What test should be ordered to detect the BCR-ABL (Breakpoint Cluster Region-Abelson) minor transcript?

Detection of BCR-ABL Minor Transcript

Qualitative RT-PCR is the mandatory test for detecting BCR-ABL minor transcripts in patients with suspected or confirmed chronic myeloid leukemia (CML). 1

Recommended Testing Approach

Qualitative RT-PCR is the only routine technique that can determine the exact BCR-ABL transcript type, including minor transcripts. This test should be performed on peripheral blood samples, which are easier to collect and correlate well with clinical response in chronic phase CML patients.

Primary Testing Strategy:

• Qualitative RT-PCR: Strongly recommended as the initial screening test and mandatory for determining BCR-ABL transcript type in all confirmed CML patients 1
  • Advantages: Only routine technique that can determine exact BCR-ABL transcript type
  • Sample type: Peripheral blood (minimum 5-10 mL, containing at least 1-2 × 10^7 nucleated cells) 1
  • Processing time: Samples should be processed within 24 hours (36 hours maximum) to prevent significant transcript degradation 1

Supporting/Confirmatory Tests:

1. Interphase FISH:

   • Recommended for initial screening
   • Picks up all BCR-ABL rearrangements regardless of breakpoint
   • Cannot identify transcript type, so positive cases need follow-up with RT-PCR 1

2. Cytogenetics:

   • Mandatory for detecting additional chromosomal abnormalities
   • Not recommended in isolation as an initial screening tool (5% of CML patients have normal karyotype) 1

3. Quantitative RT-qPCR:

   • Not generally recommended for initial diagnosis
   • Used for monitoring response to treatment and minimal residual disease 1

Technical Considerations for RT-PCR

Sample Collection and Processing:

• Use EDTA as anticoagulant 1
• Total leukocytes following red-cell lysis are preferred over Ficoll-separated fractions 1
• Process samples within 24 hours (36 hours maximum) 1

RT-PCR Methodology:

• Random primers are recommended for reverse transcription (minimum concentration 25 μM) 1
• Moloney murine leukemia virus (MMLV) or Superscript are suitable reverse transcriptases 1
• For maximum reproducibility, divide samples into two aliquots and assay both separately from the RNA extraction stage 1

Quality Control:

• Probes and primers should be RNA-specific and tested with genomic DNA 1
• Strict contamination prevention protocols must be followed 1
• Appropriate standards (RNA or DNA) should be used for calibration 1

Clinical Context and Importance

Detection of BCR-ABL minor transcripts is critical for:

1. Accurate diagnosis of CML variants
2. Selection of appropriate tyrosine kinase inhibitor therapy
3. Establishing baseline for monitoring treatment response
4. Detecting minimal residual disease during treatment

Common Pitfalls to Avoid

1. Relying solely on FISH or cytogenetics: These methods cannot determine transcript type and may miss cases with normal karyotype 1

2. Inadequate sample collection: Insufficient cell numbers can lead to false negatives; ensure minimum of 1-2 × 10^7 nucleated cells 1

3. Delayed sample processing: RNA degradation after 24-36 hours can affect test sensitivity 1

4. Using nested RT-PCR: This should be avoided due to risk of artifacts and contamination 1

5. Failing to test for atypical variants: Commercial tests may not detect all atypical BCR-ABL variants, so laboratories should ensure coverage of these variants if RT-PCR is used as primary screen 1

The European LeukemiaNet guidelines emphasize that qualitative RT-PCR is the only reliable method to determine the exact BCR-ABL transcript type, including minor transcripts, which is essential for appropriate patient management and monitoring 1.