Development and evaluation of a reagent set for quantitation of mRNA expression level of chimeric BCR::ABL1 gene

Introduction . Chronic myeloid leukemia is a myeloproliferative disease associated with a t(9;22)(q34;q11) translocation resulting in a chimeric BCR::ABL1 gene. Molecular genetic studies are used as a diagnostic method and for determination of minimal residual disease by quantification of the mRNA expression level of the chimeric BCR::ABL1 gene. Objective. To validate the developed multiplex test system for simultaneous quantitative determination of e13a2 (b2a2) and e14a2 (b3a2) transcripts of translocation p210 in comparison with the analog system registered in Russia. Materials and methods . In total, 50 peripheral blood samples were used. Of these, 39 belonged to patients diagnosed with CML; 11 blood samples from healthy people were used to confirm analytical specificity. The reagent kit includes the specifically developed primers and fluorescently labeled probes, the concentration of which was selected experimentally, as well as original reagents manufactured by the Centre for Strategic Planning, Federal Medical and Biological Agency (Russia). Statistical analysis was performed using the StatTech v.4.5.0 software (developed by StatTech, Russia). Differences were considered significant at p < 0.05. Results . The conducted analytical and diagnostic characterization of the developed kit showed the following results. The relative sensitivity determined using cell standards was 0.01 %. The reproducible analytical sensitivity was 100 copies/mL (CV = 1.86 %). In analytical specificity testing, the absence of false results was shown. When testing the reagents of the test system on clinical samples, the complete convergence (100 %) with the results obtained using the reference kit was revealed. Conclusions . Approbation of the developed reagent set showed its sufficiently high analytical sensitivity and diagnostic sensitivity. This set can be used for identification and quantitative determination of the mRNA of the chimeric BCR::ABL1 gene for the purposes of diagnosis, timely and accurate therapy, and monitoring of minimal residual disease.