IntroductionThe commonly processed nature of the control materials used in the external quality assessment (EQA) of medical laboratories requires that their suitability is evaluated against native patient samples to ensure valid evaluation of EQA results across several measurement procedures (MPs). The aim of this study was to examine the extent to which EQA analyses of a small number of native human samples in a regular EQA survey can help to assess the suitability of routinely distributed processed EQA materials (EQAMs), demonstrated by testosterone, progesterone, and 17β-estradiol measurements in human sera.MethodsLaboratory results for the native and processed EQAMs were comparatively analyzed using a mixed-effects model. This estimated the sample type-specific mean natural logarithm (ln) bias to the reference measurement value and mean differences in ln bias between the two sample types, each with a 95%-confidence interval. Applying quite strict commutability criteria defined in consideration of clinical guidelines enabled the identification of analyte/reagent manufacturer pairings exhibiting only minor differences in bias between sample types. For pairings showing differences in bias between the two sample types that exceeded the commutability criterion, efforts were made to differentiate between material- and MP-related contributions to the model outcomes – referred to as non-commutability and trueness bias, respectively.ResultsStrongly scattered bias of the native EQAMs for some MPs revealed issues with measurement performances, such as inappropriate calibration or specificity. Consistent bias but distinct differences in bias between sample types indicated issues with commutability. The two-stage assessment revealed that the processed EQAMs were suitable for EQA purposes in most pairings. In two exceptions (testosterone/DG and progesterone/BE), the processed materials showed insufficient commutability. The limited number of EQAMs may result in inconclusive commutability assessments for individual analyte/MP pairings, four in this study, but allows for targeted follow-up with additional native EQAMs or comprehensive commutability studies.ConclusionCompared to conventional commutability studies, these proposed EQA comparability studies that use comparative native EQAMs offer EQA providers a more efficient and resource-conserving approach to assess the suitability and commutability of EQAMs by leveraging existing infrastructures. Nevertheless, even this more pragmatic approach remains resource-intensive and is inherently restricted to specific questions and selected analytes.

Root observation windows (RW) installed in the field provide a tool for non-destructive monitoring of root development and rhizosphere processes. However, the highly invasive installation process, which requires cutting soil profiles, may affect plant development and, ultimately, the outcome of the experiments. This study systematically compares plant development with and without the installation of RW. Using the location of a long-term field experiment, the responses of winter wheat to different intensities of tillage, N-fertilization, and use of fungicides were compared for plants grown along root windows and in undisturbed control plots. Sampling was performed during late vegetative growth, six weeks after RW installation, with comparisons of shoot and root biomass, root length, mineral nutritional status, stress-related gene expression, stress metabolites, and the composition of microbial communities reflecting rhizosphere (RH) conditions. The presence of RW did not significantly affect most of the parameters analyzed. As an exception, RW plants showed increased foliar concentrations of several nutrients (K, Mg, Ca, Cu, Mn), potentially attributable to reduced inter-plant competition because RW were installed along border rows of the experimental plots. By contrast, N-fertilization intensity and fungicide use affected plant biomass, root growth, and fungal communities. Tillage intensity primarily affected fine-root production, the composition of RH-microbial communities, and physiological stress indicators in leaf tissue. The results suggest only a limited influence on performance of winter wheat six weeks after RW installation under the selected experimental conditions, with plant responses and experimental results comparable to undisturbed controls.

