Clinical Chemistry and Laboratory Medicine (CCLM)

IntroductionFree light chain kappa (FLC{kappa}) are a newer sensitive biomarker to detect intrathecal immunoglobulin synthesis. Here we report the study protocol of the ORCAS study which will evaluate a novel laboratory work flow recently proposed including FLC{kappa} analysis simplifying cerebrospinal fluid (CSF) analysis. MethodsThe ORCAS study is a prospective multicenter diagnostic biomarker study across at least 4 study sites. The study protocol does not specify which assays should be applied in the participating laboratories to detect oligoclonal bands (OCB) or measure FLC{kappa} or Ig synthesis to represent real world data. Primary outcome parameter is the sensitivity and negative predictive value of the absence of local FLC{kappa} synthesis for the absence of intrathecal synthesis according to OCB and/or intrathecal IgG, IgA, IgM synthesis in quotient diagrams. The reference range of FLC{kappa} will be indicated by the FLC{kappa} quotient diagram. This study was designed according to the STARD criteria. PerspectiveThe establishment of a newer biomarker in routine practice is associated with significant difficulties, such as the inconsistent comparability of different measurement platforms, the establishment of suitable cut-offs or insufficient knowledge regarding sensitivity and specificity of the biomarker. If the ORCAS study objective is achieved, the use of the proposed workflow integrating FLC{kappa} analysis in routine practice in CSF diagnostics could help to better characterize the intraindividual and disease-specific intrathecal humoral immune response in a resource-efficient manner.

BackgroundAnalysis of cerebrospinal fluid (CSF) facilitates the understanding of brain-specific molecular changes that may associate with disease progression. Proximity extension assays (PEA) have been deployed in several CSF studies, however the validation of the assay and impact of freeze-thaw cycles on the protein signal has not been documented. We sought to (1) validate the assay on the PEA platform and (2) evaluate the effect of freeze-thaw cycles on the detectability of analytes on the PEA platform. ResultsWe have validated the PEA with Next Generation Sequencing (NGS) readout assay and report on the detectability and coefficient of variation observed in CSF samples. We have also evaluated proteomic signals with a minimum of 3 and a maximum of 9 freeze thaw cycles and detected very minimal change in signal with increasing cycle number. ConclusionOur study is the first to validate PEA using NGS readout platform with CSF samples. We report lower protein detection rates and higher variability in the expansion panels compared to the original 4 panels, with acceptable variation above detectability threshold. In addition, our work demonstrates that the proteomic signal is robust and continues to be stable across multiple freeze thaw cycles. This is highly impactful to the processing and analysis of clinical samples and facilitates the investigation of samples with variable pre-analytical conditions.

ObjectivesNeurofilament light (NfL) is an established biofluid marker of neuroaxonal injury for neurological diseases. Several high-throughput and sensitive immunoassays have been developed to quantify NfL in blood and cerebrospinal fluid (CSF), facilitating the use of NfL as a biomarker in research and clinical practice. However, because of the lack of rigorous comparisons of assays, it has been difficult to determine whether data are comparable and whether assay performance differs. Here, we compared the performance of five NfL immunoassays. MethodsTo assess the five NfL immunoassays (Fujirebio, ProteinSimple, Quanterix, Roche and Siemens), we used pooled plasma or pooled CSF, as well as unique samples from 20 healthy controls and 20 individuals with El Escorial defined probable or definite amyotrophic lateral sclerosis (ALS), to evaluate precision, parallelism and/or bias. We also examined correlations between plasma and CSF NfL concentrations within and across assays and evaluated their ability to differentiate healthy controls from individuals with ALS. ResultsFour of the five assays demonstrated exemplary performance based on our analyses of precision and parallelism. Across the five assays, NfL concentrations were lower in plasma than in CSF, although they displayed a high degree of correlation. We noted bias across assays; plasma NfL concentrations were lowest for the Roche assay and highest for the ProteinSimple assay. In addition, all assays reliably distinguished healthy controls from individuals with ALS using plasma or CSF NfL. ConclusionsFour NfL assays demonstrated similar analytic performance. Alongside performance, other factors such as costs, accessibility, useability, footprint, and intended use, should be considered.

