Investigating the Current Harmonization Status of Tumor Markers Using Global External Quality Assessment Programs: A Feasibility Study.

BACKGROUND: The harmonization status of most tumor markers (TMs) is unknown. We report a feasibility study performed to determine whether external quality assessment (EQA) programs can be used to obtain insights into the current harmonization status of the tumor markers α-fetoprotein (AFP), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), cancer antigen (CA)125, CA15-3 and CA19-9. METHODS: EQA sample results provided by 6 EQA providers (INSTAND [Germany], Korean Association of External Quality Assessment Service [KEQAS, South Korea], National Center for Clinical Laboratories [NCCL, China], United Kingdom National External Quality Assessment Service [UK NEQAS, United Kingdom], Stichting Kwaliteitsbewaking Medische Laboratoriumdiagnostiek [SKML, the Netherlands], and the Royal College of Pathologists of Australasia Quality Assurance Programs [RCPAQAP, Australia]) between 2020 and 2021 were used. The consensus means, calculated from the measurement procedures present in all EQA programs (Abbott Alinity, Beckman Coulter DxI, Roche Cobas, and Siemens Atellica), was used as reference values. Per measurement procedure, the relative difference between consensus mean for each EQA sample and the mean of all patient-pool-based EQA samples were calculated and compared to minimum, desirable, and optimal allowable bias criteria based on biological variation. RESULTS: Between 19040 (CA15-3) and 25398 (PSA) individual results and 56 (PSA) to 76 (AFP) unique EQA samples were included in the final analysis. The mean differences with the consensus mean of patient-pool-based EQA samples for all measurement procedures were within the optimum bias criterion for AFP, the desirable bias for PSA, and the minimum bias criterion for CEA. However, CEA results <8 µg/L exceeded the minimum bias criterion. For CA125, CA15-3, and CA19-9, the harmonization status was outside the minimum bias criterion, with systematic differences identified. CONCLUSIONS: This study provides relevant information about the current harmonization status of 6 tumor markers. A pilot harmonization investigation for CEA, CA125, CA15-3, and CA19-9 would be desirable.

Results of German external quality assessment schemes for SARS-CoV-2 antigen detection.

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.

Classification of "Near-patient" and "Point-of-Care" SARS-CoV-2 Nucleic Acid Amplification Test Systems and a first approach to evaluate their analytical independence of operator activities.

BACKGROUND: European legislation defines as "near-patient testing" (NPT) what is popularly and in other legislations specified as "point-of-care testing" (POCT). Systems intended for NPT/POCT use must be characterized by independence from operator activities during the analytic procedure. However, tools for evaluating this are lacking. We hypothesized that the variability of measurement results obtained from identical samples with a larger number of identical devices by different operators, expressed as the method-specific reproducibility of measurement results reported in External Quality Assessment (EQA) schemes, is an indicator for this characteristic. MATERIALS AND METHODS: Legal frameworks in the EU, the USA and Australia were evaluated about their requirements for NPT/POCT. EQA reproducibility of seven SARS-CoV-2-NAAT systems, all but one designated as "POCT", was calculated from variabilities in Ct values obtained from the respective device types in three different EQA schemes for virus genome detection. RESULTS: A matrix for characterizing test systems based on their technical complexity and the required operator competence was derived from requirements of the European In Vitro Diagnostic Regulation (IVDR) 2017/746. Good EQA reproducibility of the measurement results of the test systems investigated implies that different users in different locations have no recognizable influence on their measurement results. CONCLUSION: The fundamental suitability of test systems for NPT/POCT use according to IVDR can be easily verified using the evaluation matrix presented. EQA reproducibility is a specific characteristic indicating independence from operator activities of NPT/POCT assays. EQA reproducibility of other systems than those investigated here remains to be determined.

Longitudinal Evaluation of AFP and CEA External Proficiency Testing Reveals Need for Method Harmonization.

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.

Results of the first German external quality assessment scheme for the detection of monkeypox virus DNA.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Comparison of fecal calprotectin and pancreatic elastase assays based on proficiency testing results.

