Laboratory intercomparison of gene expression assays

Abstract

The possibility of a large-scale acute radiation exposure necessitates the development of new methods that could provide rapid individual dose estimates with high sample throughput. The focus of the study was an intercomparison of laboratories' dose-assessment performances using gene expression assays. Lithium-heparinized whole blood from one healthy donor was irradiated (240 kVp, 1 Gy/min) immediately after venipuncture at approximately 37°C using single X-ray doses. Blood samples to establish calibration curves (0.25-4 Gy) as well as 10 blinded test samples (0.1-6.4 Gy) were incubated for 24 h at 37°C supplemented with an equal volume of medium and 10% fetal calf serum. For quantitative reverse transcription polymerase chain reaction (qRT-PCR), samples were lysed, stored at -20°C and shipped on ice. For the Chemical Ligation Dependent Probe Amplification methodology (CLPA), aliquots were incubated in 2 ml CLPA reaction buffer (DxTerity), mixed and shipped at room temperature. Assays were run in each laboratory according to locally established protocols. The mean absolute difference (MAD) of estimated doses relative to the true doses (in Gy) was calculated. We also merged doses into binary categories reflecting aspects of clinical/diagnostic relevance and examined accuracy, sensitivity and specificity. The earliest reported time on dose estimates was <8 h. The standard deviation of technical replicate measurements in 75% of all measurements was below 11%. MAD values of 0.3-0.5 Gy and 0.8-1.3 Gy divided the laboratories contributions into two groups. These fourfold differences in accuracy could be primarily explained by unexpected variances of the housekeeping gene (P = 0.0008) and performance differences in processing of calibration and blinded test samples by half of the contributing laboratories. Reported gene expression dose estimates aggregated into binary categories in general showed an accuracies and sensitivities of 93-100% and 76-100% for the groups, with low MAD and high MAD, respectively. In conclusion, gene expression-based dose estimates were reported quickly, and for laboratories with MAD between 0.3-0.5 Gy binary dose categories of clinical significance could be discriminated with an accuracy and sensitivity comparable to established cytogenetic assays.

Figures

FIG. 1

The box plots reflect standard deviations of cycle threshold (CT)-values from triplicate qRT-PCR measurements for each gene (x-axis labels) performed in 5 different laboratories. Dotted horizontal line in the box plot refers to the mean and the solid horizontal line to the median. Last column summarizes the distribution of all 272 measurements.

FIG. 2

Calibration curves from laboratories (labs 2–6) running either qRT-PCR (y-axis) or CLPA assay (lab 8, y-axis) are shown. Data points are fitted by a regression line of second order (labs 2–6) or connected with a spline curve (lab 8). Details are provided in parenthesis. Symbols represent mean values of triplicate measurements. Error bars represent the standard deviation and are visible when greater than the symbols. Calibration data for laboratories 1 and 7 were not reported. Either the equation or the calibration curves itself were used to calculate or extrapolate by hand corresponding dose estimates from the blinded test samples gene expression values. All laboratories ultimately only used one gene for their calibration curves as shown in Fig. 2, but one laboratory did use 4 genes (data not shown). “RD” = radiation dose (Gy).

FIG. 3

Dose estimates reported from participants with higher (white circles) or lower MAD (dark gray circles) are shown for each of the 10 blinded test samples.