Introduction: Blood neurofilament light chain (NfL) is an accessible biomarker of neuroaxonal injury across a broad range of neurological disorders, but its clinical implementation requires robust cross-platform analytical and clinical comparability. The objective of this study was to evaluate the analytical and clinical comparability of plasma NfL measurements using Simoa and Lumipulse across different neurological conditions, by assessing cross-platform agreement and the ability of both assays to distinguish neurological diseases from healthy controls. Paired CSF analyses were performed in a subset of participants to biologically anchor plasma findings to the central compartment. Methods: 383 individuals were included, comprising healthy controls and patients with neurodegenerative conditions, multiple sclerosis and stroke. Plasma NfL was measured in all participants using both Simoa and Lumipulse, with paired CSF analyses in a subset of 92 individuals The Lumipulse testing intermediate precision and between-day repeatability was assessed as by the CLSI EP15. Cross-platform agreement for plasma NfL was evaluated using correlation analyses, Passing-Bablok regression and Bland-Altman analysis. Associations between plasma/CSF NfL concentrations were assessed using Spearman's rank correlation analysis for each platform, separately. Age-adjusted cross-diagnostic differences were evaluated using permutation ANCOVA and multiple linear regression models for each platform, separately. Results: Plasma NfL measured by Simoa and Lumipulse showed strong cross-platform concordance in the whole cohort ({rho}=0.90), with similarly strong concordance observed for CSF NfL in the subset with paired samples ({rho}=0.90). Method-comparison analyses in plasma demonstrated consistent agreement between platforms, with identifiable constant and proportional bias, alongside systematically higher absolute plasma NfL values measured by Lumipulse. Within-platform analyses showed significant correlations between plasma and CSF NfL concentrations ({rho}=0.72 for Simoa; {rho}=0.78 for Lumipulse). Noteworthy, Lumipulse NfL CSF and Blood kits exhibited high precision and analytical accuracy. Across both assays, plasma NfL increased with age and was significantly elevated in patients with neurological disorders compared with healthy controls. Discussion: Simoa and Lumipulse capture a consistent biological signal in plasma across patients with neurological disorders, although their absolute NfL values differ, supporting the use of platform-specific reference ranges in clinical practice.

Serological assays for anti-SARS-CoV-2 antibodies are now of critical importance to support diagnosis, guide epidemiological intervention, and understand immune response to natural infection and vaccine administration. We developed and validated new anti-SARS-CoV-2 IgG, IgM and IgA ELISA tests (ENZY-WELL SARS-CoV-2 ELISA, DIESSE Diagnostica Senese S.p.a.) based on whole-virus antigens. We used a total of 553 serum samples including samples from COVID-19 suspected and confirmed cases, healthy donors, and patients positive for other infections or autoimmune conditions. Overall, the assays showed good concordance with the indirect immunofluorescence reference test in terms of sensitivity and specificity. Especially for IgG and IgA, we observed high sensitivity (92.5 and 93.6%, respectively); specificity was high (>96%) for all antibody types ELISAs. In addition, sensitivity was linked to the days from symptoms onset (DSO) due to the seroconversion window, and for ENZY-WELL SARS-CoV-2 IgG and IgA ELISAs resulted 100% in those samples collected after 10 and 12 DSO, respectively. The results showed that ENZY-WELL SARS-CoV-2 ELISAs may represent a valid option for both diagnostic and epidemiological purposes, covering all different antibody types developed in SARS-CoV-2 immune response.