BACKGROUND: Fecal calprotectin and fecal pancreatic elastase assays are not standardized because of a lack of suitable reference material. Laboratories may have difficulty in switching assays because different manufacturers do not compare well with each other despite having similar reference intervals. Data from proficiency testing performed in Germany (Fecal Diagnostics 01 Survey, INSTAND eV) were investigated to understand how results differed across eight calprotectin and five pancreatic elastase manufacturers. METHODS: Data were collected from participating laboratories in external quality assessment schemes from 2015 to 2020 for calprotectin and 2017 to 2020 for pancreatic elastase. The manufacturer group mean values and standard deviations were calculated. Reference points were created for each external quality assessment scheme by calculating the average of all manufacturer group means. Deming regression analyses were used to observe the differences across manufacturers. RESULTS: The slopes of the Deming regression spanned 0.37-1.91 for calprotectin and 0.84-1.33 for pancreatic elastase. The calprotectin assays had a high degree of variability in quantitative results by manufacturer. However, pancreatic elastase assays appear to be harmonized across the different manufacturer when considering the qualitative interpretation. CONCLUSIONS: Both calprotectin and pancreatic elastase assays could be improved by standardization efforts. Given the clinical utility and our data demonstrating high inter-manufacturer variability, calprotectin should be prioritized over pancreatic elastase in standardization efforts.

Epidemiology, Management, Quality of Testing and Cost of Syphilis in Germany: A Retrospective Model Analysis.

Background: A multi-dimensional model can be a useful tool for estimating the general impact of disease on the different sectors of the healthcare system. We chose the sexually transmitted disease syphilis for our model due to the good quality of reported data in Germany. Methods: The model included gender- and age-stratified incident cases of syphilis (in- and outpatients) provided by a German statutory health insurance company, as well as seroprevalence data on syphilis in first-time blood donors. Age standardized rates were calculated based on the standard German population. The test quality was assessed by extrapolating the number of false-positive and false-negative results based on data from Europe-wide external quality assessment (EQA) schemes. The model analysis was validated with the reported cases and diagnosis-related group (DRG)-statistics from 2010 to 2012. The annual direct and indirect economic burden was estimated based on the outcomes of our model. Results: The standardized results were slightly higher than the results reported between 2010 and 2012. This could be due to an underassessment of cases in Germany or due to limitations of the dataset. The number of estimated inpatients was predicted with an accuracy of 89.8 %. Results from EQA schemes indicated an average sensitivity of 92.8 % and an average specificity of 99.9 % for the recommended sequential testing for syphilis. Based on our model, we estimated a total average minimal annual burden of €20,292,110 for syphilis on the German healthcare system between 2010 and 2012. Conclusions: The linking of claims data, results from EQA schemes, and blood donor surveillance can be a useful tool for assessing the burden of disease on the healthcare system. It can help raise awareness in populations potentially at risk for infectious diseases, demonstrate the need to educate potential risk groups, and may help with predictive cost calculations and planning.

RNA reference materials with defined viral RNA loads of SARS-CoV-2-A useful tool towards a better PCR assay harmonization.

SARS-CoV-2, the cause of COVID-19, requires reliable diagnostic methods to track the circulation of this virus. Following the development of RT-qPCR methods to meet this diagnostic need in January 2020, it became clear from interlaboratory studies that the reported Ct values obtained for the different laboratories showed high variability. Despite this the Ct values were explored as a quantitative cut off to aid clinical decisions based on viral load. Consequently, there was a need to introduce standards to support estimation of SARS-CoV-2 viral load in diagnostic specimens. In a collaborative study, INSTAND established two reference materials (RMs) containing heat-inactivated SARS-CoV-2 with SARS-CoV-2 RNA loads of ~107 copies/mL (RM 1) and ~106 copies/mL (RM 2), respectively. Quantification was performed by RT-qPCR using synthetic SARS-CoV-2 RNA standards and digital PCR. Between November 2020 and February 2021, German laboratories were invited to use the two RMs to anchor their Ct values measured in routine diagnostic specimens, with the Ct values of the two RMs. A total of 305 laboratories in Germany were supplied with RM 1 and RM 2. The laboratories were requested to report their measured Ct values together with details on the PCR method they used to INSTAND. This resultant 1,109 data sets were differentiated by test system and targeted gene region. Our findings demonstrate that an indispensable prerequisite for linking Ct values to SARS-CoV-2 viral loads is that they are treated as being unique to an individual laboratory. For this reason, clinical guidance based on viral loads should not cite Ct values. The RMs described were a suitable tool to determine the specific laboratory Ct for a given viral load. Furthermore, as Ct values can also vary between runs when using the same instrument, such RMs could be used as run controls to ensure reproducibility of the quantitative measurements.

