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Test Code MISC2MAYOEEEV1 Red Blood Cell (RBC) Enzyme Evaluation, Blood


Necessary Information


Metabolic Hematology Patient Information (T810) is strongly recommended and should include clinical history. Testing may proceed without this information, however if the information requested is received, it allows for a more complete interpretation.



Specimen Required


Container/Tube: Yellow top (ACD solution B)

Specimen Volume: 12 mL

Collection Instructions: Send specimen in original tube. Do not aliquot.


Forms

1. Metabolic Hematology Patient Information (T810) is available in Special Instructions.

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

Secondary ID

607493

Useful For

Identifying defects of red cell enzyme metabolism

 

Evaluating patients with Coombs-negative hemolytic anemia

Profile Information

Test ID Reporting Name Available Separately Always Performed
EEEVI Erythrocyte Enzyme Interpretation No Yes
G6PDC G6PD Enzyme Activity, B Yes, (Order G6PD1) Yes
PKC PK Enzyme Activity, B Yes, (Order PK1) Yes
GPIC Glucose Phosphate Isomerase, B Yes, (Order GPI1) Yes
HKC Hexokinase, B Yes, (Order HK1) Yes
AKC Adenylate Kinase, B Yes, (Order AK1) Yes
PFKC Phosphofructokinase, B Yes, (Order PFK1) Yes
PGKC Phosphoglycerate Kinase, B Yes, (Order PGK1) Yes
TPIC Triosephosphate Isomerase, B Yes, (Order TPI1) Yes
GSH Glutathione, B Yes Yes
P5NT Pyrimidine 5' Nucleotidase, B Yes Yes

Testing Algorithm

This is a consultative evaluation in which the case will be evaluated at Mayo Clinic Laboratories.

Method Name

EEEVI: Medical Interpretation

G6PDC, GPIC, PKC, HKC, AKC, PFKC, PGKC, TPIC, GSH, P5NT: Kinetic Spectrophotometry

Reporting Name

RBC Enzyme Evaluation

Specimen Type

Whole Blood ACD-B

Specimen Minimum Volume

5 mL

Specimen Stability Information

Specimen Type Temperature Time Special Container
Whole Blood ACD-B Refrigerated 11 days

Reject Due To

Gross hemolysis Reject

Clinical Information

Erythrocyte enzyme deficiencies are inherited causes of hemolytic anemia. Some are very common, such as glucose-6-phosphate dehydrogenase (G6PD) deficiency, and others are very rare, found in only a few families around the world. Most are autosomal, but some are sex-linked and located on the X chromosome. Most enzyme deficiencies result in chronic nonspherocytic hemolytic anemia of variable severity; however, some, such as G6PD, can be hematologically normal with episodic acute hemolysis due to a trigger event such as medications, toxins, or some foods. The RBC enzymopathies do not typically show recurrent pathognomonic changes on the peripheral blood smear other than generic features of hemolytic anemia, although some such as pyruvate kinase deficiency can have echinocytes and pyrimidine 5' nucleotidase (P5NT) deficiency is associated with basophilic stippling. RBC enzyme activity levels are best evaluated as a panel as reticulocytosis can mask some deficient states and comparison to the background enzyme activity is useful.

 

This is a consultative evaluation of red cell enzyme activity as a potential cause of early red cell destruction.

Reference Values

Definitive results and an interpretive report will be provided.

Interpretation

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

Method Description

Glucose-6-Phosphate Dehydrogenase:

Glucose-6-phosphate dehydrogenase (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 (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 (PEP) 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 (GPI) interconverts glucose-6-P (G6P) and fructose-6-P (F6P). In this assay, the F6P is then further converted to 6-phosphogluconate (6-PG) through the G6P dehydrogenase (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 66-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)

 

Adenlyate 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 (LDH) 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 (PFK) 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:pp 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 (PGK) 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 (GAPD) 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, pp 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: 

Triosephosphate isomerase (TPI) interconverts glyceraldehyde-3-phosphate (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 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. Grune and Stratton1984; 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 (GSH). 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, ATP. 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)

Performing Laboratory

Mayo Clinic Laboratories in Rochester

Test Classification

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

CPT Code Information

82955-G6PD Enzyme Activity

84087-Glucose phosphate isomerase

84220-Pyruvate Kinase Enzyme Activity

82657-Hexokinase

82657-Adenylate Kinase

82657-Phosphofructokinase

82657-Phosphoglycerate Kinase

82657-Triosephosphate Isomerase

82978-Glutathione

83915-Pyrimidine 5’ Nucleotidase

LOINC Code Information

Test ID Test Order Name Order LOINC Value
EEEV1 RBC Enzyme Evaluation 72695-0

 

Result ID Test Result Name Result LOINC Value
PKCL PK Enzyme Activity, B 32552-2
GPICL Glucose Phosphate Isomerase, B 44050-3
G6PCL G6PD Enzyme Activity, B 32546-4
608409 Glutathione, B 2383-8
TPICL Triosephosphate Isomerase, B 44054-5
PGKCL Phosphoglycerate Kinase, B 44053-7
PFKCL Phosphofructokinase, B 72664-6
HKCL Hexokinase, B 49216-5
2734 Pyrimidine 5' Nucleotidase, B 2902-5
AKCL Adenylate Kinase, B 44051-1
608087 Erythrocyte Enzyme Interpretation 59466-3
608109 Reviewed By 18771-6

Day(s) Performed

Monday through Friday

Report Available

2 to 13 days