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Giant cell arteritis (GCA) and polymyalgia rheumatica (PMR) are closely related inflammatory disorders. Easily measurable biomarkers defining active disease and identifying patients in need of glucocorticoid (GC) sparing treatment options are highly desired. Interferon type I (IFN-I) might be involved in disease pathology, however evidence is limited. This study explores a systemic IFN-I signature and expression of IFN-I markers in GCA and PMR patients. Treatment naive GCA and PMR patients, and PMR patients with GC treatment were included. Patients suspected of but not diagnosed with GCA were used as controls. Five relevant IFN-I stimulated genes (ISGs) were identified in literature and relative expression levels were determined using RT-qPCR in PBMCs. An IFN-I score was generated. Serum levels of IFN-I induced CXCL10 and Galactin-9 were determined by multiplex immunoassay. There were no differences in IFN-I scores between the groups. An IFN-I signature was observed in 0/9 controls, 2/11 GCA patients, 4/20 treatment naive PMR patients and 2/10 PMR patients with treatment. Serum CXCL10 and Galactin-9 were not increased in GCA or PMR patients compared to control patients. Treated PMR patients had lower CXCL10 levels (423.2 pg/ml (375.1-491.1)) compared to treatment naive PMR patients (641.8 pg/ml (552.8-830.6)). An IFN-I signature does not distinguish GCA and PMR patients from controls. Also, IFN-I induced serum markers are not upregulated in GCA and PMR patients. Easily measurable IFN-I induced serum markers will therefore probably not aid in diagnosis and additional treatment options in newly diagnosed GCA and PMR patients., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Immunology.)

Objectives: Tissue transglutaminase (tTG) IgA antibodies are a hallmark for celiac disease (CD). In CD patients on gluten free diet (GFD) these antibodies are transient. Few studies are available comparing the tTG-IgA assay characteristics for monitoring response to GFD. Since discrepant results were reported in patients on GFD after switching tTG-IgA assays, we conducted a retrospective observational study to monitor GFD response using three different tTG-IgA assays., Methods: Diagnostic samples from 44 adults and 17 children with CD were included. Of most patients two follow-up samples after introduction of GFD were available. In all samples tTG-IgA were assessed using one fluorochrome-enzyme immuno-assay (FEIA) and two chemiluminescence immuno-assays (CLIA) and intestinal fatty acid binding protein (i-FABP) as surrogate marker for intestinal epithelial damage was measured., Results: Using CLIA assays, normalization of antibody levels was delayed compared to FEIA (p<0.001). Of all samples taken after at least 6 months on GFD with elevated i-FABP indicating intestinal epithelial damage, 40 % had positive tTG-IgA according to the FEIA, 85 and 90 % according to the two CLIA., Conclusions: Normalization of tTG-IgA in patients on GFD depends on the assay used. Both CLIA appear to be more sensitive in detecting suboptimal treatment response in CD-indicated by elevated i-FABP - when applying the manufacturer's recommended cut-off for the diagnosis of CD., (© 2023 Walter de Gruyter GmbH, Berlin/Boston.)

