PB2202 VALIDATION OF THE PRIMERDESIGN® QUANTITATIVE ALLELE SPECIFIC AMPLIFICATION KIT FOR THE DETECTION OF THE JAK2V617F MUTATION

Abstract

Background:Janus Kinase 2 (JAK2) is part of a group of tyrosine kinases which are involved in cellular signalling. A point mutation in codon 617 of the JAK2 gene (JAK2 V617F) causes a ‘gain of function’ effect of this process, stimulating proliferation of the myeloid cell lines, including erythrocytes and platelets, as well as causing myelofibrosis in the bone marrow. Testing for this specific mutation using an allele‐specific PCR kit at our facility (NHLS‐TAD), Pretoria] should provide quick and reliable results to investigate suspected myeloproliferative neoplasms. As part of method comparison, 15 specimens known to be positive for the JAK2V617F mutation and 19 specimens known to be JAK2 negative, as previously assessed using PCR with melting curve analysis at Charlotte Maxeke Johannesburg Academic Hospital, will be analysed using the Primerdesign Quasa test kit on the BioRad CFX 96 platform. Results of the two PCR tests will be compared to evaluate accuracy of the kit.Aims:To validate the Primerdesign® quantitative allele specific amplification (Quasa) kit for detection of the JAK2V617F mutation in DNA extracted from whole blood samples.Methods:Patients’ samples, previously tested at the National Health Laboratory Service laboratory, Charlotte Maxeke Johannesburg Academic Hospital, will be used. These comprise both whole blood collected in EDTA as well as genomic DNA. 15 specimens known to be positive for the JAK2V617F mutation as well as 19 JAK2 negative specimens will be analysed as part of method comparison. We have chosen a sample size of 34 due to the substantial cost of each kit and we are limited due to financial constraints. The WHO international reference panel for genomic JAK2V617F with proposed percentages of 0, 0.03, 1, 10.8, 29.6, 89.5 and 100% will be used for quantitative analysis of the kit. In addition, we will be analysing a diluted JAK2V617F WHO sample to confirm the manufacturer's claim of an analytical sensitivity of 0.1%. Specimens already analysed will be re‐tested using the Primerdesign® quasa kit on the CFX96 real‐time PCR platform (Bio Rad).Results:There is a 95% concordance between the results obtained from the index test (Primerdesign kit) when compared to the test of reference (LightCycler). Precision includes repeatability and reproducibility, both of which were shown in this study. We were able to detect 0.1% mutant DNA in background wild type DNA as claimed by the manufacturer. Therefore the analytical sensitivity was at 0.1%. The ability of the test to not detect the mutation in a known negative sample was at 100% concordance. Therefore analytical specificity was 100%Summary/Conclusion:The Primerdesign allele specific kit has the capability of being able to produce qualitative as well as quantitative results based on the manufacturer's instructions. Being able to provide this diagnostic test in our department will decrease turn‐around time and improve patient care.