The performance of human cytomegalovirus digital PCR reference measurement procedure in seven external quality assessment schemes over four years

A candidate digital PCR (dPCR)-based reference measurement procedure for quantification of human cytomegalovirus (hCMV) was evaluated in 10 viral load comparison schemes (seven external quality assessment (EQA) and three additional training schemes) organized by INSTAND e.V. over four years (between September 2014 and March 2018). Four metrology institutes participated in these schemes using the same extraction method and dPCR measurement procedure for the hCMV specific target sequence of UL54 gene. The calibration independent reference measurement procedure results from the metrology institutes were compared to the results of the clinical diagnostic laboratories applying hCMV qPCR measurement procedures calibrated to reference materials. While the criteria for the acceptable deviation from the target value interval for INSTAND's EQA schemes is from-0.8 log10 to +0.8 log(10), the majority of dPCR results were between-0.2 log10 to +0.2 log(10). Only 4 out of 45 results exceeded this interval with the maximum deviation of-0.542 log(10). In the training schemes containing samples with lower hCMV concentrations, more than half of the results deviated less than +/- 0.2 log(10) from the target value, while more than 95% deviated less than +/- 0.4 log(10) from the target value. Evaluation of intra-and inter-laboratory variation of dPCR results confirmed high reproducibility and trueness of the method. This work demonstrates that dPCR has the potential to act as a calibration independent reference measurement procedure for the value assignment of hCMV calibration and reference materials to support qPCR calibration as well as ultimately for routine hCMV load testing. This study was financially supported by the INFECTMET project (the EMRP project; jointly funded by the EMRP participating countries within EURAMET and the European Union, project no. HLT08); AntiMicroResist project (which has received funding from the EMPIR program co-financed by the Participating States and from the European Union’s Horizon 2020 research and innovation program, project no. 15HLT07); the Slovenian Research Agency (contract no. P4-0165 and 1000-13-0105); Ministry of Education, Science and Sport of republic of Slovenia (contract no. 3330-14-500133) and supported by the UK National Measurement System. The dPCR equipment used at NIB in this study was financed by the Metrology Institute of the Republic of Slovenia (MIRS), with financial support from the European Regional Development Fund. The funding source(s) had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. This publication reflects only the authors’ s views and EURAMET is not responsible for any use that may be made of the information it contains.