Evaluation of INSTAND e.V.'s external proficiency testing program for tetanus and diphtheria antitoxin detection: Lessons for assessing levels of immunoprotection.

OBJECTIVES: The aim of this study was to evaluate the development and status quo of the quality of high throughput in vitro diagnostic testing for tetanus and diphtheria antitoxin antibody (ATX) concentrations based on external quality assessment (EQA) data. METHODS: We analyzed manufacturer-specific data of 22 EQA surveys-each for the detection of tetanus and diphtheria ATX-to check the diagnostic strength of the corresponding in vitro diagnostic systems. RESULTS: While the results were mostly well aligned, individual surveys showed widely dispersed ATX concentrations. The medians of manufacturer collectives deviated from the overall median by up to 8.9-fold in the case of diphtheria ATX and by up to 3.5-fold in the case of tetanus ATX. Such a distribution in the results is particularly critical in the cut-off range for immunity and may lead to an incorrect assessment of vaccination status. CONCLUSION: These results were surprising as there are International Standards for both ATX; however, the results may be linked to the high ATX concentration of the reference material, which deviates considerably from clinically significant concentrations. To increase the accuracy and diagnostic strength of both assays, we recommend a recalibration of the test systems and verification of their traceability to the International Standards.

Evaluation of INSTAND e.V.'s external quality assessment for C-reactive protein and procalcitonin.

BACKGROUND: The purpose of this paper was to analyze the general diagnostic strength and performance of in vitro diagnostics for C-reactive protein and procalcitonin based on the results of external quality assessment schemes (EQAs). METHODS: We analyzed qualitative and quantitative data on both markers collected by the Society for Promotion Quality Assurance in Medical Laboratories (INSTAND e.V.) from 20 EQAs. The C-reactive protein evaluation was method-specific and the procalcitonin evaluation manufacturer-specific (pseudonymized). Coefficients of variation were determined in order to evaluate interlaboratory comparability and the performance of individual laboratories during the analyzed period was examined. RESULTS: Overall most of our participants were able to correctly distinguish the positive from the negative samples, but we occasionally observed also false-positive results for the immunological detection of C-reactive protein. For the semi-quantitative results of C-reactive protein we observed an overall median difference below 5% except for dry chemistry methods (≤ 21%). For procalcitonin two manufacturer collectives showed a good comparability, while one manufacturer detected up to 42% higher results. The coefficients of variation are promising for both analytes even though they surpass the manufacturer's indication for some collectives. The performance of individual laboratories during the analyzed period was more stable for C-reactive protein than for procalcitonin. CONCLUSION: In-vitro diagnostic testing for C-reactive protein and procalcitonin showed promising results in our EQAs but still further improvements are needed. We recommend stepping up research on reference measurement methods for both parameters to possibly enhancing the accuracy and diagnostic strength of such assays.

Evaluation of the manufacturer-dependent differences in specific immunoglobulin E results for indoor allergens.

BACKGROUND: Even though allergies are an important health issue, the detected amount of allergen-specific IgE (sIgE) has differed widely between manufacturers in the past, and even as recently as this year. These discrepancies hinder diagnostics and can even impact allergen immunotherapy. OBJECTIVE: The aim of this study was to evaluate the development and status quo of the quality of in vitro diagnostic testing for house dust mites (HDM) and cat epithelium, 2 important indoor allergen sources. METHODS: We analyzed data on the allergen sources European HDM, American HDM, and cat epithelium, which were collected by the Society for Promoting Quality Assurance in Medical Laboratories (INSTAND e.V.) during 7 years as part of External Quality Assessment schemes (EQAs). A pseudoanonymized comparison was made of the semiquantitative data and allergen-class results of the 4 main suppliers of in vitro diagnostic sIgE tests. Coefficients of variation (CV) were determined in order to evaluate interlaboratory comparability. RESULTS: In vitro allergy diagnostic testing for the major allergen sources HDM and cat epithelium still reveals manufacturer-dependent differences. Despite this, a cautious trend was found towards an alignment of the results and interlaboratory comparability, with the exception of 1 supplier. CONCLUSION: Even though these results are promising, future EQAs have to be closely monitored to ensure this positive trend is not just a snapshot.

