capillary electrophoresis, cation exchange high-performance liquid chromatography, and hemoglobin stability studies will always be performed. Reflex testing required to positively identify a hemoglobin abnormality may be added as the case requires. Osmotic fragility (OF) and eosin-5-maleimide binding band 3 flow cytometry will be performed on all cases. A normal shipping control for OF is necessary to exclude false-positive results due to preanalytic artifact.

The protein and molecular test results will be reported separately, which may result in incomplete data until all testing has been finalized.

One or more of the following molecular tests may be reflexed on this test:

-ATHAL / Alpha-Globin Gene Analysis, Varies

-WASQR / Alpha Globin Gene Sequencing, Blood

-WBSQR / Beta-Globin Gene Sequencing, Blood

-WBDDR / Beta-Globin Cluster Locus Deletion/Duplication, Blood

-WGSQR / Gamma-Globin Full Gene Sequencing, Varies

An additional comprehensive consultative interpretation that summarizes all results will be provided after all tests are completed to incorporate results into an overall evaluation.

For more information see Hereditary Hemolytic Anemia Evaluation Testing Algorithm

Hemolytic anemia (HA) is characterized by increased red blood cell (RBC) destruction and a decreased RBC life span. Patients usually have decreased hemoglobin concentration, hematocrit, and RBC count, but some can have compensated disorders, and symptoms such as reticulocytosis, pigmented gallstones, and decreased haptoglobin are factors that raise clinical suspicion. Blood smear abnormalities may include variable amounts of poikilocytosis including spherocytes, elliptocytes, schistocytes, stomatocytes, echinocytes, polychromasia, basophilic stippling, and target cells. Osmotic fragility can be increased due to the presence of spherocytes. These are all nonspecific features that can be present in both hereditary and acquired hemolytic disorders.

Inherited hemolytic disorders may include RBC membrane disorders, RBC enzyme defects, or abnormalities in the hemoglobin molecule in the RBC. This panel assesses for possible causes of congenital/hereditary causes of HA and does not evaluate for acquired causes. Therefore, the anemia should be lifelong or familial in nature. Examples of acquired HA include autoimmune HA (Coombs-positive HA, Coombs-negative autoimmune HA), cold agglutinin disease, paroxysmal nocturnal hemoglobinuria, paroxysmal cold hemoglobinuria, mechanical hemolysis (aortic stenosis or prosthetic heart valves), disseminated intravascular coagulation/thrombotic microangiopathy, and drug-induced HA.

This consultation evaluates for a hereditary cause of increased RBC destruction and includes testing for RBC membrane disorders, such as hereditary spherocytosis and hereditary pyropoikilocytosis, hemoglobinopathies, and red blood cell enzyme abnormalities.

This panel is of limited use in patients with a history of recent transfusion and should be ordered as remote a date from transfusion as possible in those patients who are chronically transfused.

Hemoglobin Variant, A2 and F Quantitation

HEMOGLOBIN A

0-30 days: 5.9-77.2%

1-2 months: 7.9-92.4%

3-5 months: 54.7-97.1%

6-8 months: 80.0-98.0%

9-12 months: 86.2-98.0%

13-17 months: 88.8-98.0%

18-23 months: 90.4-98.0%

> or =24 months: 95.8-98.0%

HEMOGLOBIN A2

0-30 days: 0.0-2.1%

1-2 months: 0.0-2.6%

3-5 months: 1.3-3.1%

> or =6 months: 2.0-3.3%

HEMOGLOBIN F

0-30 days: 22.8-92.0%

1-2 months: 7.6-89.8%

3-5 months: 1.6-42.2%

6-8 months: 0.0-16.7%

9-12 months: 0.0-10.5%

13-17 months: 0.0-7.9%

18-23 months: 0.0-6.3%

> or =24 months: 0.0-0.9%

VARIANT 1

0.0

VARIANT 2

0.0

VARIANT 3

0.0

Hemoglobin Stability

Normal (reported as normal [stable] or abnormal [unstable])

OSMOTIC FRAGILITY

> or =12 months:

0.50 g/dL NaCl (unincubated): 3-53% hemolysis

0.60 g/dL NaCl (incubated): 14-74% hemolysis

0.65 g/dL NaCl (incubated): 4-40% hemolysis

0.75 g/dL NaCl (incubated): 1-11% hemolysis

NaCl = sodium chloride

Reference values have not been established for patients who are younger than 12 months of age.

BAND 3 FLUORESCENCE STAINING RED BLOOD CELLS(RBC)

> or =12 months: Normal (reported as normal, decreased, or equivocal)

Reference values have not been established for patients who are younger than 12 months of age.

Glucose 6 Phosphate Dehydrogenase Enzyme Activity

> or =12 months of age: 8.0-11.9 U/g Hb

Reference values have not been established for patients who are younger than 12 months of age.

Pyruvate Kinase Enzyme Activity

> or =12 months of age: 5.5-12.4 U/g Hb

Reference values have not been established for patients who are younger than 12 months of age.

Glucose Phosphate Isomerase Enzyme Activity

> or =12 months of age: 40.0-58.0 U/g Hb

Reference values have not been established for patients who are younger than 12 months of age.

Hexokinase Enzyme Activity

> or =12 months: 0.7-1.7 U/g Hb

Reference values have not been established for patients who are younger than 12 months of age.