Procyanidins from plant extracts demonstrate a broad spectrum of bioactive potential. In order to establish structure-activity relationships, it is necessary to fully determine the stereochemistry and substitution pattern. Phloroglucinolysis and nuclear magnetic resonance (NMR) are conventionally employed for the analysis of galloylated B-type procyanidins (PBgals). However, these methods require substantial quantities of the compound at a high degree of purity. In this work, five dimeric galloylated procyanidins isolated from Rumex obtusifolius L. roots were analyzed using a two-pronged strategy. Initially, identification and structure elucidation were performed through the implementation of a novel miniaturized enzymatic digestion with polygalacturonase to effectively remove gallic acid residues. Subsequently, the remaining degalloylated dimeric procyanidins were compared to procyanidin B1, B2, B3, B5, and B7 reference substances based on their retention times. Thus, the R/S-configurations at the C2 and C3 positions of both monomer units and the interflavan linkage were determined as either C4→C8 or C4→C6 in a single analysis. In addition, the position of galloylation was ascertained on the upper or lower unit via high-resolution MS with collision-induced dissociation (CID) and electron-activated dissociation (EAD) fragmentation. This innovative fragmentation technology has been demonstrated to preserve the labile bonds, such as the ester bond to gallic acid. Hence, it yields novel diagnostic fragments for structure elucidation. In silico fragmentation analysis revealed that the radical fragment m/z 440.0738 only fits galloylation on the upper unit, thereby validating the results of the CID. Furthermore, phloroglucinolysis was equally miniaturized and utilized as an independent method to corroborate the results of enzymatic digestion and LC-MS/MS. Using these methods, the structural configurations of the five isolated substances were successfully and consistently identified as procyanidin B2-3-O-gallate, procyanidin B1-3-O-gallate, B2-3'-O-gallate, procyanidin B7-3-O-gallate, and procyanidin B5-3'-O-gallate. To our knowledge, this is the first time that these PBgals are reported in R. obtusifolius.

IntroductionErythropoietin (EPO) is a measurand that has not yet been sufficiently standardized in laboratory medicine. This is why quality control within the framework of external quality assessment (EQA) schemes is of great importance. Therefore, the INSTAND e.V. EPO EQA was introduced to support quality assurance of medical laboratories for this measurand. The use of commutable control material (CM) enables the comparison of various measurement systems with one another and allows to assess the level of harmonization between laboratory measurements. This study assessed the suitability of different EPO CMs for use in EPO EQA schemes and the impact of sample preparation, such as dilution, spiking and lyophilization.MethodsEleven candidate CMs in both frozen and lyophilized forms were analyzed and compared to 80 clinical samples (CS) using a chemiluminescence immunoassay (CLIA) and two different enzyme-linked immunosorbent assay (ELISA) test kits. The commercially available CMs were based on a serum pool that was either diluted with human serum albumin or enriched with various recombinant EPO (rhEPO) preparations. Commutability was assessed oriented towards the principles of difference in bias analysis based on the recommendations of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Working Group on Commutability.ResultsMost CMs remained within the range of the CS bias distribution across all paired method correlations. However, CMs exceeding the limits were also observed in some cases, even the native frozen pooled serum. Dilution and lyophilization affected the inter-method bias to varying degrees. Bias values for CMs enriched with different rhEPO preparations were mostly within the same range, though for CMs containing darbepoetin alfa in some cases higher inter-method bias was observed. Apparent differences in non-selectivity between the assays limited the ability to reliably assess commutability.ConclusionThe findings highlight current methodological challenges regarding the manufacturing of suitable materials and commutability assessment. Further investigations are needed regarding the standardization and harmonization of EPO measurements. Until further data is available, the evaluation within the EPO EQA should remain within method-specific sub-collectives.

In this Personal View, we introduce the concept of external quality assessment (EQA) super challenges, in which multiple EQA providers, at approximately the same time and in a coordinated manner, use test samples with identical characteristics in their programmes. The evaluation of test results from the resulting increase in the number of laboratories and test systems used (considering the resulting greater variety of influencing factors that apply to the analysis in the individual laboratories) enables the collection of data that reveals differences, advantages, and disadvantages of individual test systems, in addition to the extent of individual influencing factors. By comparing the analytical performance of test systems and highlighting their limitations, EQA super challenges and the examination results collected by them are valuable contributions for post-market surveillance of diagnostic tests, aid harmonisation in laboratory medicine, and help to identify areas for improvement for manufacturers, policy makers, and regulators. Especially during or in preparation for epidemics or pandemics, EQA super challenges are particularly valuable for public health institutions to quickly gain a clear picture of the testing performance.

