International external quality assurance of JAK2 V617F quantification

Abstract

External quality assurance (EQA) programs are vital to ensure high quality and standardized results in molecular diagnostics. It is important that EQA for quantitative analysis takes into account the variation in methodology. Results cannot be expected to be more accurate than limits of the technology used, and it is essential to recognize factors causing substantial outlier results. The present study aimed to identify parameters of specific importance for JAK2 V617F quantification by quantitative PCR, using different starting materials, assays, and technical platforms. Sixteen samples were issued to participating laboratories in two EQA rounds. In the first round, 19 laboratories from 11 European countries analyzing JAK2 V617F as part of their routine diagnostics returned results from in-house assays. In the second round, 25 laboratories from 17 countries participated. Despite variations in starting material, assay set-up and instrumentation the laboratories were generally well aligned in the EQA program. However, EQA based on a single technology appears to be a valuable tool to achieve standardization of the quantification of JAK2 V617F allelic burden.

Mathematical modelling of stem and progenitor cell dynamics during ruxolitinib treatment of patients with myeloproliferative neoplasms

“…In both the COMFORT-II and the RESPONSE studies, the JAK2 VAF was measured using qPCR methods ( 11 , 15 ). Though we do not know the exact size of the measurement errors, one study of qPCR methods has shown that for one particular set of equipment and techniques, the standard deviations of the measurements were 0.012 (1.2%) in a reference sample with a JAK2 VAF of 0.045 (4.5%) and 0.035 (3.5%) in a reference sample with a JAK2 of 0.13 (13%) ( 48 ). These standard deviations are close to the mean RMSE of the model fitted to the available data (0.0249), and thus the deviations between the model and the data are of a reasonable order of magnitude.…”

“…This convenient assumption makes the statistical analysis of the results simple (compared to the alternatives, see section S4 of the supplementary for more details), but it is hard to either verify or refute this assumption based on 3-8 data points per patient. The previously mentioned study of different qPCR methods suggests that the size of the measurement errors might depend on the true value of the of the JAK2 VAF ( 48 ). However, simple experiments using weighted least squares fitting gave almost identical results for all patients except patient 15, and therefore we have chosen to use the simpler ordinary least squares approach here.…”

Current status and achievements of Polish haemato-oncology

“…In 2015, the first results of the molecular characteristics of Polish patients were published [16]. The quality of JAK2 V617F quantification was assessed in an international study [98]. A comparison of qPCR and ddPCR sensitivity in the quantification of the JAK2 V617F mutation allele burden was performed in 2019 [99].…”

The Genetic Basis of Primary Myelofibrosis and Its Clinical Relevance

“…Notably, a JAK2 V617F qPCR assay was generally recommended because of its sensitivity and consistent performance across different qPCR platforms [ 78 , 79 , 80 ].…”