Proper management of chronic lymphocytic leukemia (CLL) patients requiring therapy relies on two important prognostic and theranostic molecular features: respectively, the mutational status of tumoral cells immunoglobulin heavy chain variable domain (IGHV) and the characteristics of TP53. Both these (immuno)genetic analyses require multiple time-consuming amplification and sequencing techniques by Sanger or HTS. The capture-HTS technology, allowing to select regions of interest, represents an attractive alternative and has already been applied for the detection of clonality in lymphoproliferative disorders. Here, a single-step capture design was developed to concomitantly investigate for IGHV and TP53. This was applied to a training retrospective (n=14) and a validation prospective (n=91) cohorts of CLL patients. The training cohort demonstrated the robustness of the method by comparison with the classical Sanger sequencing technology (100% identical results) for the IGHV mutational status. This consistency was confirmed for the first 59 patients of the validation cohort. Overall, the IGHV status of whole population (n=103) was accurately identified. Simultaneously, deletion or mutations of TP53 were identified from the same capture-library and HTS-sequencing run for each patient. This novel approach provides, in a single assay, useful answers about the molecular landscape of CLL patients, allowing for a documented choice of therapy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/483581v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@91685forg.highwire.dtl.DTLVardef@5b47baorg.highwire.dtl.DTLVardef@c2286corg.highwire.dtl.DTLVardef@ebd413_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundO_ST_ABSAbstractC_ST_ABSReliable SARS-CoV-2 serological assays are required for diagnosing infections, for the serosurveillance of past exposures and for assessing the response to future vaccines. In this study, the analytical and clinical performances of a chemiluminescent immunoassays for SARS-CoV-2 IgM and IgG detection (Mindray CL-1200i), targeting Nucleocapsid (N) and receptor binding domain (RBD) portion of the Spike protein, were evaluated. MethodsPrecision and linearity were evaluated using standardized procedures. A total of 157 leftover serum samples from 81 hospitalized confirmed COVID-19 patients (38 with moderate and 43 with severe disease) and 76 SARS-CoV-2 negative subjects (44 healthcare workers, 20 individuals with rheumatic disorders, 12 pregnant women) were included in the study. In an additional series of 44 SARS-CoV-2 positive, IgM and IgG time kinetics were also evaluated in a time-period of 38 days. ResultsPrecision was below or equal to 4% for both IgM and IgG, in all the studied levels, whilst a slightly significant deviation from linearity was observed for both assays in the range of values covering the manufacturers cut-off. Considering a time frame [≥] 12 days post symptom onset, sensitivity and specificity for IgM were 92.3% (95%CI:79.1%-98.4%) and 92.1% (95%CI:83.6%-97.0%). In the same time frame, sensitivity and specificity for IgG were 100% (95%CI:91.0%-100%) and 93.4% (95%CI:85.3%-97.8%). The assays agreement was 73.9% (Cohens kappa of 0.373). Time kinetics showed a substantial overlapping of IgM and IgG response, the latter values being elevated up to 38 days from symptoms onset. ConclusionsAnalytical imprecision is satisfactory as well as the linearity, particularly when taking into account the fact that both assays are claimed to be qualitative. Diagnostic sensitivity of IgG was excellent, especially considering specimens collected [≥]12 days post symptom onset. Time kinetics suggest that IgM and IgG are detectable early in the course of infection, but the role of SARS-CoV-2 antibodies in clinical practice still requires further evaluations.

There is an urgent need in rapid diagnostic test (RDT) to detect antibodies against SARS-CoV-2. We have developed a rapid and simple point-of-care lateral flow immunoassay (LFIA) detecting IgM and IgG against SARS-CoV-2 in 10 minutes. The aim of this study is to evaluate the diagnostic performance of this RDT. RT-PCR positive plasma samples (n=35) for SARS-CoV-2 and 97 negative control samples were studied. Diagnostic performance of IgG/IgM RDT was assessed using both gold standard RT-PCR and Electro-chemiluminescence immunoassay (ECLIA) Elecsys(R) Anti-SARS-CoV-2 total Ig. Overall, RDT sensitivity was 100% (95% confidence interval [95%CI]: 88-100%) and specificity 93% (95% CI: 85-97%). This IgG/IgM RDT done in plasma displays a high diagnostic accuracy for SARS-CoV-2 IgG/IgM in high COVID-19 prevalence settings. Its use could be considered in the absence of routine diagnostic serology facilities for samples collected between 10 and 180 days after symptoms onset.

