Received on January 5, 2007
Accepted on July 3, 2007

Molecular Diagnostics and Genetics

Development of an Integrated Assay for Detection of BCR-ABL RNA

¹ Cepheid, Sunnyvale, CA
² Dartmouth-Hitchcock Medical Center, Lebanon, NH
³ Molecular Diagnostics Laboratory, Johns Hopkins Medical Institutions, Baltimore, MD
⁴ Fred Hutchinson Cancer Research Center, Seattle, WA

^* To whom correspondence should be addressed. E-mail: Emily.Winn-Deen{at}cepheid.com.

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.