Abrogation of red blood cell G6PD enzyme activity through Heat treatment: development of survey material for the UK NEQAS G6PD scheme

Abstract

Introduction: Participation in external quality assessment (EQA) is central to the maintenance of high-quality laboratory results in patient diagnosis and clinical trials. Laboratories in the TAF112582 DETECTIVE study (ClinicalTrials.gov identifier: NCT01376167) are enrolled in the United Kingdom National Quality Assessment Scheme (UK NEQAS) for glucose-6-phosphate dehydrogenase (G6PD) quantitative assay, which utilizes ovine (sheep) blood as a readily available source of apparently G6PD-deficient survey material. A substitute for sheep blood was sought because some non-UK sites in the study encountered participation difficulties due to the strict regulations on the import of sheep blood into their countries.

Methods: G6PD activity in normal human donor blood was abrogated by the action of heat under controlled conditions. Residual G6PD activity in the heated samples was measured by UK NEQAS using the Trinity Biotech 345 kit (Trinity Biotech) and a Jenway 6715 UV/Vis spectrophotometer with external temperature control to monitor enzyme kinetics and linearity over a set time. Heat-treated material was also assayed for G6PD activity and assessed for its acceptability as EQA survey material by selected UK laboratories.

Results: Blood heated at 45 °C for 15 h showed a reduction in G6PD activity of 76.3 ± 4.6% (n = 6) and was considered acceptable as EQA material in terms of appearance and behaviour by the majority of UK sites in the trial.

Conclusions: We have developed a simple heat-treatment procedure to produce EQA survey material with low/intermediate G6PD activity, similar to that found in females heterozygous for G6PD deficiency.

Keywords: UK NEQAS; G6PD (Glucose-6-Phosphate Dehydrogenase); enzyme activity; heat treatment; ovine.