The reliable detection of immunoglobulin G (IgG) or total antibodies directed against the novel severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is important for clinical diagnostics and epidemiological studies. Here, we compare the diagnostic accuracy of six commercially available SARS-CoV-2 IgG (Abbott SARS-CoV-2 IgG; Diasorin Liaison(R) SARS-CoV-2 S1/2 IgG; Epitope EDI Novel Coronavirus COVID-19 IgG ELISA Kit; Euroimmun Anti-SARS-CoV-2 ELISA (IgG); Mikrogen recomWell SARS-CoV-2 IgG) or total SARS-CoV-2 antibody assays (Roche Elecsys Anti-SARS-CoV-2). The test sensitivities were analyzed with a set of 34 sera obtained from 26 patients after PCR-confirmed SARS-CoV-2 infection and varied from 76.9% (Euroimmun) to 96.2% (Abbott). The majority of assay results were confirmed in a laboratory-developed plaque reduction neutralization test and by a SARS-CoV-2 IgG-specific line assay including measurement of generally low IgG avidities (Mikrogen recomLine Coronavirus IgG [Aviditat], prototype). Moreover, 100 stored sera collected during summer 2018 (N = 50) and winter season 2018/2019 (N = 50) were included to demonstrate test specificities. These varied from 96.0% (DiaSorin) to 100% (Epitope EDI). A subset of sera were retested with a lateral flow test (STANDARD Q COVID-19 IgM/IgG Duo) and a considerably lower sensitivity was noted. Overall, the diagnostic accuracy of the six SARS-CoV-2 IgG/total antibody assays was good and varied from 92.9% (Euroimmun) to 98.4% (Abbott). Due to the different specificities, results of commercially available SARS-CoV-2 antibody tests should be interpreted with caution. A high proportion of antibody-positive patient sera demonstrated neutralizing capacity against SARS-CoV-2.

BackgroundSensitive and highly specific antibody tests are critical for detection of SARS-CoV-2 antibodies especially in populations where seroprevalence is low. AimTo set up, optimize and evaluate the analytical and clinical performance of a new in-house microsphere immunoassay for measurement of IgG antibodies to SARS-CoV-2 nucleoprotein for assessment of population seroprevalence in Finland. MethodsWe set up a new in-house microsphere immunoassay (FMIA) with SARS-CoV-2 nucleoprotein and optimized its analytical performance. For evaluation of clinical performance, we tested sera collected in a well-characterized cohort of PCR positive-confirmed SARS-CoV-2 patients (n=89) with mostly mild symptoms, and before the COVID-19 pandemic (n=402), for nucleoprotein specific IgG concentrations by FMIA and a commercial chemiluminescent immunoassay and for neutralizing antibodies by the microneutralization test. ResultsThe analytical performance of FMIA was established in terms of sensitivity, linearity and precision. FMIA discriminated between COVID-19 patient and control samples with high specificity (100%) and sensitivity (100%). We generated FMIA seropositivity cut-offs, 0.46 and 1.71 U/ml, for low- and high-seroprevalence settings, respectively. In addition, we obtained high level of agreement between FMIA results and results by the microneutralization test. ConclusionThe fluorescent microsphere immunoassay showed excellent analytical and clinical performance and is well suited for serosurveillance studies of SARS-CoV-2. However, to optimize analytical sensitivity and clinical specificity of the assay, different seropositivity thresholds depending on the intended use of the assay and the target population, may be needed.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), became a pandemic in early 2020. Lateral flow immunoassays for antibody testing have been viewed as a cheap and rapidly deployable method for determining previous infection with SARS-CoV-2; however, these assays have shown unacceptably low sensitivity. We report on nine lateral flow immunoassays currently available and compare their titer sensitivity in serum to a best-practice enzyme-linked immunosorbent assay (ELISA) and viral neutralization assay. For a small group of PCR-positive, we found two lateral flow immunoassay devices with titer sensitivity roughly equal to the ELISA; these devices were positive for all PCR-positive patients harboring SARS-CoV-2 neutralizing antibodies. One of these devices was deployed in Northern Italy to test its sensitivity and specificity in a real-world clinical setting. Using the device with fingerstick blood on a cohort of 27 hospitalized PCR-positive patients and seven hospitalized controls, ROC curve analysis gave AUC values of 0.7646 for IgG. For comparison, this assay was also tested with saliva from the same patient population and showed reduced discrimination between cases and controls with AUC values of 0.6841 for IgG. Furthermore, during viral neutralization testing, one patient was discovered to harbor autoantibodies to ACE2, with implications for how immune responses are profiled. We show here through a proof-of-concept study that these lateral flow devices can be as analytically sensitive as ELISAs and adopted into hospital protocols; however, additional improvements to these devices remain necessary before their clinical deployment.

