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

• Published in: Clinical chemistry (Baltimore, Md.) Md.), 2007-09, Vol.53 (9), p.1593-1600
• Main Authors: Winn-Deen, Emily S, Helton, Bret, Van Atta, Reuel, Wong, Wendy, Peralta, Jeffrey, Wang, James, Tsongalis, Gregory J, Belloni, Dorothy, Chan, David, Eshleman, James R, Gocke, Christopher D, Jobbagy, Zsolt, Beppu, Lan, Radich, Jerald P
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Anticoagulants; Biological and medical sciences; Blood; Blood Specimen Collection; Design; Fluorescence in situ hybridization; Fundamental and applied biological sciences. Psychology; Fusion Proteins, bcr-abl - genetics; Hematologic Tests; Humans; Investigative techniques, diagnostic techniques (general aspects); Leukemia; Leukemia, Myelogenous, Chronic, BCR-ABL Positive - genetics; Medical sciences; Methods; Microbiology; Nucleic acids; Patients; Polymerase Chain Reaction; RNA - blood; Sensitivity and Specificity; Statistical analysis; Technicians; Testing laboratories
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2007.085472

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. 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. 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. 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.