CML molecular monitoring standardisation for Latin America
medwireNews: Using secondary reference biological calibrators based on the World Health Organization (WHO) primary standards may aid chronic myeloid leukaemia (CML) molecular monitoring in Latin America, research suggests.
The WHO created the International Genetic Reference Panel for quantification of BCR–ABL1 using quantitative reverse transcription–polymerase chain reaction (RT–qPCR) but stock of this primary standard is limited, explain Michele Bianchini, from Instituto Alexander Fleming in Buenos Aires, Argentina, and co-workers.
“Our results, together with those recently reported by Cross et al., substantiate the objective initially set during the establishment of the WHO primary standards, that is, to facilitate worldwide diffusion of the IS [International Scale]”, they write in a letter to Leukemia.
The team based in Buenos Aires used a reference sample from another laboratory to derive the laboratory-specific conversion factor (CF) necessary to convert a PCR result to the IS, reaching a figure of 0.7.
The researchers then developed and validated secondary reference materials using lyophilized mixes of Philadelphia chromosome-positive and negative cell lines with an assigned IS% value for each batch. Stability studies showed that the BCR–ABL1 to ABL1 ratio was stable for up to 6 months across a range of temperatures.
These calibrated secondary reference standards were sent to 18 laboratories in seven Latin–American countries and assessed in four independent runs on different days.
Results from the 1312 RT-qPCR assays gave average raw percentage ratios consistent within a 10-fold serial dilution that were linear for the first four of the five levels. The assays also have “uniform” bias, allowing calculation of a valid CF that nullified the residual mean bias.
The laboratories also used raw BCR–ABL1 to ABL1 ratios to demonstrate a corresponding level-specific coefficient of variation of between 8% and 58%, with 12 of the laboratories achieving a coefficient of less than 30%.
The authors note that BCR–ABL1 in the lowest positive samples – the fifth calibrator – was inconsistently detected, at a rate of 12.5–100.0% across the 10 participating laboratories; just eight sites could reproducibly detect BCR–ABL1 in the fifth calibrator samples.
The researchers highlight the importance of confirming laboratory agreement after IS conversion. They swapped 41 whole blood samples from CML patients with one of the participating laboratories. The concordance in molecular response IS between the reference and external laboratory was 88% after conversion.
“For the first time in Latin America, this study provides a platform on which to assess the performance of distinct clinical BCR-ABL1 tests and confirm the utility of secondary reference materials to further improve IS accuracy and inter-laboratory precision”, Bianchini et al write.
“This effort will continue in the future by providing secondary reference material to the centers involved in this project and potential new participants; moreover, due to its higher precision and absolute quantification capability, we are evaluating the possibility of including digital PCR as the calibration method for the future.”
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