Objectives: Celiac disease (CD) is an immune-mediated enteropathy driven by gluten intake. Presence of tTG-IgA antibodies is important for the diagnosis. However, different tTG-IgA assays are used and test performance may vary. Therefore, a retrospective multicenter study was performed to compare the diagnostic performance of three assays., Methods: The fluorescence enzyme-linked immunoassay (FEIA) EliA Celikey IgA (Phadia), the chemiluminescence immunoassays (CLIA) h-tTG IgA QUANTA Flash ® (Inova Diagnostics) and the anti-tTG ChLIA IgA (Euroimmun) were compared. Diagnostic samples from CD cases (95 adults; 65 children) and controls (479 adults; 253 children) were included. Samples were blinded and reanalyzed on all platforms., Results: A high quantitative correlation between platforms was found (p<0.0001). Both CLIA were more sensitive (adults 100%; children 100%) compared to the FEIA (adults 88.4%; children 96.6%). Specificity of all assays was high (≥97.6%) with the FEIA having the highest specificity. A cut-off based on receiver operator characteristic analysis (6.5 U/mL) improved the sensitivity of the FEIA (adults 95.8%; children 100%) without affecting specificity. Cut-off values for the CLIA assays did not need further optimization. With the FEIA, 71% of pediatric cases had a tTG-IgA level ≥10× upper limit of normal compared to 91 and 92% with QUANTA Flash and ChLIA, respectively., Conclusions: All platforms have high diagnostic accuracy. The CLIA assays are more sensitive compared to the FEIA assay. A lower cut-off for the FEIA improves diagnostic performance, particularly in adult cases that, as demonstrated in this study, present with lower tTG-IgA levels compared to pediatric cases., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Background: Bronchoalveolar (BAL) cellular analysis can be supportive in the diagnosis of interstitial lung disease. The flow cytometric analysis of BAL fluid cells is complicated by cell fragility and adherence and autofluorescence of macrophages, making conventional analysis of BAL fluid cells as done in external quality schemes (EQA) for blood lymphocyte subsets, not representative. Following a procedure for stabilized BAL cells, a separate EQA was set up. The results of 20 years' experience are presented., Methods: From each round between 2000 and 2020 the following flow cytometric parameters were recorded from each participant: total lymphocyte population (TLY), CD3+ lymphocytes, CD3+ CD4+ lymphocytes, CD3+ CD8+ lymphocytes, CD3- CD16+/56+ lymphocytes, CD19+ lymphocytes and CD103 + CD3+ lymphocytes. In addition, the eosinophils and neutrophils were recorded. The mean and standard deviation of each parameter per round were calculated. The 40 rounds were divided in four respective groups of 10 in order to compare the results as function of time. In addition the interpretation of the results of participants was scored., Results: The median SD in the four groups was below 10% for all parameters except for TLY and the CD103+ CD3+ lymphocytes. No improvement in time was observed for any (sub)population except for the CD3+ CD4+ subset. Interpretation of the results varied based on disease, with greatest consensus for sarcoidosis cases and lowest for nonspecific interstitial lung disease cases., Conclusions: A dedicated EQA for BAL fluid cellular analysis appears to be justified as the test material is substantially different from that of peripheral blood. We show that adequate analytical and post-analytical quality control can be achieved., (© 2022 International Clinical Cytometry Society.)

Background: Measuring calprotectin concentration in stool is increasingly important in monitoring disease activity and treatment response in inflammatory bowel disease. This study evaluates the impact of preanalytical storage conditions on reliability of calprotectin testing using 5 different calprotectin immunoassays., Methods: Aliquots of homogenized fresh fecal samples in untreated or extracted form were stored at room temperature or 4°C. Calprotectin concentration was measured day 0 to 4 and 8. Five different immunoassays and accompanying extraction buffers were used (CALiaGold, Phadia EliA, Bühlmann fCal turbo, ELISA Bühlmann, Inova Quanta Flash). Repeated measurements of change from baseline calprotectin levels over time were analyzed using a mixed model analysis., Results: Calprotectin concentrations declined over time under all preanalytical conditions with all assays, except for extracted feces stored at 4°C. The rate of decline was greatest in untreated stool kept at room temperature, reaching significant difference from baseline already after 1 day (P &lt; 0.001). In extracted feces kept at room temperature, significant difference from baseline was reached after 2 days, and in untreated feces at 4°C, after 4 days. However, the results differed significantly between assays. After 4 days of storage at room temperature, the mean calprotectin decline from baseline differed between 30% and 60%, dependent on the assay used., Conclusions: Fecal calprotectin concentration in stool samples declines over time, and the rate of decline is greater at higher temperatures. In extracted feces stored at 4°C, calprotectin is most stable. It is assay-dependent how long extracted feces stored at 4°C give reliable test results., Competing Interests: Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: None declared. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: Sysmex Europe GmbH facilitated the design of the study. Sysmex Nederland B.V. and Abbott Laboratories funded the kits used in the present study. These organizations played no role in analysis and interpretation of data, writing of the report, or in the decision to submit the findings for publication. P.F.v.R. received funding from Bühlmann laboratories for other projects. Other authors have no financial disclosures or conflicts of interest. Expert Testimony: None declared. Patents: None declared., (© American Association for Clinical Chemistry 2022.)

