Table 1 Summary phenotypic tests

Direct tests

Spectrophotometry

Quantitative gross enzymatic activity assay. The initial reaction velocity of the G6PD reaction is measured spectrophotometrically as an increase in absorbance at 340 nm. Despite standardized methods variations in results have been observed between sites.

Requires a biochemistry laboratory and may vary with ambient temperature and humidity.

[31–34]

”Beutler’s” fluorescent spot test

A popular screening test in which a drop of blood incubated with G6PD reaction substrates is placed on filter paper and illuminated with UV light, the presence or absence of fluorescence provides a categorical measure of G6PD activity.

Recommended as the most suitable method for screening in the field, despite the need for an UV lamp, water bath incubator, and micropipette [34].

[35]

Indirect tests

Methaemoglobin reduction test (MRT)

G6PD activity is assessed by first treating RBCs with nitrite and then examining the rate of NADPH-dependent methaemoglobin reduction in the presence of an appropriate redox catalyst and substrate (glucose).

Requires a biochemistry laboratory.

[36, 37]

Brilliant cresyl blue, resazurin, or formazan ring tests

Indirect assays of G6PD activity translate NADPH production into a colorimetric readout using chromophores. It takes several hours to process these assays.

Requires a biochemistry laboratory.

[35, 38–41]

Cytochemical typing

Methaemoglobin elution test

RBCs are labelled according to their relative methaemoglobin content based on MRT.

Requires a biochemistry laboratory.

[42]

Cytofluorometric assay

Quenching of glutaraldehyde-induced autofluorescence by formazan is detected by cytofluometry. Cytofluorometric assays can provide valuable data based on individual cells, which neither genotyping nor biochemical assays can provide but require considerable resources.

Requires a laboratory with experience in cytofluometry.

[43, 44]

Rapid tests

Hirono – 1-methoxy PMS Sephadex method

Substrate mixture (G6P, NADP, saponine) and MTT-PMS mixture are dissolved in water and mixed with Sephadex gel.

Requires a biochemistry laboratory and requires a skilled technician.

[45]

WST8/1-methoxy PMS method

An enzymatic method which utilizes a tetrazolium salt WST8, and a PMS hydrogen carrier, 1-methoxy PMS.

The quantitative, colorimetric nature, the reduced light sensitivity and the possibility of using this method with dried bloodspots make it more suitable for field use than the Hirono method.

[38, 46]

Rapid, point-of-care tests

BinaxNow^® G6PD test

A qualitative enzyme chromatographic test distinguishes accurately between samples with G6PD activity less than 4.0 U/g of haemoglobin and those with greater enzyme activity. Approved by the United States Federal Drug Administration and rather costly. Requires an operating temperature 18° to 25°C, a cold chain for reagents and pipettes. Sensitivity 98%, Specificity 98%.

Specifically developed as a point of care test.

[47]

CareStart^® G6PD deficiency screening test

A qualitative enzyme chromatographic test, distinguishes between samples with G6PD activity less than 2.7 U/g of haemoglobin and those with greater enzyme activity. In development, not yet commercially available. Sensitivity 68%, Specificity 100%.

Is being developed as a point of care test and may become useful as a public health tool.

[46, 48]