Optimizing Measurement and Interpretation of the G6PD/Hb Ratio

Abstract

Abstract Background Glucose-6-phosphate dehydrogenase (G6PD)/hemoglobin (Hb) ratio helps detect G6PD deficiency, an X-linked disorder that can be asymptomatic or cause acute hemolytic anemia and chronic hemolysis. We investigated preanalytical, analytical, and postanalytical aspects to optimize G6PD/Hb measurement and interpretation. Methods G6PD was measured with the Pointe Scientific assay and Hb with Drabkin’s reagent on Alinity c® (Abbott Diagnostics). Stability of G6PD/Hb was assessed after 7 and 14 days while stored at 2–8 °C. Stability of hemolysate prepared for G6PD analysis was assessed using QC and patient samples up to 4 h at room temperature or 2–8 °C. Analytical performance specifications including precision, method comparison, linearity, LOQ, and carry-over were established for the enzymatic reaction of G6PD and spectrophotometric reading of Hb. G6PD/Hb reference interval and cut-offs were established indirectly using truncated maximum likelihood method (TML) using retrospective data (n = 4715 patient data points). Results Samples were stable after 7 days at 2–8°C, unless grossly hemolyzed. Hemolysate prepared for G6PD measurement remained stable for up to 4 h for QC at room temperature and 2–8°C, but up to 30 min–1 h at room temperature and 1–2 h at 2–8 °C for patient samples. Precision, linearity, LOQ, and carryover were acceptable. G6PD/Hb cut-offs were <3.3, ≥3.3, 3.3–8.9, and ≥8.9 U/g Hb for deficient males/females, normal males, intermediate females, and normal females, respectively. Conclusions In vitro hemolysis and delayed hemolysate analysis significantly reduce G6PD/Hb stability. QC material cannot detect the impact of delayed hemolysate analysis. These findings were foundational for optimizing G6PD/Hb protocols for a new platform and establishing laboratory-specific G6PD/Hb cut-offs.