Laboratory intercomparison of gene expression assays

TY - JOUR

T1 - Laboratory intercomparison of gene expression assays

AU - Badie, C

AU - Kabacik, S

AU - Balagurunathan, Y

AU - Bernard, N

AU - Brengues, M

AU - Faggioni, G

AU - Greither, R

AU - Lista, F

AU - Peinnequin, A

AU - Poyot, T

AU - Herodin, F

AU - Missel, A

AU - Terbrueggen, B

AU - Zenhausern, F

AU - Rothkamm, K

AU - Meineke, V

AU - Braselmann, H

AU - Beinke, C

AU - Abend, M

PY - 2013/8

Y1 - 2013/8

N2 - 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.

AB - 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.

KW - Adult

KW - Biological Assay/methods

KW - Dose-Response Relationship, Radiation

KW - Electrophoresis, Capillary/methods

KW - Gene Expression/radiation effects

KW - Humans

KW - Laboratory Proficiency Testing

KW - Leukocytes/radiation effects

KW - Male

KW - Microspheres

KW - Nucleic Acid Amplification Techniques/methods

KW - Radiation Injuries/diagnosis

KW - Radioactive Hazard Release

KW - Radiometry/methods

KW - Reproducibility of Results

KW - Reverse Transcriptase Polymerase Chain Reaction/methods

KW - Sensitivity and Specificity

KW - Single-Blind Method

KW - Time Factors

KW - Triage

U2 - 10.1667/RR3236.1

DO - 10.1667/RR3236.1

M3 - SCORING: Journal article

C2 - 23886340

VL - 180

SP - 138

EP - 148

IS - 2

ER -