2.1 G6PD phenotype assigned based on activity
G6PD activity testing was performed as an in-house clinical laboratory test with a same day turnaround for a subset of patients who underwentG6PD genotyping. G6PD enzyme activity was measured in erythrocytes using a quantitative spectrophotometric assay,12,13 and tests were ordered as part of clinical screening, generally for those in whom rasburicase might be needed (e.g. newly diagnosed leukemia patients) or for those with anemia. Whole blood samples were collected in EDTA-containing tubes. Because a high WBC count may result in an artefactually elevated G6PD activity result, samples with a WBC count greater than 100 x 103cells/mm3 had the buffy coat removed prior to assaying to minimize the potential for interference from G6PD content contributed by WBCs. Samples were prepared with Lyse reagent and assayed immediately on a CobasĀ® 6000 c501 analyzer.
A result below the lower limit of normal (<6.3 units/g Hgb for the St. Jude analytical assay) was in the deficient range and a problem list entry of G6PD deficiency was placed into the electronic health record (EHR); other results were considered normal. Because the G6PD activity test can be affected by hematologic parameters, G6PD activity results were assessed for interferences prior to phenotype assignment as shown in Figure 1. Provisional deficient activity was assigned when a G6PD activity result was in the deficient range but in the setting of a critically low hemoglobin (<7 g/dL at St. Jude). Provisional normal activity was assigned when a G6PD activity result was in the normal range but in the setting of an RBC transfusion within the past 60 days,14 elevated reticulocyte count (>0.085 x 106 cells/mm3at St. Jude),8 or WBC count greater than 100 x 103 cells/mm3 and the buffy coat-free procedure was not used. Follow-up testing was recommended when a provisional phenotype was assigned (Fig. 1). Follow-up testing included G6PD genotype or a repeat activity test when known interferences were no longer present.