ObjectiveNeurofilaments are key axonal proteins, with neurofilament light (NfL) and heavy (NfH) chain recognized as promising biomarkers for neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). However, neurofilament medium chain (NfM) remained previously underexplored due to a lack of quantitative assays. In this study, we developed a sensitive immunoassay to measure NfM in cerebrospinal fluid (CSF) and analyzed its levels in ALS, Alzheimers disease (AD), frontotemporal dementia (FTD), and Lewy body dementia (LBD). Correlations among neurofilaments and their diagnostic performance were also evaluated. MethodsIn this study CSF levels of three neurofilament proteins were measured in 271 participants, including patients with ALS (n=91), AD (n=25), FTD (n=38), LBD (n=18), non-neurodegenerative controls (CTRL, n=51), and 48 individuals initially evaluated for ALS but ultimately diagnosed with other conditions (CTRL.DD). ResultsAll three neurofilaments were significantly elevated in ALS compared to CTRL and CTRL.DD groups (p<0.0001 for both), with NfM and NfL also increased in FTD (p<0.0001 for both) and AD (NfM, p=0.0017; NfL, p=0.0135 ) compared to CTRL. NfH demonstrated the greatest distinction between ALS and FTD (p<0.0001). Strong correlations were observed among neurofilament subunits, particularly between NfM and NfL (r=0.94, 95% CI: 0.93-0.96, p<0.0001). All neurofilaments effectively distinguished ALS from CTRL and CTRL.DD, with AUC values ranging from 0.92 to 0.99. NfM and NfL showed high accuracy in differentiating AD (NfM, AUC: 0.89; NfL, AUC: 0.90) and FTD (NfM, AUC: 0.91; NfL, AUC: 0.92) from CTRL, while NfH best separated ALS from FTD (AUC: 0.96). ConclusionThis study provides the first quantitative comparison of NfM with NfL and NfH in a neurodegenerative cohort, highlighting its potential diagnostic value. Further research with larger cohorts, longitudinal studies, and investigations into neurofilament distribution in different compartments is needed to clarify the distinct roles of NfM, NfL, and NfH in the diagnosis and treatment of neurological diseases.