For the external quality assessment (EQA) of glucose point-of-care testing (POCT), a high-quality, suitable, and practicable control material for assessing measurement accuracy has been lacking so far. As both the analyte glucose and the matrix whole blood are not stable without stabilizers, the current EQA surveys are based on glucose measurement in processed plasma. However, the manufacturer generally designed the devices for POCT glucose measurement for capillary blood. For this reason, INSTAND e.V. recently presented a new concept for control material in glucose POCT for EQA systems: The Whole Blood Beads model (WBB) [1]. The WBB model is also being subjected to a Europe-wide study on the commutability and suitability of control materials as part of COMET. COMET is an EU-funded project that has been set up to develop new certified reference materials and EQA materials to calibrate and verify the performance of in vitro diagnostics. In this work, we present new data from recent EQA survey using the WBB model to investigate its suitability as an EQA control material. Furthermore, we address issues that have been encountered in its use, drawing on the recent results of the EQA survey and the experience of our participants. In this context, we also suggest possible solutions to improve the performance of the WBB model. The WBB model relies on two components: A solid bead containing a defined amount of glucose and a glucose-free whole blood matrix. The EQA survey participant places the bead in the whole blood matrix immediately before performing the POCT. We evaluated its suitability as an EQA material based on the results of the recent EQA survey. The glucose target value was determined using the reference measurement procedure and served as an evaluation criterion for the accuracy of the results of the EQA survey. Limitations of the WBB model were addressed based on the latest EQA survey results and the experiences of participants. Based on the reference measurement value for glucose, the results of the EQA survey showed an acceptable pass rate (approx. 65 %) for the participating POCT devices, with low inaccuracy (approx. -2 % bias) but a need for improvement in reducing imprecision (variation coefficient of approx. 13 %). Accordingly, new tubes with transparent labels for performing the EQA survey were introduced, as the variations in the results of the participants were associated with insufficient mixing of glucose beads and whole blood matrix. The WBB model performs well in the latest EQA survey in terms of accuracy, representing a promising option for whole blood-based accuracy control for glucose POCT analyses. Continuous improvement of the WBB model regarding sample handling aims to reduce the current imprecision, enable a participant-friendly application in the EQA surveys, and thus ensure the accuracy and reliability of POCT glucose measurements for patient care. [1] Kaiser P, Kramer U, Rosenthal H, Genz C, Weiss N, Schellenberg I, Spannagl M. New concept for control material in glucose point-of-care-testing for external quality assessment schemes. Clinical Chemistry and Laboratory Medicine (CCLM). 2025;63(3): 552-558.

Microbiome-based solutions are regarded key for sustainable agroecosystems. However, it is unclear how agricultural practices affect the rhizosphere microbiome, plant-microorganism interactions and crop performance under field conditions. Therefore, we installed root observation windows in a winter wheat field cultivated either under long-term mouldboard plough (MP) or cultivator tillage (CT). Each tillage practice was also compared at two nitrogen (N) fertilization intensities, intensive (recommended N-supply with pesticides/growth regulators) or extensive (reduced N-supply, no fungicides/growth regulators). Shoot biomass, root exudates and rhizosphere metabolites, physiological stress indicators, and gene expression were analyzed together with the rhizosphere microbiome (bacterial/archaeal 16S rRNA gene, fungal ITS amplicon, and shotgun metagenome sequencing) shortly before flowering. Compared to MP, the rhizosphere of CT winter wheat contained more primary and secondary metabolites, especially benzoxazinoid derivatives. Potential copiotrophic and plant-beneficial taxa (e.g. Bacillus, Devosia, and Trichoderma) as well as functional genes (e.g. siderophore production, trehalose synthase, and ACC deaminase) were enriched in the CT rhizosphere, suggesting that tillage affected belowground plant-microorganism interactions. In addition, physiological stress markers were suppressed in CT winter wheat compared to MP. In summary, tillage practice was a major driver of crop performance, root deposits, and rhizosphere microbiome interactions, while the N-fertilization intensity was also relevant, but less important.

Experiences and challenges for EQA providers in assessing the commutability of control materials in accuracy-based EQA programs

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.

