title: An international study to standardize the detection and quantitation of BCR-ABL transcripts from stabilized peripheral blood preparations by quantitative RT-PCR.
abstract: Due to the lack of comparability of BCR-ABL mRNA quantification results generated by various methodologies in different laboratories, an international multicenter trial was started with the participation of six laboratories (platforms: LightCycler LC, n=3; TaqMan TM, n=3). One hundred and eighty-six PB samples derived from healthy donors were spiked with serial dilutions (1:20 to 1:2x10(6)) of b2a2, b3a2 or e1a2 BCR-ABL positive white blood cells (WBC) from leukemic patients. After PAXgene stabilization, blinding, freezing and distribution, standardized RNA extraction, cDNA synthesis, PCR protocols and data evaluation were carried out. There was no significant difference in the results achieved using LC and TM technologies, but a considerable overall variation (CV=0.74 for ratios BCR-ABL/ABL). Up to a dilution of 1:1,000, 27/30 of the 2.5 mL samples tested positive. For higher dilutions, a PB volume of 5 or 10 ml was required to improve sensitivity. The study showed the feasibility of RQ-PCR standardization independent of the PCR machine used.
authors.affiliation: III. Medizinische Universitätsklinik, Medizinische Fakultät Mannheim der Universität Heidelberg, Mannheim, Germany. mueller@uni-hd.de
authors.forename: Martin C
authors.initials: MC
authors.lastname: Müller
authors.forename: Guiseppe
authors.initials: G
authors.lastname: Saglio
authors.forename: Feng
authors.initials: F
authors.lastname: Lin
authors.forename: Heike
authors.initials: H
authors.lastname: Pfeifer
authors.forename: Richard D
authors.initials: RD
authors.lastname: Press
authors.forename: Raymond R
authors.initials: RR
authors.lastname: Tubbs
authors.forename: Peter
authors.initials: P
authors.lastname: Paschka
authors.forename: Enrico
authors.initials: E
authors.lastname: Gottardi
authors.forename: Steven G
authors.initials: SG
authors.lastname: O'Brien
authors.forename: Oliver G
authors.initials: OG
authors.lastname: Ottmann
authors.forename: Hubertus
authors.initials: H
authors.lastname: Stockinger
authors.forename: Lothar
authors.initials: L
authors.lastname: Wieczorek
authors.forename: Kirsten
authors.initials: K
authors.lastname: Merx
authors.forename: Heiko
authors.initials: H
authors.lastname: König
authors.forename: Uwe
authors.initials: U
authors.lastname: Schwindel
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Hehlmann
authors.forename: Andreas
authors.initials: A
authors.lastname: Hochhaus
mesh_terms: D016044:Fusion Proteins, bcr-abl / Q000032:analysis / Q000235:genetics*; D006801:Humans; D007391:International Cooperation; D007938:Leukemia / Q000097:blood / Q000175:diagnosis; D012334:RNA, Neoplasm / Q000032:analysis*; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*
chemical_list: D012334:RNA, Neoplasm; D016044:Fusion Proteins, bcr-abl