Identification of Factors Contributing to Variability in a Blood-Based Gene Expression Test

by Michael R. Elashoff, Rachel Nuttall, Philip Beineke, Michael H. Doctolero, Mark Dickson, Andrea M. Johnson, Susan E. Daniels, Steven Rosenberg, James A. Wingrove

Background

Corus CAD is a clinically validated test based on age, sex, and expression levels of 23 genes in whole blood that provides a score (1–40 points) proportional to the likelihood of obstructive coronary disease. Clinical laboratory process variability was examined using whole blood controls across a 24 month period: Intra-batch variability was assessed using sample replicates; inter-batch variability examined as a function of laboratory personnel, equipment, and reagent lots.

Methods/Results

To assess intra-batch variability, five batches of 132 whole blood controls were processed; inter-batch variability was estimated using 895 whole blood control samples. ANOVA was used to examine inter-batch variability at 4 process steps: RNA extraction, cDNA synthesis, cDNA addition to assay plates, and qRT-PCR. Operator, machine, and reagent lots were assessed as variables for all stages if possible, for a total of 11 variables. Intra- and inter-batch variations were estimated to be 0.092 and 0.059 Cp units respectively (SD); total laboratory variation was estimated to be 0.11 Cp units (SD). In a regression model including all 11 laboratory variables, assay plate lot and cDNA kit lot contributed the most to variability (p = 0.045; 0.009 respectively). Overall, reagent lots for RNA extraction, cDNA synthesis, and qRT-PCR contributed the most to inter-batch variance (52.3%), followed by operators and machines (18.9% and 9.2% respectively), leaving 19.6% of the variance unexplained.

Conclusion

Intra-batch variability inherent to the PCR process contributed the most to the overall variability in the study while reagent lot showed the largest contribution to inter-batch variability.

For the full article visit: Identification of Factors Contributing to Variability in a Blood-Based Gene Expression Test
Syndicated from:PLoS ONE

Article is licensed under a Creative Commons Attribution License.