Identification of factors contributing to variability in a blood-based gene expression test.
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.


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.


CONCLUSIONS
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.
CardioDx, Inc., Palo Alto, California, United States of America.
Michael R
MR
Elashoff
Rachel
R
Nuttall
Philip
P
Beineke
Michael H
MH
Doctolero
Mark
M
Dickson
Andrea M
AM
Johnson
Susan E
SE
Daniels
Steven
S
Rosenberg
James A
JA
Wingrove
D003324:Coronary Artery Disease / Q000175:diagnosis* / Q000235:genetics; D018076:DNA, Complementary / Q000096:biosynthesis; D020869:Gene Expression Profiling / Q000379:methods / Q000592:standards*; D006801:Humans; D059232:Laboratory Personnel; D012313:RNA / Q000302:isolation & purification; D011933:Reagent Kits, Diagnostic / Q000592:standards*; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D015203:Reproducibility of Results
D018076:DNA, Complementary; D011933:Reagent Kits, Diagnostic; D012313:RNA