BackgroundHuntingtons disease (HD) is caused by a cytosine adenine guanine-repeat expansion in the huntingtin gene. This results in the production of toxic mutant huntingtin protein (mHTT), which has an elongated polyglutamine (polyQ) stretch near the proteins N-terminal end. The pharmacological lowering of mHTT expression in the brain targets the underlying driver of HD and is one of the principal therapeutic strategies being pursued to slow or stop disease progression. This report describes the characterisation and validation of an assay designed to quantify mHTT in the cerebrospinal fluid of individuals with HD, for use in registrational clinical trials. MethodsThe assay was optimised, and its performance was characterised with recombinant huntingtin protein (HTT) varying in overall and polyQ-repeat length. ResultsThe assay was successfully validated by two independent laboratories in regulated bioanalytical environments and showed a steep signal increase as the polyQ stretch of recombinant HTTs pivoted from wild-type to mutant protein forms. Linear mixed effects modelling confirmed highly parallel dose-response curves for HTTs, with only a minor impact of individual slopes of the dose-response for different HTTs (typically <5% of the overall slope). This implies an equivalent quantitative signal behaviour for HTTs with differing polyQ-repeat lengths. ConclusionThe reported method may be a reliable biomarker tool with relevance across the spectrum of HD mutations, which can facilitate the clinical development of HTT-lowering therapies in HD. Trial registrationNot applicable. FundingF. Hoffmann-La Roche Ltd.

Here we describe a novel immunogenic method to detect COVID-19. The method is a chromogenic magnetic bead-based ELISA which allows inexpensive and quantitative detection of human IgG or IgM antibodies against SARS-CoV-2 in serum or whole blood samples in just 12 minutes. As a proof of concept, we compared the performance of our new method to classical ELISA. Person correlation between optical densities obtained using the two methods was 0.98, the color intensity observed in the novel method correlated with antibody titers determined by classical ELISA. The novel magnetic bead-based ELISA correctly classified all 6 positive COVID-19 samples tested and showed 100% specificity as judged by the analysis of a cohort of 26 negative samples. The magnetic bead-based ELISA performed better than classic ELISA to discriminate COVID-19 positive serum with low antibody titer. The chromogenic magnetic bead-based ELISA method described here can be applied to both point of care and high throughput analysis. The method is readily adaptable to be used with other protein and peptide-based antigens.

BackgroundPrior research conducted in model rats of CAA Type 1 (rTg-DI) identified a range of cerebrospinal fluid biomarker candidates associated with sCAA pathology. This list of potential biomarkers includes the lysosomal proteases cathepsins B and S (CTSB/CTSS) and hexosaminidase B (HEXB). It is yet unknown if these findings obtained in rTg-DI rats translate to differential protein levels and/or enzyme activities in cerebrospinal fluid (CSF) of sCAA patients. In this study, we attempted to validate CTSB, CTSS and HEXB in CSF as potential biomarkers for sCAA in a human population. Materials and methodsWe have included sCAA patients (n = 34) and control participants (n = 27) from our BIONIC/CAFE cohort. We analysed the CSF of these participants with ELISA for protein levels of CTSB and CTSS. Additionally, we used in-house enzyme assays to determine activity levels of total hexosaminidase and hexosaminidase A (HEXA) in CSF. The proportion of HEXA activity to total HEX activity was used as a proxy for HEXB activity. ResultsCSF CTSB and CTSS protein levels were not significantly different between sCAA and controls (p = 0.21 and p = 0.34). Total HEX activity was unaltered as well (p = 0.11), whereas a significant decrease was observed in HEXA activity levels (p = 0.05). HEXA / total HEX activity levels (as a proxy for HEXB activity) were unaltered between sCAA patients and controls (p = 0.19). Additionally, CTSB and CTSS protein levels positively associated with total HEX activity (rsp = 0.37, p = 0.005; rsp = 0.40, p = 0.003). ConclusionThe contrasting results between biomarker discovery in rats and validation in human participants highlight the challenges and complexities of biomarker research. These findings offer valuable insights into the nuances of disease and the difficulties in translating laboratory findings using animal models to clinical practice. Understanding these discrepancies is essential for improving the precision of biomarker translation, ensuring clinical relevance, and developing comprehensive biomarker panels for CAA and related conditions.

