title: 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.
authors.affiliation: Public Health England, Centre for Radiation, Chemical and Environmental Hazards, Chilton, Didcot, Oxon OX11 0RQ, United Kingdom.
authors.forename: C
authors.initials: C
authors.lastname: Badie
authors.forename: S
authors.initials: S
authors.lastname: Kabacik
authors.forename: Y
authors.initials: Y
authors.lastname: Balagurunathan
authors.forename: N
authors.initials: N
authors.lastname: Bernard
authors.forename: M
authors.initials: M
authors.lastname: Brengues
authors.forename: G
authors.initials: G
authors.lastname: Faggioni
authors.forename: R
authors.initials: R
authors.lastname: Greither
authors.forename: F
authors.initials: F
authors.lastname: Lista
authors.forename: A
authors.initials: A
authors.lastname: Peinnequin
authors.forename: T
authors.initials: T
authors.lastname: Poyot
authors.forename: F
authors.initials: F
authors.lastname: Herodin
authors.forename: A
authors.initials: A
authors.lastname: Missel
authors.forename: B
authors.initials: B
authors.lastname: Terbrueggen
authors.forename: F
authors.initials: F
authors.lastname: Zenhausern
authors.forename: K
authors.initials: K
authors.lastname: Rothkamm
authors.forename: V
authors.initials: V
authors.lastname: Meineke
authors.forename: H
authors.initials: H
authors.lastname: Braselmann
authors.forename: C
authors.initials: C
authors.lastname: Beinke
authors.forename: M
authors.initials: M
authors.lastname: Abend
mesh_terms: D000328:Adult; D001681:Biological Assay / Q000379:methods*; D004307:Dose-Response Relationship, Radiation; D019075:Electrophoresis, Capillary / Q000379:methods; D015870:Gene Expression / Q000528:radiation effects*; D006801:Humans; D059021:Laboratory Proficiency Testing; D007962:Leukocytes / Q000528:radiation effects* / Q000648:ultrastructure; D008297:Male; D008863:Microspheres; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011832:Radiation Injuries / Q000175:diagnosis / Q000235:genetics; D018788:Radioactive Hazard Release; D011874:Radiometry / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D016037:Single-Blind Method; D013997:Time Factors; D014218:Triage