Characterization of a reference material for BCR-ABL (M-BCR) mRNA quantitation by real-time amplification assays: towards new standards for gene expression measurements.
Monitoring of BCR-ABL transcripts has become established practice in the management of chronic myeloid leukemia. However, nucleic acid amplification techniques are prone to variations which limit the reliability of real-time quantitative PCR (RQ-PCR) for clinical decision making, highlighting the need for standardization of assays and reporting of minimal residual disease (MRD) data. We evaluated a lyophilized preparation of a leukemic cell line (K562) as a potential quality control reagent. This was found to be relatively stable, yielding comparable respective levels of ABL, GUS and BCR-ABL transcripts as determined by RQ-PCR before and after accelerated degradation experiments as well as following 5 years storage at -20 degrees C. Vials of freeze-dried cells were sent at ambient temperature to 22 laboratories on four continents, with RQ-PCR analyses detecting BCR-ABL transcripts at levels comparable to those observed in primary patient samples. Our results suggest that freeze-dried cells can be used as quality control reagents with a range of analytical instrumentations and could enable the development of urgently needed international standards simulating clinically relevant levels of MRD.
Saldanha
J
J
1National Institute of Biological Standards and Controls, South Mimms, UK.
Silvy
M
M
Beaufils
N
N
Arlinghaus
R
R
Barbany
G
G
Branford
S
S
Cayuela
J-M
JM
Cazzaniga
G
G
Gonzalez
M
M
Grimwade
D
D
Kairisto
V
V
Miyamura
K
K
Lawler
M
M
Lion
T
T
Macintyre
E
E
Mahon
F-X
FX
Muller
M C
MC
Ostergaard
M
M
Pfeifer
H
H
Saglio
G
G
Sawyers
C
C
Spinelli
O
O
van der Velden
V H J
VH
Wang
J Q
JQ
Zoi
K
K
Patel
V
V
Phillips
P
P
Matejtschuk
P
P
Gabert
J
J
D005612:Freeze Drying; D016044:Fusion Proteins, bcr-abl; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards*; D006801:Humans; D007202:Indicators and Reagents; D020014:K562 Cells; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000235:genetics*; D011786:Quality Control; D012333:RNA, Messenger / Q000032:analysis*; D012015:Reference Standards
D007202:Indicators and Reagents; D012333:RNA, Messenger; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl