Molecular Tests Evaluated for G6PD Deficiency
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzyme defect, for which many different laboratory tests are available.
Fluorescent spot test method for glucose-6-phosphate dehydrogenase deficiency
(Photo courtesy of Dr. Tundun Williams)
Most individuals with G6PD deficiency are asymptomatic, but a deficiency in this enzyme can result in acute haemolytic anaemia after exposure to oxidative agents, such as the consumption of fava beans or antimalarial drugs.
An international team of scientists led by those at Mahidol University (Bangkok, Thailand) investigated four G6PD activity detections methods including fluorescence spot (FS), methemoglobin reduction (MR), biochemical and cytochemical test. These methods accompanied with mutation analysis of blood samples were taken from 295 apparently healthy individuals aged 18 to 50 years. There were 67 males and 228 females all with unknown G6PD deficiency status.
The MR test was performed with minor modifications, and the FS was performed on the heparinized blood using a commercial kit (R&D Diagnostics; Cholargos, Greece). For the quantitative evaluation of G6PD activity, the G6PD kit from Biolabo SA (Maizy, France) was used. A cytochemical assay of cellular G6PD activity was performed with a minor modification and cells were analyzed for formazan presence by light microscopy.
DNA was extracted from G6PD-deficient blood samples by using Qiaquick Blood DNA extraction kit (Qiagen; Hilden, Germany). DNA sequencing was carried out initially in one direction in all exons, using either the forward or the reverse primer. Fluorescence-based cycle labeling of polymerase chain reaction (PCR) products was performed using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems; Foster City, CA, USA).
At least one G6PD mutation was identified in 42 of the 295 (14.2%) individuals and G6PD Viangchan was the most common and was detected in 36/42 (83.3%). The prevalence of G6PD deficiency was 6.1% by FS test and 7.1% by MR test. G6PD activity measured as international units per gram hemoglobin (IU/gHb) and was 11 ± 2.5 IU/gHb in nondeficient females, and 10.9 ± 0.6 IU/gHb in nondeficient males. The upper and lower limit cut-off points for partial deficiency in adults was 5.7 IU/gHb, 60% of the normal mean, and for severe deficiency it was 0.95 IU/gHb , 10% of the normal mean.
The authors concluded that using a cut-off number of negative cell of greater than 20% in the cytochemical assay to define G6PD deficiency, the prevalence of G6PD deficiency was closest to the molecular analysis of 12.9% G6PD-deficient compared to the others methods. Therefore, the cytochemical method is a significant predictor of this disease, while FS and MR test are recommended for the detection of severe G6PD deficiency in developing countries. G6PD deficiency is present in more than 400 million people worldwide.
The study was published on August 21, 2013, in the Malaria Journal.