A novel point-of-care device for measuring glucose-6-phosphate dehydrogenase enzyme deficiency

Abstract

Extreme hyperbilirubinemia can cause bilirubin neurotoxicity. Infants with glucose-6-phosphate dehydrogenase (G6PD) deficiency can develop hemolysis and thus are at high risk. We evaluated a device that quantitatively measures G6PD activity kinetically using digital microfluidics (DMF). Intra- and inter-instrument and -day imprecision (CVs) were first assessed. G6PD activity in 86 samples was then measured and compared between DMF and 2 reference methods. Overall DMF reproducibility was 3.8% over 5 days by 2 operators on 2 instruments. Mean intra- and inter-instrument variabilities were 3.6% and 3.9%, respectively (n = 28), with a user variability of 4.3%. Mean G6PD activity was 6.40±4.62 and 6.37±4.62 U/g hemoglobin for DMF and Reference Methods 1 (n = 46) and 12.15±3.86 and 11.48±1.55 for DMF and 2 (n = 40), respectively, and strongly correlated (r = 0.95 and 0.95) with mean biases of +0.04±2.90 and +0.67±1.55 for methods 1 and 2, respectively. The novel device could be used for early newborn G6PD screening.

Fig. 1.. Pentose phosphate pathway.

The enzyme glucose-6-phosphate dehydrogenase (G6PD) is involved in the pentose phosphate shunt that generates NADPH in mature red blood cells, which protects the cell membrane from oxidative damage; and if deficient, hemolysis can occur.

Fig, 2.. Digital microfluidics (DMF) method.

(A) The prototype point-of-care device is designed on a DMF platform; (B) DMF is based on electric fields to manipulate droplets of blood or “electrowetting”; and (C) Disposable “chip” cartridge is tightly regulated at 37°C and contains all the required reagents in dried form, which are then reconstituted once sample analysis is initiated. The assay requires ~50 μL of whole blood.

Fig. 3.. Device performance comparison with Reference Methods: linear correlation.

(A) When measurements using the digital microfluidics (DMF) method were compared to those of Reference Method 1, a correlation (r) of 0.95, with a slope of 0.95 and y-intercept of 0.34 was found. The majority of glucose-6-phosphate dehydrogenase (G6PD)-deficient samples (88.2%) were from males (15/17). (B) When measurements using the DMF were compared to those of Reference Method 2, a correlation (r) of 0.95, with a slope of 1.08 and y-intercept of −0.32 was found. The majority of samples had normal G6PD enzyme activity.

Fig. 4.. Device performance comparison with Reference Methods: bias and imprecision.

Bland-Altman plot showed that the bias and imprecision of the digital microfluidics (DMF) method to: (A) Reference Method 1, which showed only +0.04 with an imprecision of 2.90 U/g hemoglobin (Hb) of n = 46 subjects; (B). Reference Method 2, which showed that the bias of the DMF method was +0.67 U/g Hb and standard deviation of the difference was 1.55 U/g Hb of n = 40 subjects.