Quality assurance in RT-PCR-based BCR/ABL diagnostics – results of an interlaboratory test and a standardization approach

Prospective BCR-ABL analysis by polymerase chain reaction (RT-PCR) in adult acute B-lineage lymphoblastic leukemia: reliability of RT-nested-PCR and comparison to cytogenetic data

“…Only few t(9;22) translocations detectable by cytogenetic analysis were missed by RT-nested-PCR, although others reported up to 14% false negative PCR products. 12,20 Our study finally showed ambiguous RT-PCR results in 3% of all cases, but initially had up to 24% false positives in the first RT-nested-PCR (153/640) despite scrupulous precautions being taken to minimize contaminations. These data are in accordance with a recent interlaboratory test for quality assurance of BCR-ABL RT-PCR, which detected false positive results in 30% of the 20 participating laboratories.…”

“…These data are in accordance with a recent interlaboratory test for quality assurance of BCR-ABL RT-PCR, which detected false positive results in 30% of the 20 participating laboratories. 20 Thus, the possibility of a false positive result is a major concern in ALL patients due to the extraordinary sensitivity of BCR-ABL RT-PCR 33 and RT-PCR protocols are recommended to include a number of specific procedures to decrease PCR product carryover of amplified DNA. 34 In our experience, at least a second RT-nested-PCR or RT-single-step-PCR is warranted to guarantee reliable conclusions from the highly sensitive BCR-ABL RT-PCR approach.…”

Prophylactic transfer of BCR-ABL–, PR1-, and WT1-reactive donor T cells after T cell–depleted allogeneic hematopoietic cell transplantation in patients with chronic myeloid leukemia

“…For all samples, real-time quantitative PCR was performed using the published EAC quantitative PCR primers and probes, 31 with minor modifications and detection on an ABI7500 system (Applied Biosystems). In brief, white blood cells were obtained after erythrocyte lysis from peripheral blood and bone marrow samples, with approximately 5 ϫ 10 5 to 1 ϫ 10 7 cells being used for RNA extraction by the RNeasy Blood kit (QIAGEN).…”

Early reduction of BCR-ABL mRNA transcript levels predicts cytogenetic response in chronic phase CML patients treated with imatinib after failure of interferon α

“…3 This discrepancy may be due to different technologies used. In addition to RT-PCR strategies, which are widely comparable 25 and standardized, 26 preanalytical conditions such as the amount of PB and number of leukocytes processed, methods of RNA extraction and cDNA synthesis are considered crucial for the final sensitivity of the assay. Thus, we analyzed cell lysates derived from 20 ml PB corresponding to 6 × 10 7 nucleated cells in a patient in good hematologic response with leukocyte counts of 3 000/l.…”