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An international study to standardize the detection and quantitation of BCR-ABL transcripts from stabilized peripheral blood preparations by quantitative RT-PCR

From the III. Medizinische Universitätsklinik, Medizinische Fakultät Mannheim der Universität Heidelberg, Mannheim, Germany (MM, PP, KM, HK, US, RH, AH); Azienda Ospedaliera S. Luigi Gonzaga, Orbassano, Italy (GS, EG); School of Clinical and Laboratory Sciences, University of Newcastle, UK (FL); II. Medizinische Klinik, Universität Frankfurt, Germany (HP, OGO); Dept. of Pathology, Oregon Health and Science University, Portland OR, USA (RDP); Dept. of Clinical Pathology, Cleveland Clinic Lerner College of Medicine, Cleveland, OH, USA (RRT); Roche Diagnostics, Penzberg, Germany (HS)

Correspondence: Martin C. Müller, III. Medizinische Universitätsklinik, Medizinische Fakultät Mannheim der Universität Heidelberg, Wiesbadener Str. 7-11, 68305 Mannheim, Germany. E-mail: mueller{at}uni-hd.de

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^TM, LC, n=3; TaqMan^TM, TM, n=3). One hundred and eighty-six PB samples derived from healthy donors were spiked with serial dilutions (1:20 to 1:2x10⁶) of b2a2, b3a2 or e1a2 BCR-ABL positive white blood cells (WBC) from leukemic patients. After PAXgene^TM 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.

Key words: standardization, quantitative PCR, BCR-ABL, chronic myelogenous leukemia.

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