OBJECTIVES:Until now, the external quality assessment (EQA) of glucose point-of-care testing (POCT) has lacked a high quality, suitable and commutable control material to assess measurement accuracy. Here we present a concept for determining the accuracy of glucose measurements, which uses human whole blood and does not require stabilising agents. METHODS:This new generation of quality control samples uses a bead that contains a specific amount of glucose. The bead is then dissolved in a whole blood matrix by the EQA participant immediately before the POCT. We analysed its suitability as an EQA material with respect to its reproducibility, homogeneity and stability, and applied it in an EQA pilot study. The glucose target value was determined using the reference measurement procedure and served as an evaluation criterion for the accuracy of the EQA survey results. RESULTS:The homogeneity and stability of the new control material fulfilled the quality requirements of ISO 17043. Based on the reference measurement value for glucose, the results of the pilot EQA scheme showed a pass rate of 84.6 % for the participating POCT devices. The acceptance limit was a 15 % permitted deviation from the target value according to Rili-BAEK. All of the device collectives deviated from the target value by 0-4.4 % with the exception of one device type, which deviated by 21 %. CONCLUSIONS:The new concept offers, for the first-time, whole blood-based trueness controls for glucose POCT analysis for external quality assurance. The concept does not require the addition of any stabilising reagent and is easy to use.

Introduction: This study presents a longitudinal analysis of external quality assessment (EQA) results for erythropoietin (EPO) determinations conducted between 2017 and 2022 with a continuously increasing number of participating laboratories. The aim of this work was to evaluate participant performance and methodological aspects.Methods: In each of the eleven EQA surveys, a blinded sample set of lyophilized human serum containing one sample with lower EPO concentrations (L) and one with higher EPO concentrations (H) was sent to the participating laboratories.Results: A total of 1,256 measurements were included. The median (interquartile range) fraction of participants not meeting the criteria of acceptance set at 20% around the robust mean of the respective survey was 9.5% (6.1%-10.7%) (sample L) and 9.1% (5.8%-11.8%) (sample H) but lacked a clear trend in the observed period. Some surveys exhibited unusually high interlaboratory variation, suggesting interfering components in the EQA samples. Different immunological methods and reagent manufacturers also showed variability in measurement outcomes to some extent.Conclusion: These findings highlight the need for continuous quality assessment in EPO measurements to ensure patient safety and identify areas for further research and investigation.

Background:Quality control (QC), quality assurance, and standardization are crucial for modern diagnostic testing in the field of medical microbiology. The need for efficient QC to ensure accurate laboratory results, treatment, and infection prevention has led to significant efforts in standardizing assay reagents and workflows. External quality assessment (EQA) schemes, like those offered by INSTAND, play a vital role in evaluating in-house and commercial routine diagnostic assays, regarded as mandatory by national and global guidelines. The recent impact of polymerase chain reaction/nucleic acid amplification technology (PCR/NAAT) assays in medical microbiology requires that high-performing assays be distinguished from inadequately performing ones, especially those made by inexperienced suppliers.Objectives:The study assesses the evolving diagnostic performance trends over 2 decades for the detection of EHEC/STEC, Borrelia (B.) burgdorferi, and MRSA/cMRSA. It explores the historical context of assay utilization, participant engagement, and rates of correct results in EQA schemes. The research seeks to identify patterns in assay preferences, participant proficiency, and the challenges encountered in detecting emerging variants or clinical strains.Results:The study highlights the decline in in-house PCR assay usage, the emergence of new diagnostic challenges, and educational aspects within EQA schemes. Specific examples, such as the inclusion, in certain EQA surveys, of EHEC strains carrying stx-2f or B. miyamotoi, highlight the role of EQAs in increasing awareness and diagnostic capabilities. Advancements in MRSA detection, especially through the adoption of commercial assays, demonstrate the impact that technology evolution has had on diagnostic performance.Conclusion:Achieving excellence in diagnostic molecular microbiology involves a multifaceted approach, including well-evaluated assays, careful instrumentation selection, and structured training programs. EQA schemes contribute significantly to this pursuit by providing insights into the evolving diagnostic landscape and identifying areas for improvement in the diagnostic workflow as well as in PCR/NAAT assay design.