BackgroundWe evaluate the diagnostic performance of dried blood microsampling combined with a high-throughput microfluidic nano-immunoassay (NIA) for the identification of anti-SARS-CoV-2 Spike IgG seropositivity. MethodsWe conducted a serological study among 192 individuals with documented prior SARS-CoV-2 infection and 44 SARS-CoV-2 negative individuals. Participants with prior SARS-CoV-2 infection had a long interval of 11 months since their qRT-PCR positive test. Serum was obtained after venipuncture and tested with an automated electrochemiluminescence anti-SARS-CoV-2 S total Ig reference assay, a commercial ELISA anti-S1 IgG assay, and the index test NIA. 109 participants from the positive cohort and 44 participants from the negative cohort also participated in capillary blood collection using three microsampling devices: Mitra, repurposed glucose test strips, and HemaXis. Samples were dried, shipped by regular mail, extracted, and measured with NIA. FindingsUsing serum samples, we achieve a clinical sensitivity of 98{middle dot}33% and specificity of 97{middle dot}62% on NIA, affirming the high performance of NIA in participants 11 months post infection. Combining microsampling with NIA, we obtain a clinical sensitivity of 95{middle dot}05% using Mitra, 61{middle dot}11% using glucose test strips, 83{middle dot}16% using HemaXis, and 91{middle dot}49% for HemaXis after automated extraction, without any drop in specificity. InterpretationHigh sensitivity and specificity was demonstrated when testing micro-volume capillary dried blood samples using NIA, which is expected to facilitate its use in large-scale studies using home-based sampling or samples collected in the field. FundingSwiss National Science Foundation NRP 78 Covid-19 grant 198412 and Private Foundation of the Geneva University Hospital. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSSerological surveillance is of importance to better understand the evolution and spread of SARS-CoV-2 and adapt public health measures. We identified multiple studies conducting such serological surveys using decentralized collection of capillary blood, facilitating the logistics and reducing burden on participants and healthcare facilities. To perform the detection of anti-SARS-CoV-2 antibodies with a high-throughput and at low-cost, a microfluidic nano-immunoassay (NIA) was developed which requires ultra-low sample volumes and minimizes reagent consumption. Added value of this studyIn this study we showed the possibility of combining capillary microsampling with NIA. We validated the use of NIA in serum samples obtained 11 months after infection and show the good clinical performance of the assay in samples with waning antibody titers. Using three different microsampling device, namely Mitra, repurposed glucose test strips, and HemaXis, we implemented a protocol using dried blood sample collection, shipping, extraction, and testing on the microfluidic assay. The sensitivity and specificity were measured and are presented when using the different microsampling devices. Implications of all the available evidenceWe show that the performance of NIA is good when using serum samples, but also in combination with microsampling. Facilitated logistics and increased convenience of microsampling, together with high-throughput and low-cost testing on a microfluidic assay should facilitate the conduction of serological surveys.

BackgroundDetection of viral RNA by nucleic acid amplification testing (NAAT) remains the gold standard for diagnosis of SARS-CoV-2 infection but is limited by high cost and other factors. Whether serology-based assays can be effectively incorporated into a diagnostic algorithm remains to be determined. Herein we describe the development of a serology-based testing algorithm for SARS-CoV-2 infection. Patients and MethodsBetween July 2020 and February 2021, we included symptomatic unvaccinated patients evaluated in the Emergency Department of our institution for suspected SARS-CoV-2. All patients had testing by real-time Reverse Transcription Polymerase Chain Reaction. The performance characteristics of five commercial enzymatic serology assays testing for different antibody isotypes were evaluated in a derivation cohort and the assay with the best performance was further tested on a validation cohort. Optimal cut-off points were determined using receiver operating characteristic (ROC) curves and further tested using logistic regression. ResultsThe derivation and validations cohorts included 72 and 319 patients, respectively. Based on its initial performance, the Elecsys Anti-SARS-CoV-2 assay (Roche Diagnostics) was further tested in the validation cohort. Using ROC curve analysis, we estimated the diagnostic performance for different cut-off points assuming a prevalence of positive tests of 5%. At any given cut-off point the NPV was over 97%. DiscussionThis study suggests that an initial diagnostic strategy using the Elecsys Anti-SARS-CoV-2 serology test in symptomatic unvaccinated patients could help to rule out an acute SARS-CoV2 infection and potentially lead to appropriately tailored infection control measures or rational guidance for further testing with a potential cost reduction and increased availability.

