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.
Highlights
The discovery of the c.1849G>T mutation leading to the p.Val617Phe (V617F) substitution in JAK2 [1,2,3,4] has been a landmark in molecular diagnosis of the myeloproliferative neoplasms (MPN) polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF)
To identify the parameters of specific importance for causing outliers in a JAK2 V617F External quality assurance (EQA) where a quantitative value of mutation burden is determined by quantitative polymerase chain reaction (qPCR), different starting materials, different qPCR assays, and different technical platforms were included
In QA1, samples were divided into four groups based on the reference levels of JAK2 V617F as determined by ddPCR: < 2% (n = 4), 2–10% (n = 3), 10–20% (n = 2), and > 30% (n = 2)
Summary
The discovery of the c.1849G>T mutation leading to the p.Val617Phe (V617F) substitution in JAK2 [1,2,3,4] has been a landmark in molecular diagnosis of the myeloproliferative neoplasms (MPN) polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Quantification of the mutation has shown that mutation burden could reflect different subtypes of MPN. Quantification of the allelic burden in JAK2 V617F-positive patients is increasingly. It has been recommended that the assay should be sensitive enough to detect a mutant burden around 1% [10]. The combination of a sensitive detection and reproducible quantification of JAK2 V617F challenges the methodology used in a routine setting. Conventional Sanger sequencing does not show the required sensitivity in cases with low mutation burden, and methodologies involving generation sequencing are unnecessarily labor intensive and expensive for mutation detection of a single nucleotide substitution. As a step towards standardization of reliable molecular diagnostics, the European Leukemia Net (ELN) and MPN&MPNr-EuroNet have evaluated performance of different allele-specific (AS)-qPCR assays [8].
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