Cautions

This assay is comprehensive for detecting BCR::ABL1 kinase domain (KD) mutations but does not detect all possible mutations in ABL1; therefore, a negative result by this assay does not exclude the presence of a rare, less-well characterized, or unknown mutation that could be associated with some degree of tyrosine kinase inhibitor resistance. The clinical significance of such rarely occurring mutations is, however, uncertain.

The quantitative level of BCR::ABL1 transcript is critical for a successful assay mutation analysis because the amplification efficiency for a longer messenger RNA (mRNA) template is decreased with a low abundance of target. If the BCR::ABL1 quantitative polymerase chain reaction (PCR) level is too low, reverse transcription-PCR amplification of BCR::ABL1 may be unsuccessful to yield product for sequencing. Although laboratory standards are yet to be developed, a BCR::ABL1/ABL1 quantitative level above 0.1% is generally considered to be required to detect KD mutations by this assay.

Subclonal mutations may be difficult to identify by Sanger sequencing method, even if the BCR::ABL1 mRNA amplification was successful. This is due to the inherit sensitivity level limit of sequencing, which is typically around 15% to 20% mutant allele in a wild-type background.

EDTA blood specimens are preferred for testing. Bone marrow specimens are acceptable; there occasionally are specimen failures from bone marrow RNA, for reasons that are not completely understood. Heparin anticoagulant cannot be used due to PCR inhibition.

Assay precision does not appear to be significantly affected by specimen transport or moderate delays in processing. However, in specimens with lower levels of BCR::ABL, these conditions may cause sufficient RNA degradation to produce false-negative results. Thus, specimens should be shipped as quickly as possible. Ambient specimens over 3 days old and refrigerate specimens over 5 days old at the time of receipt are unacceptable.