New insights in the composition of extracellular vesicles from pancreatic cancer cells: implications for biomarkers and functions.

BACKGROUND: Pancreatic cancer development is associated with characteristic alterations like desmoplastic reaction and immune escape which are mediated by the cell-cell communication mechanism and by the microenvironment of the cells. The whole of released components are important determinants in these processes. Especially the extracellular vesicles released by pancreatic cancer cells play a role in cell communication and modulate cell growth and immune responses. RESULTS: Here, we present the proteomic description of affinity purified extracellular vesicles from pancreatic tumour cells, compared to the secretome, defined as the whole of the proteins released by pancreatic cancer cells. The proteomic data provide comprehensive catalogues of hundreds of proteins, and the comparison reveals a special proteomic composition of pancreatic cancer cell derived extracellular vesicles. The functional analysis of the protein composition displayed that membrane proteins, glycoproteins, small GTP binding proteins and a further, heterogeneous group of proteins are enriched in vesicles, whereas proteins derived from proteasomes and ribosomes, as well as metabolic enzymes, are not components of the vesicles. Furthermore proteins playing a role in carcinogenesis and modulators of the extracellular matrix (ECM) or cell-cell interactions are components of affinity purified extracellular vesicles. CONCLUSION: The data deepen the knowledge of extracellular vesicle composition by hundreds of proteins that have not been previously described as vesicle components released by pancreatic cancer cells. Extracellular vesicles derived from pancreatic cancer cells show common proteins shared with other vesicles as well as cell type specific proteins indicating biomarker candidates and suggesting functional roles in cancer cell stroma interactions.

A soluble form of the giant cadherin Fat1 is released from pancreatic cancer cells by ADAM10 mediated ectodomain shedding.

In pancreatic cancer, there is a clear unmet need to identify new serum markers for either early diagnosis, therapeutic stratification or patient monitoring. Proteomic analysis of tumor cell secretomes is a promising approach to indicate proteins released from tumor cells in vitro. Ectodomain shedding of transmembrane proteins has previously been shown to contribute significant fractions the tumor cell secretomes and to generate valuable serum biomarkers. Here we introduce a soluble form of the giant cadherin Fat1 as a novel biomarker candidate. Fat1 expression and proteolytic processing was analyzed by mass spectrometry and Western blotting using pancreatic cancer cell lines as compared to human pancreatic ductal epithelial cells. RNA expression in cancer tissues was assessed by in silico analysis of publically available microarray data. Involvement of ADAM10 (A Disintegrin and metalloproteinase domain-containing protein 10) in Fat1 ectodomain shedding was analyzed by chemical inhibition and knockdown experiments. A sandwich ELISA was developed to determine levels of soluble Fat1 in serum samples. In the present report we describe the release of high levels of the ectodomain of Fat1 cadherin into the secretomes of human pancreatic cancer cells in vitro, a process that is mediated by ADAM10. We confirm the full-length and processed heterodimeric form of Fat1 expressed on the plasma membrane and also show the p60 C-terminal transmembrane remnant fragment corresponding to the shed ectodomain. Fat1 and its sheddase ADAM10 are overexpressed in pancreatic adenocarcinomas and ectodomain shedding is also recapitulated in vivo leading to increased Fat1 serum levels in some pancreatic cancer patients. We suggest that soluble Fat1 may find an application as a marker for patient monitoring complementing carbohydrate antigen 19-9 (CA19-9). In addition, detailed analysis of the diverse processed protein isoforms of the candidate tumor suppressor Fat1 can also contribute to our understanding of cell biology and tumor behavior.