Modified Fluorescent Spot Test Improves Detection of Enzyme Deficiency

The glucose-6-phosphate dehydrogenase (G6PD) fluorescent spot test (FST) is a useful screening test for G6PD deficiency, but is unable to detect heterozygote G6PD-deficient females.

A modified semiquantitative in-house method has been applied to cord blood samples involving a three-scale reading when reporting results of FST: normal, intermediate or deficient and then compared to a commercial quantitative enzyme assay.

Geneticists at the University Malaya (Kuala Lumpur, Malaysia) analyzed 1,266 cord blood samples, of which 87 samples were found to be intermediate or deficient by FST, with 49 deficient and 38 intermediate. Of the 49 deficient samples, 48 had G6PD enzyme activity of ≤ 9.5 U/g Hb and one sample had normal enzyme activity. Samples identified as showing absent or intermediate fluorescence on FST were analyzed for the presence of G6PD mutations using genotyping assays and direct nucleotide sequencing.

All the 38 intermediate samples were from female and of these, 21 had G6PD activity of between 20% and 60%, and 17 samples showed normal G6PD activity. Twenty-seven of the 38 samples were available for mutation analysis of which 13 had normal G6PD activity. Eleven of the 13 samples with normal G6PD activity had identifiable G6PD mutations.

Molecular identification using rapid real-time polymerase chain reaction (PCR) methods supplemented by conventional sequencing is feasible for confirmation of G6PD variants. Ninety-four percent (30/32) of affected males were found to have G6PD mutations using a limited set of primers designed to detect common polymorphisms in the population. The commercial enzyme assay used for comparison was manufactured by R&D Diagnostics (Papagou, Greece) and amplification of DNA and real-time detection was performed on the Rotorgene 3000 (Corbett Research; Mortlake, NSW, Australia).

The authors concluded that the FST is effective in identifying deficient hemizygote males and is also able to identify mildly deficient heterozygote females, provided diminished fluorescence is distinguished as a separate category. The FST will continue to have an important role to play in screening for G6PD deficiency in view of its relatively good diagnostic efficiency coupled with its ease of use and low cost. Glucose-6-phosphate dehydrogenase deficiency is the most common human enzyme defect, being present in more than 400 million people worldwide. This X-linked inherited disorder most commonly affects persons of African, Asian, Mediterranean, or Middle Eastern descent. The study was published in the October 2011 edition of the International Journal of Laboratory Hematology.

Related Links:
University Malaya
R&D Diagnostics
Corbett Research