Background: The metabolic health index (MHI) is a biomarker-based model that objectively assesses the cumulative impact of comorbidities type 2 diabetes mellitus, hypertension and dyslipidemia on the health state of bariatric patients. The MHI was developed on a single-center cohort using a fully laboratory data-driven approach, resulting in a MHI score on a range from 1 to 6. To show universal applicability in clinical care, the MHI was validated externally and potential laboratory-related shortcomings were evaluated., Methods: Retrospective laboratory and national bariatric quality registry data were collected from five Dutch renowned bariatric centers (n = 11 501). MHI imprecision was derived from the cumulative effect of biological and analytical variance of the individual input variables of the MHI model. The performance of the MHI (model) was assessed in terms of discrimination and calibration., Results: The cumulative imprecision in MHI was 0.25 MHI points. Calibration of the MHI model diverged over the different centers but was accounted for by misregistration of comorbidity after cross-checking the data. Discriminative performance of the MHI model was consistent across the different centers., Conclusions: The MHI model can be applied in clinical practice of bariatric centers, regardless of patient mix and analytical platform. Because the MHI is based on objective parameters, it is insensitive to diverging clinical definitions of comorbidities. Therefore, the MHI can be used to objectify severity of metabolic comorbidities in bariatric patients. The MHI can support the patient-selection process for surgery and objectively assessing the effect of surgery on the metabolic health state., Competing Interests: Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest: Employment or Leadership: None declared. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: This study and A.-K. Boer were supported by the Catharina Research Fund. The authors received no financial support for the authorship, and/or publication of this article. Expert Testimony: None declared. Patents: None declared., (© American Association for Clinical Chemistry 2022. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Almost 2 years from the beginning of the coronavirus disease 2019 (COVID-19) pandemic, there is still a lot unknown how the humoral response affects disease progression. In this study, we investigated humoral antibody responses against specific SARS-CoV2 proteins, their strength of binding, and their relationship with COVID severity and clinical information. Furthermore, we studied the interactions of the specific receptor-binding domain (RBD) in more depth by characterizing specific antibody response to a peptide library., Materials and Methods: We measured specific antibodies of isotypes IgM, IgG, and IgA, as well as their binding strength against the SARS-CoV2 antigens RBD, NCP, S1, and S1S2 in sera of 76 COVID-19 patients using surface plasmon resonance imaging. In addition, these samples were analyzed using a peptide epitope mapping assay, which consists of a library of peptides originating from the RBD., Results: A positive association was observed between disease severity and IgG antibody titers against all SARS-CoV2 proteins and additionally for IgM and IgA antibodies directed against RBD. Interestingly, in contrast to the titer of antibodies, the binding strength went down with increasing disease severity. Within the critically ill patient group, a positive association with pulmonary embolism, d-dimer, and antibody titers was observed., Conclusion: In critically ill patients, antibody production is high, but affinity is low, and maturation is impaired. This may play a role in disease exacerbation and could be valuable as a prognostic marker for predicting severity., Competing Interests: Patents: Provisional patent is filed based on the data generated from this work. Provisional title: Strength of Binding of Antibodies Against Infectious Diseases Measured With Evanescent Field Based Biosensors. COVID-19 Patients With High Titers of Low Affinity Antibodies Are Associated With Disease Severity. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hendriks, Schasfoort, Koerselman, Dannenberg, Cornet, Beishuizen, van der Palen, Krabbe, Mulder and Karperien.)

Surface Plasmon Resonance imaging (SPRi) was used to determine the presence and strength of binding of IgG, IgM and IgA against the Receptor Binding Domain (RBD) of SARS-CoV-2 in sera of 119 CoViD-19 patients. The SPRi assay measures the antibody isotype levels and the strength of binding to the RBD of ultimate 384 patient samples in one run. It turns out that during the course of the disease, the IgG levels and strength of binding increased while generally the IgM and IgA levels go down. Recovered patients all show high strength of binding of the IgG type to the RBD protein. The anti-RBD immunoglobulins SPRi assay provides additional insights in the immune status of patients recovering from CoViD-19 and this new method can furthermore be applied for the assessment of the quality of the immune reaction of healthy individuals to SARS-CoV-2 in vaccination programs., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Flowcytometric analysis of lymphocytes and their subpopulations in bronchoalveolar lavages (BAL) can support the diagnosis of interstitial lung diseases. This analysis should be done within 4 hr after lavage due to rapid cell deterioration. We tested three methods in order to stabilize for at least 28 days the BAL cell populations to allow delayed flowcytometric analysis in order to facilitate external quality assurance (EQA)., Methods: We compared an in-house, dual-step stabilization method for BAL cells with results of two different commercial available stabilization reagents: TransFix® and Streck Cell Preservative™. All three methods were compared with native BAL cells as reference. BAL samples from six patients were tested on six occasions following stabilization from 1 to 28 days by flow cytometry., Results: Following stabilization and storage at 4°C, BAL cell suspensions had stable light scatter patterns and lymphocyte subsets. As expected, rapid deterioration of cells was seen with native BAL cells. The stabilized lavages showed more stable counts of WBC and lymphocyte populations with only minor differences found between the three methods., Conclusions: If analysis of the BAL cells is performed more than 24 hr after the lavage, stabilized BAL cells are superior to native cells. The in-house method can be used for EQA purposes with stability for at least 28 days. The TransFix and Streck methods might be useful for postponed diagnostic analysis of lavage cells but did not meet our 28 days criterion defined needed for EQA purposes., (© 2020 International Clinical Cytometry Society.)