BackgroundTo accurately measure seroprevalance in the population, both the expected immune response as well as the assay performances have to be well characterised. Here, we describe the collection and initial characterisation of a blood and saliva biobank obtained after the initial peak of the SARS-CoV-2 pandemic in Switzerland. MethodsTwo laboratory ELISAs measuring IgA & IgG (Euroimmun), and IgM & IgG (Epitope Diagnostics) were used to characterise the biobank collected from 349 re- and convalescent patients from the canton of Basel-Landschaft. FindingsThe antibody response in terms of recognized epitopes is diverse, especially in oligosymptomatic patients, while the average strength of the antibody response of the population does correlate with the severity of the disease at each time point. InterpretationThe diverse immune response presents a challenge when conducting epidemiological studies as the used assays only detect[~] 90% of the oligosymptomatic cases. This problem cannot be rectified by using more sensitive assay setting as they concomitantly reduce specificity. FundingFunding was obtained from the "Amt fur Gesundheit" of the canton Basel-Landschaft, Switzerland.

BackgroundThe Elecsys(R) Anti-SARS-CoV-2 immunoassay (Roche Diagnostics) was developed to provide an accurate and reliable method for the detection of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluated the specificity of the Elecsys Anti-SARS-CoV-2 immunoassay in prepandemic sample cohorts across five sites in Germany, Austria and Switzerland. MethodsSpecificity of the immunoassay was evaluated using anonymised, frozen, residual serum and/or plasma samples from blood donors or routine diagnostic testing. All samples were collected before September 2019 and therefore presumed negative for SARS-CoV-2-specific antibodies. Cohorts included samples from blood donors, pregnant women and paediatric patients. Point estimates and 95% confidence intervals (CIs) were calculated. ResultsOverall specificities for the Elecsys Anti-SARS-CoV-2 immunoassay in 9575 samples from blood donors (n = 6714) and diagnostic specimens (n = 2861) were 99.82% (95% CI 99.69-99.91) and 99.93% (95% CI 99.75-99.99), respectively. Among 2256 samples from pregnant women, specificity was 99.91% (95% CI 99.68-99.99). Among 205 paediatric samples, specificity was 100% (95% CI 98.22-100). ConclusionThe Elecsys Anti-SARS-CoV-2 immunoassay demonstrated a very high specificity across blood donor samples and diagnostic specimens from Germany, Austria and Switzerland. Our findings support the use of the Elecsys Anti-SARS-CoV-2 immunoassay as a potential tool for determination of an immune response following previous exposure to SARS-CoV-2 in the general population, including in blood donors, pregnant women and paediatric populations.

Multiplexed imaging approaches of various molecular modalities in tissues are becoming increasingly adopted in discovery and translational studies. For clinical implementation, novel instrumentation, complex analysis workflows and high costs per sample are bottlenecks that hinders broader introduction in the clinical setting. Here, we demonstrate a cost efficient integrated workflow that combines multiplexed immunofluorescence of a handful of protein markers, with in situ proximity ligation assay, to detect direct protein interactions between neighboring cells. As a proof of concept case of relevance for clinical adaptation, we target the major immunotherapy signalling axis of programmed death receptor 1 (PD-1) and its ligand PD-L1, to demonstrate the interaction between immune cells in germinal centers of tonsil tissue and in a tertiary lymphoid structure in bladder cancer tissue, respectively, from a patient treated with immunotherapy.