Development of an integrated assay for detection of BCR-ABL RNA.
BACKGROUND
Current practice guidelines for managing patients with chronic myelogenous leukemia (CML) call for monitoring BCR-ABL transcript concentrations with a quantitative reverse transcription-PCR (qRT-PCR) assay. Because the available laboratory-developed assays lack consensus on the appropriate design, reporting of results, and reference intervals, we developed and evaluated an integrated BCR-ABL assay that yields standardized results for any laboratory and can be performed by technicians with no specialized training.


METHODS
We used the Cepheid Xpert BCR-ABL Monitor assay to measure both BCR-ABL and ABL (endogenous control) transcripts in blood samples from CML patients and healthy individuals. The assay involves 8 manual pipetting steps, fully automated nucleic acid purification, a nested qRT-PCR step, and data analysis.


RESULTS
The BCR-ABL assay requires approximately 2 h 20 min and covers a 5-log concentration range with a lower detection limit for the BCR-ABL:ABL ratio of approximately 0.005%. Assay results were negative for 100% of the 56 known CML-negative samples (12 patients with other hematologic disorders and 44 healthy blood donors). Testing of CML-positive patients undergoing disease monitoring showed 85% agreement with negative results (17 of 20) and 100% agreement with positive results (26 of 26). An imprecision/portability study revealed no differences in performance between sites, days, instruments, and operators.


CONCLUSIONS
The Xpert BCR-ABL Monitor assay provides a robust and reproducible alternative to laboratory-developed assays. Its ease of use may allow more laboratories to offer BCR-ABL testing for patients, and the short assay time enables same-day results for treating physicians.
Winn-Deen
Emily S
ES
Cepheid, Sunnyvale, CA, USA. Emily.Winn-Deen@cepheid.com
Helton
Bret
B
Van Atta
Reuel
R
Wong
Wendy
W
Peralta
Jeffrey
J
Wang
James
J
Tsongalis
Gregory J
GJ
Belloni
Dorothy
D
Chan
David
D
Eshleman
James R
JR
Gocke
Christopher D
CD
Jobbagy
Zsolt
Z
Beppu
Lan
L
Radich
Jerald P
JP
D000925:Anticoagulants; D001800:Blood Specimen Collection; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D006403:Hematologic Tests; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012313:RNA / Q000097:blood*; D012680:Sensitivity and Specificity
D000925:Anticoagulants; D012313:RNA; D016044:Fusion Proteins, bcr-abl