title: Quality control methods for optimal BCR-ABL1 clinical testing in human whole blood samples.
abstract: 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 10(3) 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 10(3) GUSB in whole blood is obtained when gene-specific primers are used in RT and samples are analyzed within 24 hours after blood collection.
authors.affiliation: Department of Medicine, University of Toledo Health Sciences Campus, Toledo, OH, USA.
authors.forename: Lauren M
authors.initials: LM
authors.lastname: Stanoszek
authors.forename: Erin L
authors.initials: EL
authors.lastname: Crawford
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Blomquist
authors.forename: Jessica A
authors.initials: JA
authors.lastname: Warns
authors.forename: Paige F S
authors.initials: PF
authors.lastname: Willey
authors.forename: James C
authors.initials: JC
authors.lastname: Willey
mesh_terms: D019411:Clinical Laboratory Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D016044:Fusion Proteins, bcr-abl / Q000097:blood / Q000235:genetics*; D005966:Glucuronidase / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000097:blood / Q000235:genetics*; D011786:Quality Control; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: C581634:BCR-ABL1 fusion protein, human; D017931:DNA Primers; D018076:DNA, Complementary; D012313:RNA; D016044:Fusion Proteins, bcr-abl; D005966:Glucuronidase
references.citation: Blood. 2008 Oct 15;112(8):3330-8
references.pmid: 18684859
references.citation: Clin Chem. 2008 Sep;54(9):1568-71
references.pmid: 18641042
references.citation: Cell. 1984 Jan;36(1):93-9
references.pmid: 6319012
references.citation: Int J Food Microbiol. 1992 Sep;17(1):37-45
references.pmid: 1476866
references.citation: J Clin Microbiol. 1998 Sep;36(9):2443-6
references.pmid: 9705371
references.citation: Nucleic Acids Res. 1991 Nov 11;19(21):6051
references.pmid: 1719488
references.citation: Leukemia. 2004 Apr;18(4):884-6
references.pmid: 14961029
references.citation: Best Pract Res Clin Haematol. 2009 Sep;22(3):355-65
references.pmid: 19959086
references.citation: Haematologica. 2007 Jul;92(7):970-3
references.pmid: 17606448
references.citation: Nucleic Acids Res. 1993 Feb 25;21(4):1047
references.pmid: 8451177
references.citation: Bioinformatics. 2007 Nov 1;23(21):2947-8
references.pmid: 17846036
references.citation: Blood. 2010 Nov 25;116(22):e111-7
references.pmid: 20720184
references.citation: Leukemia. 2003 Dec;17(12):2474-86
references.pmid: 14562124
references.citation: BMC Genomics. 2011 Feb 18;12:118
references.pmid: 21332979
references.citation: N Engl J Med. 2006 Dec 7;355(23):2408-17
references.pmid: 17151364
references.citation: Lancet. 1989 Apr 29;1(8644):928-9
references.pmid: 2565420
references.citation: Methods Mol Biol. 2004;258:13-41
references.pmid: 14970455
references.citation: Best Pract Res Clin Haematol. 2009 Jun;22(2):181-90
references.pmid: 19698927
references.citation: Nat Methods. 2005 Oct;2(10):731-4
references.pmid: 16179916
references.citation: Expert Rev Mol Diagn. 2005 Jan;5(1):43-52
references.pmid: 15723591
references.citation: Nature. 1973 Jun 1;243(5405):290-3
references.pmid: 4126434
references.citation: Lancet Oncol. 2011 Sep;12(9):841-51
references.pmid: 21856226
references.citation: Clin Lab Haematol. 2002 Dec;24(6):337-41
references.pmid: 12452813
references.citation: Physiol Genomics. 2004 Nov 17;19(3):247-54
references.pmid: 15548831
references.citation: N Engl J Med. 1999 Jul 15;341(3):164-72
references.pmid: 10403855
references.citation: Int J Oncol. 2006 May;28(5):1099-103
references.pmid: 16596225
references.citation: Clin Lymphoma Myeloma. 2009;9 Suppl 4:S391-4
references.pmid: 20007108
references.citation: Nature. 1982 Dec 23;300(5894):765-7
references.pmid: 6960256
references.citation: J Mol Diagn. 2006 Nov;8(5):551-8
references.pmid: 17065423
references.citation: Proc Natl Acad Sci U S A. 2006 Feb 21;103(8):2794-9
references.pmid: 16477019
references.citation: J Mol Diagn. 2006 Jul;8(3):385-9
references.pmid: 16825513
references.citation: J Forensic Sci. 1994 Mar;39(2):362-72
references.pmid: 8195750
references.citation: Biochem J. 1999 Jan 15;337 ( Pt 2):231-41
references.pmid: 9882620
references.citation: Blood. 2009 May 7;113(19):4497-504
references.pmid: 19264678
references.citation: Blood. 2012 Feb 2;119(5):1123-9
references.pmid: 22160483
references.citation: Leukemia. 2000 Oct;14(10):1850-6
references.pmid: 11021760
references.citation: Nature. 1983 Nov 17-23;306(5940):239-42
references.pmid: 6316147
references.citation: Expert Rev Hematol. 2010 Dec;3(6):665-73
references.pmid: 21091142
references.citation: J Natl Compr Canc Netw. 2009 Jul;7 Suppl 4:S1-34, quiz S35-6
references.pmid: 19635230
references.citation: N Engl J Med. 2002 Feb 28;346(9):683-93
references.pmid: 11870247
references.citation: J Clin Oncol. 2010 Jan 20;28(3):398-404
references.pmid: 20008620
references.citation: Leuk Res. 2011 Jul;35(7):e121-2
references.pmid: 21489625
references.citation: Blood. 1993 Jan 1;81(1):158-65
references.pmid: 8417787
references.citation: BMC Genomics. 2005 Nov 02;6:150
references.pmid: 16266432
references.citation: N Engl J Med. 2010 Jun 17;362(24):2260-70
references.pmid: 20525995
references.citation: Blood. 2006 Jul 1;108(1):28-37
references.pmid: 16522812
references.citation: J Mol Diagn. 2007 Sep;9(4):421-30
references.pmid: 17690211
references.citation: N Engl J Med. 2003 Oct 9;349(15):1423-32
references.pmid: 14534335
references.citation: J Clin Microbiol. 2001 Feb;39(2):485-93
references.pmid: 11158094
references.citation: N Engl J Med. 2001 Apr 5;344(14):1031-7
references.pmid: 11287972
references.citation: J Mol Diagn. 2006 May;8(2):231-9
references.pmid: 16645210
references.citation: J Clin Oncol. 2010 Jan 20;28(3):392-7
references.pmid: 20008621
references.citation: Tohoku J Exp Med. 2008 Feb;214(2):97-104
references.pmid: 18285666