Quality Control Methods for Optimal BCR-ABL1 Clinical Testing in Human Whole Blood Samples
2013; Elsevier BV; Volume: 15; Issue: 3 Linguagem: Inglês
10.1016/j.jmoldx.2013.02.004
ISSN1943-7811
AutoresLauren M. Stanoszek, Erin L. Crawford, Thomas Blomquist, Jessica A. Warns, Paige Willey, James C. Willey,
Tópico(s)Chronic Lymphocytic Leukemia Research
ResumoReliable breakpoint cluster region (BCR)–Abelson (ABL) 1 measurement is essential for optimal management of chronic myelogenous leukemia. There is a need to optimize quality control, sensitivity, and reliability of methods used to measure a major molecular response and/or treatment failure. The effects of room temperature storage time, different primers, and RNA input in the reverse transcription (RT) reaction on BCR-ABL1 and β-glucuronidase (GUSB) cDNA yield were assessed in whole blood samples mixed with K562 cells. BCR-ABL1 was measured relative to GUSB to control for sample loading, and each gene was measured relative to known numbers of respective internal standard molecules to control for variation in quality and quantity of reagents, thermal cycler conditions, and presence of PCR inhibitors. Clinical sample and reference material measurements with this test were concordant with results reported by other laboratories. BCR-ABL1 per 103 GUSB values were significantly reduced (P = 0.004) after 48-hour storage. Gene-specific primers yielded more BCR-ABL1 cDNA than random hexamers at each RNA input. In addition, increasing RNA inhibited the RT reaction with random hexamers but not with gene-specific primers. Consequently, the yield of BCR-ABL1 was higher with gene-specific RT primers at all RNA inputs tested, increasing to as much as 158-fold. We conclude that optimal measurement of BCR-ABL1 per 103 GUSB in whole blood is obtained when gene-specific primers are used in RT and samples are analyzed within 24 hours after blood collection. Reliable breakpoint cluster region (BCR)–Abelson (ABL) 1 measurement is essential for optimal management of chronic myelogenous leukemia. There is a need to optimize quality control, sensitivity, and reliability of methods used to measure a major molecular response and/or treatment failure. The effects of room temperature storage time, different primers, and RNA input in the reverse transcription (RT) reaction on BCR-ABL1 and β-glucuronidase (GUSB) cDNA yield were assessed in whole blood samples mixed with K562 cells. BCR-ABL1 was measured relative to GUSB to control for sample loading, and each gene was measured relative to known numbers of respective internal standard molecules to control for variation in quality and quantity of reagents, thermal cycler conditions, and presence of PCR inhibitors. Clinical sample and reference material measurements with this test were concordant with results reported by other laboratories. BCR-ABL1 per 103 GUSB values were significantly reduced (P = 0.004) after 48-hour storage. Gene-specific primers yielded more BCR-ABL1 cDNA than random hexamers at each RNA input. In addition, increasing RNA inhibited the RT reaction with random hexamers but not with gene-specific primers. Consequently, the yield of BCR-ABL1 was higher with gene-specific RT primers at all RNA inputs tested, increasing to as much as 158-fold. We conclude that optimal measurement of BCR-ABL1 per 103 GUSB in whole blood is obtained when gene-specific primers are used in RT and samples are analyzed within 24 hours after blood collection. Chronic myelogenous leukemia (CML) represents 15% of all adult leukemias in Western populations.1Faderl S. Talpaz M. Estrov Z. O'Brien S. Kurzrock R. Kantarjian H.M. The biology of chronic myeloid leukemia.N Engl J Med. 1999; 341: 164-172Crossref PubMed Scopus (1080) Google Scholar This malignancy is caused by a chromosomal translocation, t(9:22)(q34;q11); a segment of the breakpoint cluster region (BCR) gene from chromosome 22 (region q11) is fused to a site within the Abelson (ABL1) gene from chromosome 9 (region q34).2Nowell P.C. Hungerford D.A. 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Another potential source of inconsistency in BCR-ABL1 measurement is variation in the efficiency of mRNA-to-cDNA conversion during reverse transcription (RT). Blood-specific inhibitors of RT may be present within RNA extracted from whole blood, including heme, IgG, leukocyte genomic DNA, and the anticoagulants, EDTA and heparin.33Akane A. Matsubara K. Nakamura H. Takahashi S. Kimura K. Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification.J Forensic Sci. 1994; 39: 362-372Crossref PubMed Google Scholar, 34Al-Soud W.A. Radstrom P. Purification and characterization of PCR-inhibitory components in blood cells.J Clin Microbiol. 2001; 39: 485-493Crossref PubMed Scopus (719) Google Scholar, 35Rossen L. Norskov P. Holmstrom K. Rasmussen O.F. 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However, even in such tubes, relative representation of certain genes may change as early as minutes after venipuncture because of an altered regulation and/or degradation rate.40Pahl A. Gene expression profiling using RNA extracted from whole blood: technologies and clinical applications.Expert Rev Mol Diagn. 2005; 5: 43-52Crossref PubMed Scopus (16) Google Scholar, 41Tanner M.A. Berk L.S. Felten D.L. Blidy A.D. Bit S.L. Ruff D.W. Substantial changes in gene expression level due to the storage temperature and storage duration of human whole blood.Clin Lab Haematol. 2002; 24: 337-341Crossref PubMed Scopus (61) Google Scholar Thus, there is a need to identify the optimal RT priming method for both BCR-ABL1 and the reference gene, GUSB, under the most optimal storage time. The optimal amount of whole blood RNA to include in the RT reaction may be affected by each of the previously mentioned factors. In an effort to establish optimal conditions for measurement of BCR-ABL1, we did the following: i) developed a quality-controlled, quantitative PCR method, ii) measured the effect of total RNA input or type of RT primers on RT efficiency, and iii) assessed the effect of storage time on BCR-ABL1 measurement. Three proficiency samples were obtained from the College of American Pathologists (CAP; Northfield, IL). Sample minimal residual disease (MRD)-04 was RNA extracted from the K562 cell line that expresses the b3a2 BCR-ABL1 transcript. Sample MRD-05 was RNA extracted from a BCR-ABL1–negative cell line. Sample MRD-06 was RNA extracted from MRD-04 diluted 1:10,000 in BCR-ABL1–negative RNA (MRD-05).42MRD BCR-ABL p210 Survey Participant Summary. College of American Pathologists, Northfield, IL2011Google Scholar Each RNA sample was DNase treated with DNA-free DNAse Treatment and Removal Reagent (Life Technologies, Grand Island, NY). All blood samples were collected under protocols approved by the University of Toledo (Toledo, OH) Medical Center Institutional Review Board. Whole blood samples were collected in 4-mL dipotassium EDTA Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) at ambient temperature at the University of Toledo Medical Center. Blood used to mix with K562 cells (described later) was obtained through the Department of Pathology, University of Toledo Medical Center, from patients undergoing routine venipuncture for laboratory testing. Leftover blood not needed for standard-of-care purposes from patients with a normal complete blood cell count was anonymized and provided to the research laboratory. Peripheral blood was obtained from three patients with CML [two with chronic-phase (CP) CML newly diagnosed and one with CP-CML at MMR under treatment] at the same time blood was drawn for shipment to ARUP Laboratories (Salt Lake City, UT) for BCR-ABL1 measurement and conversion of results to the International Scale. The K562 cell line, expressing the BCR-ABL1 b3a2 fusion transcript, was purchased from ATCC (Manassas, VA) and incubated in RPMI 1640 medium + 10% fetal bovine serum at 37°C, in an atmosphere containing 5% CO2 and 90% humidity. K562 cells were isolated from culture at 80% confluency. In each of the two experiments, whole blood samples were collected in EDTA tubes from three anonymized individuals, and each sample was spiked with a known number of K562 cells suspended in PBS. In the first experiment (samples A, B, and C), the concentration of K562 cells in each whole blood sample was 5.8 × 105 cells/mL (2.32 × 106 cells total), and in the second experiment (samples D, E, and F), the concentration in each sample was 4.7 × 106 K562 cells/mL (1.88 × 107 cells total). Each whole blood/K562 cell sample was incubated in 15-mL conical tubes at room temperature for various amounts of time. For each sample, the time course was initiated within 6 hours of venipuncture. At each time point, individual conical tubes were inverted three times before RNA extraction. Known numbers of K562 cells