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We have developed an one step assay (OSMMR2000) for the determination of Glucose-6-Phosphate Dehydrogenase (G-6-PD) activity in neonates, which includes our “Hemoglobin Normalization” invention. This invention allows the direct expression of results in U/g Hb while it normalizes the blood content of all samples against the control. The assay is performed in microtiter plates using dried blood spots of any size or a quantity of whole blood or red blood cells (usually 5 microliters). It should be noted that dried blood spots and whole blood samples can be tested in the same microplate with the same control. After the elution a portion of the eluate is transferred to a clean microplate well and the reagent mixture (containing a substrate, coenzyme and buffer) is added. G-6-PD present in the specimen converts NADP to NADPH which is detected by a kinetic reading at 340 nm. G-6-PD activity in U/g Hb is calculated from the rate of a control. After termination of the kinetic readings the microplate is read at a suitable wavelength where haemoglobin (Hb) is measured. We strongly recommend 405 nm as the best wavelength (highest peak for Hb) although alternative wavelengths (e.g. 570 nm) can be used. Correlation between the Sigma assay and OSMMR2000 results was very good. Results from 14.192 neonates screened in Porto Allegre, Brazil and 12.500 neonates screened in Athens, Greece showed remarkably similar results (range : 0.4 – 18 U/g Hb; mean value: 11.5 U/g Hb). A cut-off point of 2,5 U/g Hb was selected for total deficiency whereas samples with an activity between 2,5 and 6,5 U/g Hb were classified as being partially deficient. In contrast 5.115 neonates screened in a pilot study in the New York State revealed higher activity (mean value 20,5+5,0) while the cut-off points was set to 4,1 and 12,2 U/g Hb for the totally deficient and partially deficient populations respectively. The prevalence of this disorder was 1.5, 1.6 and 1.8% for total deficiency (USA, Brazil and Greece respectively) and 4.2, 5.0 and 5.7 % for the two forms combined (Brazil, Greece and USA respectively). The new kit proved to be quick, easy to perform and reliable with a good reproducibility. The entire procedure (Hb normalization and elution time included) takes less than 40 minutes and uses standard laboratory equipment.

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