Background: Nucleic acid amplification tests (NAATs) assist in the diagnosis of numerous infectious diseases. They are typically sensitive and specific and can be quickly developed and adapted. Far more challenging is the development of standards to ensure NAATs are performing within specification; reference materials take time to develop and suitable reference measurement procedures (RMPs) have not been available. This study investigated digital PCR (dPCR) RMP delivery of traceability for NAAT external quality assessment (EQA). Methods: Three National Metrology Institutes (NMIs) applied reverse transcription (RT)-dPCR as a candidate RMP to estimate the RNA quantity in 32 independent severe acute respiratory syndrome coronavirus 2 materials. The results were combined to value assign the respective materials: 21 materials were used in 6 rounds of EQA over 17 months for 61 laboratories for COVID-19 testing results compared with reference values. Results: The agreement between the 3 NMIs showed <2-fold difference between laboratories. EQA laboratory reverse transcription quantitative PCR (RT-qPCR) values estimation of viral RNA quantity showed good median agreement with RT-dPCR reference value; however, RT-qPCR differences were generally between 10- and 50-fold between laboratories. Conclusion: This work demonstrates how RT-dPCR can provide reference values for whole virus materials for NAAT quality assurance. RT-dPCR values guided EQA control material selection and provided EQA participants with traceability to RNA copy number delivered through the RMP. This approach can be used to support routine reference material use as well as to standardize quality assurance for NAATs where established reference materials are not available, such as in disease outbreaks.

The glycoproteins alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) have long been approved as biomarkers for diagnosing and monitoring tumors. International Reference Preparations (IRPs) have been around since 1975. Nevertheless, manufacturer-dependent differences have been reported, indicating a lack of harmonization. This paper analyzes data from 15 external quality assessment (EQA) surveys conducted worldwide between 2018 and 2022. The aim was to gain insight into the longitudinal development of manufacturer-dependent differences for CEA and AFP. In each survey, participating laboratories received two samples with different tumor marker levels. Inter- and intra-assay variability was analyzed and the mean 80% and 90% of the manufacturer collectives were compared to the evaluation criteria of the German Medical Association (RiliBÄK). The median EQA results for CEA revealed manufacturer-dependent differences between the highest and lowest collective of up to 100%; for AFP, the median differences mostly remained below 40%. The coefficients of variation were predominantly low for both markers. We concluded that the current assays for AFP and CEA detection are better harmonized than previously reported. The assays displayed a good robustness; however, a narrowing of the current assessment limits in EQA schemes could further enhance the quality of laboratory testing.

As hormonal disorders are linked to several diseases, the accurate quantitation of steroid hormone levels in serum is crucial in order to provide patients with a reliable diagnosis. Mass spectrometry-based methods are regarded as having the highest level of specificity and sensitivity. However, immunoassays are more commonly used in routine diagnostics to measure steroid levels as they are more cost effective and straightforward to conduct. This study analyzes the external quality assessment results for the measurement of testosterone, progesterone and 17β-estradiol in serum using immunoassays between early 2020 and May 2022. As reference measurement procedures are available for the three steroid hormones, the manufacturer-specific biases were normalized to the reference measurement values. The manufacturer-specific coefficients of variation were predominantly inconspicuous, below 20% for the three hormones when outliers are disregarded, however there were large differences between the various manufacturer collectives. For some collectives, the median bias to the respective reference measurement value was repeatedly greater than ±35%, which is the acceptance limit defined by the German Medical Association. In the case of testosterone and progesterone determination, some collectives tended to consistently over- or underestimate analyte concentrations compared to the reference measurement value, however, for 17β-estradiol determination, both positive and negative biases were observed. This insufficient level of accuracy suggests that cross-reactivity continues to be a fundamental challenge when antibody detection is used to quantify steroids with a high structural similarity. Distinct improvements in standardization are required to provide accurate analysis and thus, reliable clinical interpretations. The increased accuracy of the AX immunoassay for testosterone measurement, as observed in the INSTAND EQAs between 2020 and 2022, could be the result of a recalibration of the assay and raises hope for further improvement of standardization of immunoassay-based steroid hormone analyses in the coming years.

In May 2022, the monkeypox virus (MPXV) spread into non-endemic countries and the global community was quick to test the lessons learned from the SARS-CoV-2 pandemic. Due to its symptomatic resemblance to other diseases, like the non-pox virus varicella zoster (chickenpox), polymerase chain reaction methods play an important role in correctly diagnosing the rash-causing pathogen. INSTAND quickly established a new external quality assessment (EQA) scheme for MPXV and orthopoxvirus (OPXV) DNA detection to assess the current performance quality of the laboratory tests. We analyzed quantitative and qualitative data of the first German EQA for MPXV and OPXV DNA detection. The survey included one negative and three MPXV-positive samples with different MPX viral loads. The threshold cycle (Ct) or other measures defining the quantification cycle (Cq) were analyzed in an assay-specific manner. A Passing Bablok fit was used to investigate the performance at laboratory level. 141 qualitative datasets were reported by 131 laboratories for MPXV detection and 68 qualitative datasets by 65 laboratories for OPXV detection. More than 96% of the results were correctly identified as negative and more than 97% correctly identified as positive. An analysis of the reported Ct/Cq values showed a large spread of these values of up to 12 Ct/Cq. Nevertheless, there is a good correlation of results for the different MPXV concentrations at laboratory level. Only a few quantitative results in copies/mL were reported (MPXV: N = 5; OPXV: N = 2), but the results correlated well with the concentration differences between the EQA samples, which were to a power of ten each. The EQA results show that laboratories performed well in detecting both MPXV and OPXV. However, Ct/Cq values should be interpreted with caution when conclusions are drawn about the viral load as long as metrological traceability is not granted.

The COVID-19 pandemic illustrated the important role of diagnostic tests, including lateral flow tests (LFTs), in identifying patients and their contacts to slow the spread of infections. INSTAND performed external quality assessments (EQA) for SARS-CoV-2 antigen detection with lyophilized and chemically inactivated cell culture supernatant of SARS-CoV-2 infected Vero cells. A pre-study demonstrated the suitability of the material. Participants reported qualitative and/or quantitative antigen results using either LFTs or automated immunoassays for five EQA samples per survey. 711 data sets were reported for LFT detection in three surveys in 2021. This evaluation focused on the analytical sensitivity of different LFTs and automated immunoassays. The inter-laboratory results showed at least 94% correct results for non-variant of concern (VOC) SARS-CoV-2 antigen detection for viral loads of ≥ 4.75 × 10⁶ copies/mL and SARS-CoV-2 negative samples. Up to 85% had success for a non-VOC viral load of ~ 1.60 × 10⁶ copies/mL. A viral load of ~ 1.42 × 10⁷ copies/mL of the Delta VOC was reported positive in > 96% of results. A high specificity was found with almost 100% negative SARS-CoV-2 antigen results for HCoV 229E and HCoV NL63 positive samples. Quantitative results correlated with increasing SARS-CoV-2 viral load but showed a broad scatter. This study shows promising SARS-CoV-2 antigen test performance of the participating laboratories, but further investigations with the now predominant Omicron VOC are needed.