First-Line Test for BCR-ABL
Quantitative reverse transcription polymerase chain reaction (RT-qPCR) on peripheral blood is the first-line test for BCR-ABL, as it serves dual purposes: confirming diagnosis by detecting BCR-ABL1 transcripts and establishing a baseline for subsequent molecular monitoring. 1
Recommended Diagnostic Approach
Primary Testing Strategy
RT-qPCR (quantitative) should be performed at initial workup to establish the presence of quantifiable BCR-ABL1 mRNA transcripts, which serves as the baseline for all future monitoring 1
Bone marrow aspirate and biopsy with conventional cytogenetics must be performed concurrently to detect additional chromosomal abnormalities (ACAs) in Philadelphia chromosome-positive cells, which have prognostic significance 1, 2
The combination of cytogenetic analysis plus RT-PCR is mandatory for comprehensive initial assessment, as cytogenetics alone may miss 5% of CML cases with normal karyotype or cryptic translocations 2
Specimen Requirements
Peripheral blood is the preferred specimen for RT-qPCR, avoiding the need for invasive bone marrow procedures for molecular testing 1
Bone marrow is required for cytogenetic analysis with a minimum of 20 metaphases analyzed (ideally 25) to ensure detection of ACAs 1, 2
Alternative When Bone Marrow Unavailable
FISH with dual-color probes for BCR and ABL1 genes on peripheral blood is acceptable if bone marrow evaluation is not feasible, though it has a false-positive rate of 1-5% depending on the probe used 1
Double-fusion FISH is preferred over extra signal (ES) probes as it has lower false-positive rates and can detect variant translocations 1, 2
Critical Technical Considerations
Transcript Type Identification
Qualitative RT-PCR must determine the exact BCR-ABL1 transcript type (e13a2, e14a2, or atypical variants) before starting treatment to facilitate accurate monitoring 1, 3
Most CML cases (>90%) express e13a2 and/or e14a2 transcripts, but screening only for these variants will miss approximately 2% of cases with atypical fusions 1
If BCR-ABL1 is unexpectedly low or undetectable at diagnosis, qualitative RT-PCR for atypical transcripts should be performed 1
Sensitivity Requirements
RT-qPCR assays must have sensitivity of at least 4-log reduction from the standardized baseline to adequately monitor deep molecular responses 1
RT-qPCR can detect one CML cell in 100,000 normal cells, making it the most sensitive assay available for BCR-ABL1 measurement 1
International Scale Reporting
Results must be reported on the International Scale (IS), where the standardized baseline (from the IRIS trial) is set to 100% 1
The IS uses BCR-ABL1 transcript ratio to control genes (ABL1, BCR, or GUSB) multiplied by a laboratory-specific conversion factor 1
Common Pitfalls to Avoid
False-Negative Results
Interphase FISH has a 1-5% false-positive rate depending on probe selection, making it less reliable than RT-qPCR for initial diagnosis 1
FISH cannot detect all variant translocations unless dual-fusion probes are used 1
Up to 5% of CML cases have normal karyotype, requiring molecular confirmation even when cytogenetics appear normal 2
Inadequate Baseline Assessment
Failure to establish baseline BCR-ABL1 levels prevents accurate interpretation of early molecular response at 3 months 1
Not identifying the specific transcript type at diagnosis makes subsequent monitoring unreliable or impossible 1, 3
Monitoring Limitations
FISH is inadequate for monitoring minimal residual disease and should not be used for response assessment if RT-qPCR is available 1
Conventional cytogenetics becomes unnecessary after complete cytogenetic response is achieved, as RT-qPCR provides superior sensitivity 1
Additional Baseline Testing
For Adult ALL or MPAL
FISH for t(9;22)(q34.1;q11.2)/BCR-ABL1 is required as part of the initial diagnostic panel 1
RT-PCR for BCR-ABL1 fusion products should be performed for monitoring minimal residual disease 1