BackgroundC-reactive protein (CRP) is an established serum biomarker for different pathologies such as tissue injury and inflammatory events. One rising area of interest is the incorporation of low concentrations of CRP, so called high-sensitive (hs-) CRP, in the risk assessment and treatment monitoring of cardiovascular diseases (CVDs). Many research projects and the resulting meta-analyses have reported controversial results for the use of hs-CRP, especially in the risk assessment of CVDs. However, since these analyses used different assays to detect hs-CRP, it is important to assess the current level of assay harmonization.MethodsThis paper analyzes data from 17 external quality assessment (EQA) surveys for hs-CRP conducted worldwide between 2018 and 2023. Each EQA survey consisted of two blinded samples. In 2020 the sample material changed from pooled serum to single-donor samples. The aim was to assess the current status of assay harmonization by a manufacturer-based approach, taking into consideration the clinical decision limits for hs-CRP risk-stratification of CVDs as well as the scatter of results.ResultsOur analyses show that harmonization has increased in recent years from median differences of up to 50% to below 20%, with one exception that showed an increasing bias throughout the observed period. After changing sample materials from pools to single-donor samples, the coefficient of variation decreased to below 10% with one exception. Nevertheless, even these differences in the clinical setting could lead to disparate classification of patients depending on the assay used.ConclusionWhile there was a positive trend towards harmonization, meta-analysis of different risk-score publications should stratify their analysis by assay to account for the manufacturer-specific differences observed in this paper. Furthermore, assays are currently traceable to different international standard preparations, which might have a negative impact on future harmonization.

BackgroundTumor markers are established laboratory tools that help to diagnose, estimate prognosis, and monitor the course of cancer. For meaningful decision-making in patient care, it is essential that methods and analytical platforms demonstrate high sensitivity, specificity, precision, and comparability. Regular participation at external quality assessment (EQA) schemes is mandatory for laboratories. Here, a longitudinal evaluation of EQA data was performed to assess the performance of tumor marker assays over time.MethodsLongitudinal data of the cancer antigens (CA) 15-3 (n = 5,492), CA 19-9 (n = 6,802), and CA 125 (n = 5,362) from 14 INSTAND EQAs conducted between 2019 and 2023 were evaluated. A median of 197, 244 and 191 laboratories participated at the EQAs for CA 15-3, CA 19-9 and CA 125, respectively. Data evaluation encompasses intra- and inter-manufacturer specific variations over time, assay precision, and adherence to the EQA limits of ±24% for CA 15-3, ±27% for CA 19–9 and ±36% for CA 125.ResultsThe study showed median manufacturer-dependent differences of up to 107% for CA 15-3, 99% for CA 125, and even 549% for CA 19-9 between the highest and the lowest methods over the studied period. Regarding the normalized median of all methods, the values of the most deviant methods were 0.42 for CA 15-3, 7.61 for CA 19-9, and 1.82 for CA 125. Intra-manufacturer variability was generally low, with median coefficients of variation (CV) below 10%. As the methods were evaluated according to method-specific consensus values, most participants passed the EQAs within the acceptance criteria. When the criteria were consistently set at 24%, the central 90% of participants passed the EQAs in 78.6%–100% for CA 15-3 (with exception of AX), 89.3%–100% for CA 125, and 64.3%–100% for CA 19-9.ConclusionWhile intra-method precision of most analytical platforms is acceptable for all three tumor markers, considerable inter-method variability was observed over the whole studied period demonstrating the necessity for better standardization and harmonization of the methods, development of international reference materials, and comprehensive commutability studies with patient samples.

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.

During an epidemic, individual test results form the basis of epidemiological indicators such as case numbers or incidence. Therefore, the accuracy of measures derived from these indicators depends on the reliability of individual results. In the COVID-19 pandemic, monitoring and evaluating the performance of the unprecedented number of testing facilities in operation, and novel testing systems in use, was urgently needed. External quality assessment (EQA) schemes are unique sources of data reporting on testing performance, and their providers are recognised contacts and support for test facilities (for technical-analytical topics) and health authorities (for planning the monitoring of infection diagnostics). To identify information provided by SARS-CoV-2 genome detection EQA schemes that is relevant for public health microbiology, we reviewed the current literature published in PubMed between January, 2020, and July, 2022. We derived recommendations for EQA providers and their schemes for best practices to monitor pathogen-detection performance in future epidemics. We also showed laboratories, test facilities, and health authorities the information and benefits they can derive from EQA data, and from the non-EQA services of their providers.

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.