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Clinical characteristics and treatment outcome of pediatric patients with chronic myeloid leukemia

Diagnostic process and molecular analysis methodology for detection and quantification of the BCR/ABL gene rearrangement. The diagnosis for the 12 patients was made on evaluation of the bone marrow aspirate and trephine biopsy. Bone marrow samples were subjected to routine Hematoxylin-Eosin (H&E) and immunohistochemical staining in addition to flow-cytometry to determine the immunophenotypes of the cells. While this was suggestive of the diagnosis, confirmation of Ph+ chronic myeloid leukemia was made only after availability of cytogenetic and/or molecular genetic studies that showed the presence of the characteristic t(9;22)(q34;q11) translocation involving the BCR and ABL genes. Molecular analysis for BCR/ABL gene rearrangement was performed on extracted RNA which is subjected to real-time reverse transcription-polymerase chain reaction (RT-PCR). The generated cDNA is amplified with forward primer located in the BCR exon b2 to combine with the reverse primer in the ABL exon a4. The fluorescent hybridization probe pair is located in the ABL exon a3. The location of primers and hybridization probes also allows the amplification and detection of the b2a2 and b3a2 fusion transcripts. The amount of fluorescence resulting from the two probes is proportional to the amount of PCR product. The method uses 5 standards of 10 to10 dilution of k562 RNA in normal RNA which is diluted fresh and undergoes RT in each run. The limit of detection is 1/10000 and any result <1/10000 undergoes nested PCR before being considered negative. The sensitivity of the nested PCR is expected to be at the 10-10 level. Clinical characteristics and treatment outcome of pediatric patients with chronic myeloid leukemia