Workflow for the use of the lyophilized control with a glucose-6-phosphate dehydrogenase (G6PD) rapid diagnostic test.

<p>Workflow for the use of the lyophilized control with a glucose-6-phosphate dehydrogenase (G6PD) rapid diagnostic test.</p

Titration of lyophilized human recombinant glucose-6-phosphate dehydrogenase (r-G6PD).

<p>Lyophilized human r-G6PD (blue line) was titrated and tested in the Trinity Biotech quantitative assay alongside the Trinity controls (grey line). From these data, three lyophilized concentrations were chosen to represent normal, intermediate, and deficient enzyme activity.</p

Stability of the reconstituted lyophilized recombinant glucose-6-phosphate dehydrogenase (r-G6PD).

<p>Lyophilized controls were reconstituted under four conditions to determine stability after rehydration. Three concentrations were assessed: normal (blue), high intermediate (purple), and intermediate (green). The controls were rehydrated in protein storage buffer (PSB) or deionized (DI) water and kept on ice or at room temperature after rehydration: PSB on ice (filled squares), PSB at room temperature (filled circles), DI water on ice (filled triangles), and DI water at room temperature (filled diamonds). Activity was measured on the Trinity Biotech quantitative assay at time zero, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and overnight 24 hours later. Thresholds are shown to indicate whether each measurement is still within an acceptable range of G6PD enzyme activity based on the ranges of Trinity controls: lower acceptable normal (blue dotted line), upper intermediate (black dotted line), lower intermediate (green dotted line), and upper deficient (red dotted line).</p

Target product profile for quality control reagents to support point-of-care diagnostic tests for glucose-6-phosphate dehydrogenase (G6PD) deficiency.

<p>Minimal and optimal requirements are described for different product specifications. The unitized recombinant G6PD tubes are expected to meet most specifications with the exception of the shelf-life of the reconstituted reagents (not achieved).</p

Stability of lyophilized human recombinant glucose-6-phosphate dehydrogenase (r-G6PD) reagent panel: Normal (blue), intermediate (green), and deficient (red).

<p>Lyophilized panel of three concentrations of human r-G6PD were stored for 12 months at five different temperatures: -80°C filled squares, 4°C filled circles, 30°C filled diamonds, 45°C filled triangles, and 55°C crosses. Activity was measured using the Trinity Biotech quantitative assay. The graph shows the stability of the panel over time. Stability starts to decline after approximately one month at a high temperature of 55°C. Thresholds are shown to indicate whether each measurement is still within an acceptable range of G6PD enzyme activity based on the ranges of Trinity controls: lower acceptable normal (blue dotted line), upper intermediate (black dotted line), lower intermediate (green dotted line), and upper deficient (red dotted line).</p

Performance of human recombinant glucose-6-phosphate dehydrogenase (r-G6PD) controls on qualitative tests for G6PD.

<p>Two lots of three concentrations (normal, intermediate, and deficient) of human r-G6PD were tested on two qualitative assays for G6PD: the fluorescent spot test (panels A and B) and a novel prototype rapid diagnostic test for G6PD (panels C and D). In panels A and B, the top row represents the Trinity normal controls for reference; the second, third, and fourth rows show the signals for the normal, intermediate, and deficient human r-G6PD controls, respectively. In panels C and D, the line intensity of test output correlates with expected enzyme activity, referring to normal, intermediate, and deficient activity. The signal for one lot of freshly lyophilized human r-G6PD (A and C) is compared to a second lot that had been stored at 4°C for more than one year (B and D).</p

Comparison of freshly lyophilized recombinant glucose-6-phosphate dehydrogenase (r-G6PD) versus year-old lyophilized r-G6PD and control.

<p>Human r-G6PD was lyophilized and assayed as “fresh” (grey bars) and compared to lyophilized r-G6PD samples from a lot that had been stored at 4°C for one year (striped bars). The Trinity controls were run at the same time for comparison of activity levels (black bars).</p

Performance of the positive controls on a qualitative glucose-6-phosphate dehydrogenase (G6PD) test.

<p><b>A.</b> Evaluation of human recombinant glucose-6-phosphate dehydrogenase (r-G6PD) standards. The lyophilized r-G6PD controls were tested in the newly developed rapid diagnostic test for G6PD. Line intensity versus G6PD activity was generated using over 20 replicates for each enzyme control. The data show that there are significant differences when comparing intermediate and deficient results to normal. B. Representative images of visual results with r-G6PD standards. Visual results presented here show that the control lines gave similar output across the standards tested. Differences were seen between all groups however, the intermediate high and low test line intensities were similar to the trained eye.</p

Descriptive statistics for data illustrated in Fig 6.

<p>Testing of the rapid diagnostic test with recombinant human glucose-6-phosphate dehydrogenase (G6PD) standards, with test-line intensities being measured using a low-cost hand-held reader. Mean difference in readings and lower to upper bounds for a 95% confidence interval (95% CI) as determined by the Dunnetts multiple comparisons test are shown for each control compared to the Normal control readings.</p