Surface Plasmon Resonance imaging (SPRi) was used to determine the presence and strength of binding of IgG, IgM and IgA against the Receptor Binding Domain (RBD) of SARS-CoV-2 in sera of 102 CoViD-19 and non-CoViD-19 patients. The SPRi assay simultaneously measures the antibody isotype levels and the strength of binding to the RBD of ultimate 384 patient samples in one run. It turns out that during the course of the disease, the IgG levels and strength of binding increased while generally the IgM and IgA levels go down. Recovered patients all show high strength of binding of the IgG type to the RBD protein. The anti-RBD immunoglobulins SPRi assay provides additional insights in the immune status of patients recovering from CoViD-19. This new high throughput method can be applied for the assessment of the quality of the immune reaction of healthy individuals to SARS-CoV-2 and its mutants in vaccination programs.•Surface Plasmon Resonance imaging is an unprecedented technology for high throughput screening of antibody profiling of CoViD19 patients.•Fingerprinting of isotypes IgM, IgG and IgA can be performed for 384 patients in one run.•An affinity maturation effect was shown for patients recovering from CoViD19., (© 2021 Published by Elsevier B.V.)

Idiopathic inflammatory myopathies (IIM) are a heterogeneous group of connective tissue diseases, collectively known as myositis. Diagnosis of IIM is challenging while timely recognition of an IIM is of utter importance considering treatment options and otherwise irreversible (severe) long-term clinical complications. With the EULAR/ACR classification criteria (2017) considerable advancement has been made in the diagnostic workup of IIM. While these criteria take into account clinical parameters as well as presence of one autoantibody, anti-Jo-1, several autoantibodies are associated with IIM and are currently evaluated to be incorporated into classification criteria. As individual antibodies occur at low frequency, the development of line blots allowing multiplex antibody analysis has improved laboratory diagnostics for IIM. The Euroline myositis line-blot assay (Euroimmun) allows screening and semi-quantitative measurement for 15 autoantibodies, i.e . myositis specific antibodies (MSA) to SRP, EJ, OJ, Mi-2α, Mi-2β, TIF1-γ, MDA5, NXP2, SAE1, PL-12, PL-7, Jo-1 and myositis associated antibodies (MAA) to Ku, PM/Scl-75 and PM/Scl-100. To evaluate the clinical significance of detection and levels of these autoantibodies in the Netherlands, a retrospective analysis of all Dutch requests for extended myositis screening within a 1 year period was performed. A total of 187 IIM patients and 632 non-IIM patients were included. We conclude that frequencies of MSA and MAA observed in IIM patients in a routine diagnostic setting are comparable to cohort-based studies. Weak positive antibody levels show less diagnostic accuracy compared to positive antibody levels, except for anti-NXP2. Known associations between antibodies and skin involvement (anti-MDA5, anti-TIF1-γ), lung involvement (anti-Jo-1), and malignancy (anti-TIF1-γ) were confirmed in our IIM study population. The availability of multiplex antibody analyses will facilitate inclusion of additional autoantibodies in clinical myositis guidelines and help to accelerate diagnosing IMM with rare but specific antibodies., (© 2019 The Authors.)

Background: Hantavirus infection is an uncommon cause of acute renal failure with massive proteinuria. Serology tests to support a presumptive diagnosis usually take a few days. During the initial work-up, autoimmune causes including anti-glomerular basement membrane (GBM) glomerulonephritis need to be excluded, because these require urgent therapy. In this case the delay in serological testing caused a dilemma in treatment initiation., Case Presentation: An 18-year-old patient was admitted to the hospital with acute renal failure, erythrocyturia and massive proteinuria. Routine blood analysis showed leucocytosis (40,5 × 109/l) and a serum creatinine of 233 μmol/l. Infectious causes, e.g. leptospirosis or hantavirus infection, or an autoimmune disease, e.g., AAV or anti-GBM glomerulonephritis was the most feasible underlying diagnosis. Before hantavirus serology results were known, anti-GBM antibodies were positive. Treatment for anti-GBM glomerulonephritis was withheld, because of the absence of other signs and symptoms of the disease and slight improvement of renal function. The diagnosis of acute hantavirus infection was later on confirmed, by seroconversion of a follow-up serum sample. Without further intervention renal function recovered and anti-GBM antibodies disappeared., Conclusion: Hantavirus infection may induce anti-GBM antibodies, falsely suggestive of anti-GBM glomerulonephritis. Anti-GBM antibodies are supposed to be 100% specific. No earlier reports of false positive anti-GBM titers were reported. Nevertheless, the anti-GBM antibodies in this case were seen as an innocent bystander effect. Considering the need of urgent initiation of plasmapheresis and administration of immunosuppressants it may lead to diagnostic dilemmas with crucial therapeutic consequences. Knowledge of this anomaly when diagnosing acute renal failure, is very important.