Adenylate Kinase Enzyme Activity

> or =12 months: 195-276 U/g Hb

Reference values have not been established for patients who are younger than 12 months of age.

Phosphofructokinase Enzyme Activity

> or =12 months of age: 5.8-10.9 U/g Hb

Reference values have not been established for patients who are younger than 12 months of age.

Phosphoglycerate Kinase Enzyme Activity

> or =12 months: 142-232 U/g Hb

Reference values have not been established for patients who are younger than 12 months of age.

Triosephosphate Isomerase Enzyme Activity

> or =12 months of age: 1033-1363 U/g Hb

Reference values have not been established for patients who are younger than 12 months of age.

Glutathione

> or =12 months: 46.9-90.1 mg/dL RBC

Reference values have not been established for patients who are younger than 12 months of age.

Pyrimidine 5' Nucleotidase

Normal

A hematopathologist expert in these disorders evaluates the case, appropriate tests are performed, and an interpretive report is issued.

Recent transfusion may cause unreliable results.

A normal shipping control for osmotic fragility (OF) is necessary to exclude false-positive results due to preanalytical artifact. OF and eosin-5-maleimide binding testing will be canceled if no shipping control is received or if the shipping control is abnormal.

This panel is most effectively interpreted in the context of clinical information and the peripheral blood morphology. Fill out the Metabolic Hematology Patient Information (T810) to maximize the interpretive capabilities of the panel.

This group of tests should not ordinarily be requested in patients who are likely to have immune hemolytic anemia (HA), such as that due to either warm or cold antibodies or to paroxysmal nocturnal hemoglobinurias. Coombs tests, tests for cold agglutinins, sucrose hemolysis, and Hams and Crosby tests are not part of the HA evaluation. In general, the foregoing tests should have been performed and found to be negative prior to requesting an HA evaluation. Since Wilson disease is another rare cause for acute intermittent hemolysis, testing for Wilson disease also may be appropriate prior to requesting an HA evaluation.

1. Steiner LA, Gallagher PG. Erythrocyte disorders in the perinatal period. Semin Perinatol. 2007;31(4):254-261

2. Beutler E: Glucose-6-phosphate dehydrogenase deficiency and other enzyme abnormalities. In: Beutler E, Lichtman MA, Coller BS, Kipps TJ, eds. Hematology. 5th ed. McGraw-Hill Book Company; 1995:564-581

3. Hoyer JD, Hoffman DR: The thalassemia and hemoglobinopathy syndromes. In: McClatchey KD, Amin HM, Curry JL, eds. Clinical Laboratory Medicine. 2nd ed. Lippincott, Williams and Wilkins; 2002:866-895

4. King MJ, Garcon L, Hoyer JD, et al. International Council for Standardization in Haematology. ICSH guidelines for the laboratory diagnosis of nonimmune hereditary red cell membrane disorders. Int J Lab Hematol. 2015;37(3):304-325

5. Lux SE: Anatomy of the red cell membrane skeleton: unanswered questions. Blood. 2016;127(2):187-199. doi:10.1182/blood-2014-12-512772

6. Gallagher PG. Abnormalities of the erythrocyte membrane. Pediatr Clin North Am. 2013;60(6):1349-1362

7. Bianchi P, Fermo E, Vercellati C, et al. Diagnostic power of laboratory tests for hereditary spherocytosis: a comparison study in 150 patients grouped according to molecular and clinical characteristics. Haematologica. 201297(4):516-523

8. Cappellini MD, Fiorelli G. Glucose-6-phosphate dehydrogenase deficiency. Lancet. 2008;371:64-74

9. Glader B: Hereditary hemolytic anemias due to red blood cell enzyme disorders. In: Greer JP, Arber DA, Glader B, et al, eds. Wintrobe's Clinical Hematology. 13th ed. Wolters Kluwer/Lippincott, Williams and Wilkins; 2014:728

10. Gallagher PG. Diagnosis and management of rare congenital nonimmune hemolytic disease. Hematology Am Soc Hematol Educ Program. 2015;2015:392-399

11. Koralkova P, van Solinge WW, van Wijk R. Rare hereditary red blood cell enzymopathies associated with hemolytic anemia - pathophysiology, clinical aspects, and laboratory diagnosis. Int J Lab Hematol. 2014;36(3):388-397

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 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: 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 degrees C. Washed erythrocytes are hemolyzed and cleared by centrifugation. The hemolysate is incubated at 50 degrees 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 degrees 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 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; 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.

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This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. It has not been cleared or approved by the US Food and Drug Administration.

83020-26-Hemolytic Anemia Interpretation

82657-Hexokinase, B

82955-G6PD Enzyme Activity, B

83020-Hemoglobin electrophoresis

83021-High-Performance Liquid Chromatography (HPLC)

83068-Hemoglobin Stability

84087-Glucose phosphate isomerase, B

84220-Pyruvate Kinase Enzyme Activity, B

82657-Adenylate Kinase, B

82657-Phosphofructokinase, B

82657-Phosphoglycerate Kinase, B

82657-Trisephosphate Isomerase, B

85060-26 -Morphology review

85557-Osmotic fragility

88184-Band 3 Fluorescence Staining, RBC

83915-Pyrimidine 5' Nucleotidase

82978-Glutathione, B

83789 (if appropriate)

82664 (if appropriate)

88184 (if appropriate)