Lauren M. Stanoszek, Erin L. Crawford, Thomas M. Blomquist, Jessica A

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Presentation transcript:

Quality Control Methods for Optimal BCR-ABL1 Clinical Testing in Human Whole Blood Samples Lauren M. Stanoszek, Erin L. Crawford, Thomas M. Blomquist, Jessica A. Warns, Paige F.S. Willey, James C. Willey The Journal of Molecular Diagnostics Volume 15, Issue 3, Pages 391-400 (May 2013) DOI: 10.1016/j.jmoldx.2013.02.004 Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Interlaboratory comparison of BCR-ABL1 measurement. A: Log10 reduction of BCR-ABL1 per 103 GUSB measurement in CAP MRD-B–BCR/ABL1 p210 2011 Survey measured at the University of Toledo Medical Center (UTMC) compared with median log10 reduction values of survey participants. B: Comparison of BCR-ABL1 per 103 GUSB measurement to International Scale percentage values reported by ARUP Laboratories in three CML patient blood samples (two with CP-CML newly diagnosed without treatment and one with CP-CML at MMR under treatment). Gene-specific RT primers were used for newly diagnosed CML patient samples, and RH RT primers were used for the MMR CML patient sample. The UTMC data presented are means for three or more replicate measurements. Error bars, SD of the means. The Journal of Molecular Diagnostics 2013 15, 391-400DOI: (10.1016/j.jmoldx.2013.02.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Effect of storage time on BCR-ABL1 measurement and RIN score. Effect of incubation at room temperature on BCR-ABL1 per 103 GUSB measurement and RIN score value relative to baseline time equal to 0 hours. RNA was extracted from whole blood collected in EDTA tubes, mixed with K562 cells, and then incubated at room temperature for 3 days. The random hexamers were used for reverse transcription. Asterisks denote statistical significance (P < 0.01) from baseline measurement at 0 hours. The results are means for samples from six donors, with three or more replicate measurements of each sample. The Journal of Molecular Diagnostics 2013 15, 391-400DOI: (10.1016/j.jmoldx.2013.02.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Effect of RT primer and RNA input on measured transcript abundance. A: Effect of RT priming method on yield of BCR-ABL1 b3a2 and GUSB molecules/PCR assay and BCR-ABL1 b3a2 per 103 GUSB measurement after quantitative RT-PCR of RNA extracted from whole blood/K562 mixture with high level of BCR-ABL1 transcript. GSP-19, gene-specific (19-base) primer. B: Effect of blood/K562 cell mixture RNA concentration in a 30-μL RT reaction and RT priming method on yield of BCR-ABL1 b3a2 or GUSB cDNA relative to RH-primed baseline (0.9 μg RNA/RT). Asterisk denotes baseline measurements on graph from which fold-changes were determined. GSP-RT is gene-specific primed RT reaction, and RH-RT is RH-primed RT reaction. C: Effect of RNA input on RH-primed RT efficiency. RT efficiency is ERCC 171/113 measurement normalized to ERCC 171/113 measurement for 0 μg RNA input. The results are means for samples with three or more replicate measurements of each sample. Error bars, SD of the means. The Journal of Molecular Diagnostics 2013 15, 391-400DOI: (10.1016/j.jmoldx.2013.02.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Effect of extended BCR-ABL1 gene-specific RT primer on target specificity. A: Agilent Bioanalyzer electropherograms demonstrating (top line) off-target reverse transcription priming with 19-base BCR-ABL1 gene-specific RT primer with sample having low level BCR-ABL1. NT and IS product peaks are outcompeted in PCR by RANBP3 transcript isoforms (bottom line), presence of clean NT and IS peaks with 29-base BCR-ABL1 gene-specific RT primer. B: Effect of RT priming method on BCR-ABL1 b3a2 per 103 GUSB measurement after reverse transcription of low level of BCR-ABL1 transcript in a blood background. GSP-29, gene-specific primer (29 bases). The data presented are means for three or more replicate measurements. Error bars, SD of the means. The Journal of Molecular Diagnostics 2013 15, 391-400DOI: (10.1016/j.jmoldx.2013.02.004) Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions