A simple G6PD deficiency diagnostic test goes a long way...
Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme that is rate limiting in the pentose phosphate pathway and the metabolism of glutathione (figure1). The enzyme is present in all cells, including red blood cells (RBCs) and shields them from oxidative damage triggered by certain medications, diseases, and foods [1,2]. G6PD deficiency is an X-linked recessive hereditary disorder, and it exposes RBCs to become more vulnerable to hemolysis under certain conditions. It affects an estimated 400 million people worldwide, making it the most common genetic abnormality [2, 3]. It is usually asymptomatic, but clinical manifestations of G6PD deficiency include neonatal jaundice, acute haemolytic anaemia (AHA) triggered by certain drugs, infections, or fava beans and chronic non-spherocytic haemolytic disease (CNSHD) . It is commonly found in people from malaria-endemic countries, notably in people from the Mediterranean Basin, the Middle East, South-East Asia, and West Africa. The G6PD gene is highly polymorphic, as more than 400 allelic variants of the gene exist and in heterozygous females, there is variable phenotypic expression . Many malaria-endemic countries such as Singapore and Malaysia have implemented neonatal screening for G6PD deficiency [6, 7].
In newborns with severe jaundice, G6PD deficiency is a notable cause of hyperbilirubinemia and kernicterus, a significant type of brain damage . Accordingly, it is necessary to have diagnostic tests that measure the G6PD status in newborns in hospitals to guide clinicians and parents. In Malaysia, where 3.1% of the males have G6PD deficiency, every hospital performs the semi quantitative fluorescent spot test (FST) [9, 10]. The FST can distinguish severe deficiencies and can accurately differentiate hemizygous-deficient males and homozygous- or heterozygous-deficient females . Typically, the activity of G6PD in this population is less 20-30% of the mean normal activity. This is acceptable for males with a deficient (<30% of the normal activity) or a normal (>80% normal) G6PD activity. But the test is ineffectual for classifying the G6PD activity in females, who can be G6PD intermediate (between 30 and 80% activity) in addition to deficient and normal. Thus, there is a need for quantitative tests, as they have the capacity to impact health on many levels, including decreasing neonatal mortality and morbidity.
The G6PD deficiency is diagnosed by measuring G6PD activity, adjusted for temperature, in blood normalised for either haemoglobin or RBC count. Therefore, it is often necessary to independently count the number of RBCs in the sample where the assay is performed. The influence of temperature on the enzyme’s activity is such that only reference or specialized laboratories used to estimate G6PD activity using a complex assay on a temperature-controlled instrument. Ideally, a G6PD quantitative test should be simple to perform, fast, sensitive, and usable with simple equipment and its performance should be comparable to a reference assay selected by the WHO or other organizations. The G6PD quantitative assay kit developed by R&D Diagnostics has been evaluated against various reference assays, initially the G6PDH quantitative test by Trinity Biotech, which production was subsequently suspended, then by Pointe Scientific reference assay [12, 13].
The R&D Diagnostics G6PD quantitative assays measure G6PD activity and haemoglobin (Hb) concentration simultaneously [http://www.rddiagnostics.com/g6pd_disorder.htm]. This allows direct expression of results in U/g Hb. In fact, this assay correlates the enzymatic activity to the amount of Hb in the sample, namely it allows Hb normalization. Hb normalization eliminates the need for a separate Hb determination, thus it is a simpler and more effective test. Normalization of Hb automatically corrects sample volume errors since the results are expressed as a ratio of two parameters measured from the same sample. This also ensures an accuracy difficult to achieve when measuring activity and Hb (or RBCs) in two separate operations. In a study performed in Malaysia neonates, Azma et al. demonstrated that the test could provide rapid and reproducible results in a screening of cord blood for G6PD deficiency. Moreover, the one-step implementation was simple, just needing a spectrophotometer or a microplate reader . The test is also more sensitive than the semiquantitative FST as it could identify partial deficiency observed in most females, previously missed by the FST.
A study by LaRue et al. compared this assay with a reference quantitative assay and confirmed that the R&D assay is sensitive enough to measure severe G6PD deficiencies. However, the authors argued that different assays provide different mean activity, a fact observed in another comparative study . Thus, it is necessary to be consistent in using a reference assay in each setting. Nevertheless, the R&D test is ideal for labs performing 20-2000 tests per day. It is very easily automated but works equally well manually, either on a dried blood spot or whole blood. It is also particularly suitable for screening of G6PD deficiency in newborns, and in adults requiring anti-malaria treatments and in the prevention to adverse drug reactions [13,14].
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