were spiked into anonymized whole blood samples collected in EDTA tubes from one anonymized individual (K). The concentration of K562 cells in sample K was 5.8 × 105 cells/mL (2.32 × 106 cells total). Known numbers of K562 cells were spiked into anonymized whole blood samples collected in EDTA tubes from each of three individuals (G, H, and I) for the random hexamer (RH)–primed RT efficiency study. The concentration of K562 cells was 4.4 × 106 K562 cells/mL (1.76 × 107 cells total) in samples G and H and 5.0 × 106 K562 cells/mL (2.00 × 107 cells total) in sample I. For the gene-specific primed RT efficiency study, blood from one anonymized individual (J) was used. The concentration of K562 cells was 4.9 × 106 K562 cells/mL (1.76 × 107 cells total). In vitro–transcribed synthetic, alien RNA standards developed by the External RNA Control Consortium (ERCC), termed ERCC 171 and 113, were donated by Dr. Marc Salit, National Institute for Standards and Technology (Gaithersburg, MD).43Baker S.C. Bauer S.R. Beyer R.P. Brenton J.D. Bromley B. Burrill J. et al.The External RNA Controls Consortium: a progress report.Nat Methods. 2005; 2: 731-734Crossref PubMed Scopus (251) Google Scholar, 44External R.N.A. Controls Consortium. Proposed methods for testing and selecting the ERCC external RNA controls.BMC Genomics. 2005; 6: 150Crossref PubMed Scopus (100) Google Scholar, 45Devonshire A.S. Elaswarapu R. Foy C.A. Applicability of RNA standards for evaluating RT-qPCR assays and platforms.BMC Genomics. 2011; 12: 118Crossref PubMed Scopus (31) Google Scholar RNA stocks from ERCC 113 and 171 standards were first diluted in RNase-free water, and the RNA concentration was measured using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE). ERCC 113 and 171 standards were each secondarily diluted in 100 ng/μL salmon sperm DNA to a final concentration of 1.0 (10−10) mol/L (Invitrogen, Carlsbad, CA). The ERCC 113 standard was reverse transcribed into cDNA, and the cDNA was quantified using the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA). A reverse transcription standards mixture (RTSM) was prepared by combining known molar amounts of ERCC 171 RNA and ERCC 113 cDNA (previously reverse transcribed). A 1-μL aliquot of RTSM was included in each RT reaction to assess RT efficiency. The ERCC 171 RNA was reverse transcribed into cDNA, along with other RNA species in the RT reaction, whereas the ERCC 113 cDNA remained unchanged. Yield of ERCC 171 cDNA was a measure of RT efficiency, and ERCC 113 cDNA was included as a sample loading control. Each 1-μL aliquot of RTSM contained 1.42 × 104 (±6.7 × 103) ERCC 171 cDNA molecules and 1.68 × 104 (±7.4 × 103) ERCC 113 cDNA molecules when measured in RT reactions with no background RNA. After RT, an aliquot of RT product, cDNA, was subjected to PCR in the presence of a known number of IS molecules for ERCC 171 and ERCC 113. ERCC 171 RNA RT efficiency was determined by measuring the ERCC 171 cDNA native template (NT)/IS PCR product ratio over the ERCC 113 NT/IS product ratio. In this way, normalization of ERCC 171 cDNA molecules to ERCC 113 cDNA molecules controlled for variation in RTSM sample loading, enabling reliable measurement of RT efficiency. PCR products associated with the background noise peaks were isolated using an E-Gel SizeSelect 2% gel with the E-Gel iBase Power System (Invitrogen), and tailed BCR-ABL1 b3a2 sequencing primers corresponding to Ion Torrent Amplicon Sequencing adapters were used to amplify both isolated native and off-target products (Ion Torrent Amplicon Application Note, April 4, 2011; Amplicon Sequencing, South San Francisco, CA). Amplified products with attached Ion Torrent Amplicon Sequencing adapters were gel purified using E-Gel Size Select 2% and sent to the Ohio University Genomics Facility for Ion Torrent 314 DNA Chip Sequencing Service (Ohio University, Athens, OH). Sequencing data captured internally to the sequencing reagents were used to build consensus sequences using ClustalX 2.1 (Conway Institute UCD, Dublin, Ireland).46Larkin M.A. Blackshields G. Brown N.P. Chenna R. McGettigan P.A. McWilliam H. Valentin F. Wallace I.M. Wilm A. Lopez R. Thompson J.D. Gibson T.J. Higgins D.G. Clustal W.and Clustal X. version 2.0.Bioinformatics. 2007; 23: 2947-2948Crossref PubMed Scopus (22841) Google Scholar These consensus sequences were th
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