An international study to standardize the detection and quantitation of BCR-ABL transcripts from stabilized peripheral blood preparations by quantitative RT-PCR.
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.
Müller
Martin C
MC
III. Medizinische Universitätsklinik, Medizinische Fakultät Mannheim der Universität Heidelberg, Mannheim, Germany. mueller@uni-hd.de
Saglio
Guiseppe
G
Lin
Feng
F
Pfeifer
Heike
H
Press
Richard D
RD
Tubbs
Raymond R
RR
Paschka
Peter
P
Gottardi
Enrico
E
O'Brien
Steven G
SG
Ottmann
Oliver G
OG
Stockinger
Hubertus
H
Wieczorek
Lothar
L
Merx
Kirsten
K
König
Heiko
H
Schwindel
Uwe
U
Hehlmann
Rüdiger
R
Hochhaus
Andreas
A
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*
D012334:RNA, Neoplasm; D016044:Fusion Proteins, bcr-abl