Advantages of the WRAIR whole blood cholinesterase assay: comparative analysis to the micro-Ellman, Test-mate ChE, and Michel (DeltapH) assays.

Red blood cell AChE (RBC-AChE) and plasma BChE can be used as sensitive biomarkers to detect exposure to OP nerve agents, pesticides, and cholinergic drugs. In a comparative study, RBC-AChE and serum BChE activities in whole blood was obtained from forty seven healthy male and female human volunteers, and then exposed separately ex vivo to three OP nerve agents (soman (GD), sarin (GB) and VX) to generate a wide range of inhibition of AChE and BChE activity (up to 90% of control). These samples were measured using four different ChE assays: (i) colorimetric microEllman (using DTNB at 412 nm), (ii) Test-mate ChE field kit (also based on the Ellman assay), (iii) Michel (delta pH), and (iv) the Walter Reed Army Institute of Research Whole Blood (WRAIR WB) cholinesterase assay. The WRAIR assay is a modified Ellman method using DTP at 324 nm (which minimizes hemoglobin interference and improves sensitivity), and determines AChE and BChE in a small whole blood sample simultaneously. Scatter plots of RBC-AChE activities were determined using the WRAIR ChE assay versus the micro-Ellman, Test-mate and Michel after exposure to varying concentrations of soman, sarin and VX. Regression analyses yielded mostly linear relationships with high correlations (r2 = 0.83-0.93) for RBC-AChE values in the WRAIR assay compared to the alternate methods. For the plasma BChE measurements, individual human values were significantly more variable (as expected), resulting in lower correlations using WRAIR ChE versus the alternate assays (r2 values 0.5 - 0.6). To circumvent the limitations of simple correlation analysis, Bland and Altman analysis for comparing two independent measurement techniques was performed. For example, a Bland and Altman plot of the ratio of the WRAIR whole blood AChE and Michel AChE (plotted on the y-axis) vs. the average of the two methods (x-axis) shows that the majority of the individual AChE values are within +/- 1.96 S.D. of the mean difference, indicating that the two methods may be used interchangeably with a high degree of confidence. The WRAIR ChE assay can be thus be used as a reliable inter-conversion assay when comparing results from laboratory-based (Michel) and field-based (Test-mate ChE kit), which use different methodology and report in different units of AChE activity.