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Background and Aims: Rheumatoid arthritis (RA) manifests through various symptoms and systemic manifestations. Diagnosis involves serological markers like rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA). Past studies have shown the added value of likelihood ratios (LRs) in result interpretation. LRs can be combined with pretest probability to estimate posttest probability for RA. There is a lack of information on pretest probability. This study aimed to estimate pretest probabilities for RA., Materials and Methods: This retrospective study included 133 consecutive RA patients and 651 consecutive disease controls presenting at a rheumatology outpatient clinic. Disease characteristics, risk factors associated with RA and laboratory parameters were documented for calculating pretest probabilities and LRs., Results: Joint involvement, erosions, morning stiffness, and positive CRP, ESR tests significantly correlated with RA. Based on these factors, probabilities for RA were estimated. Besides, LRs for RA were established for RF and ACPA and combinations thereof. LRs increased with antibody levels and were highest for double high positivity. Posttest probabilities were estimated based on pretest probability and LR., Conclusion: By utilizing pretest probabilities for RA and LRs for RF and ACPA, posttest probabilities were estimated. Such approach enhances diagnostic accuracy, offering laboratory professionals and clinicians insights in the value of serological testing during the diagnostic process., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [XB received lecture fees from Thermo Fisher and Werfen and has been a consultant for Werfen. LVH received lecture fees from Thermo Fisher and Werfen andhas been a consultant for Thermo Fisher]., (Copyright © 2024. Published by Elsevier B.V.)

Introduction: Proper implementation of Point-of-Care testing (POCT) for C-reactive protein (CRP) in primary care can decrease the inappropriate use of antibiotics, thereby tackling the problem of growing antimicrobial resistance., Objective: The analytical performance and user-friendliness of four POCT-CRP assays were evaluated: QuikRead go easy, LumiraDx, cobas b 101 and Afinion 2., Materials and Methods: Imprecision was evaluated using plasma pools in addition to manufacturer-specific control material. Trueness was assessed by verification of traceability to ERM-DA474/IFCC in parallel to method comparison towards the central laboratory CRP method (cobas c 503) using i) retrospectively selected plasma samples (n = 100) and ii) prospectively collected capillary whole blood samples (n = 50). User-friendliness was examined using a questionnaire., Results: Between-day imprecision on plasma pools varied from 4.5 % (LumiraDx) to 11.5 % (QuikRead). Traceability verification revealed no significant difference between cobas c 503 CRP results and the ERM-DA474/IFCC certified value. cobas b 101 and Afinion achieved the best agreement with the central laboratory method. LumiraDx and QuikRead revealed a negative mean difference, with LumiraDx violating the criterion of > 95 % of POCT-CRP-results within ± 20 % of the comparison method. Regarding user-friendliness, Afinion obtained the highest Likert-scores., Conclusion: The analytical performance and user-friendliness of POCT-CRP devices varies among manufacturers, emphasizing the need for quality assurance supervised by a central laboratory., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Autoantibodies are important laboratory markers to support diagnosis of autoimmune diseases. Interpretation of autoantibodies is classically done in a dichotomous way (positive versus negative). Yet, interpretation of autoantibody test results can be improved by reporting likelihood ratios. Likelihood ratios convey information on how much more/less likely a test result is in individuals with the disease compared to individuals without the disease. It incorporates information on the antibody level (the higher the antibody level, the higher the association with the disease), which is helpful for (differential) diagnosis. Likelihood ratios are unit-independent and allow users to harmonize test result interpretation. When the likelihood ratio is combined with information on the pre-test probability, post-test probability can be appraised. In this review, the applicability of likelihood ratio in autoimmune diagnostics will be reviewed from the perspective of the clinician, the laboratory professional and the in vitro diagnostic industry., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xavier Bossuyt reports a relationship with Thermo Fisher Scientific Inc. that includes: speaking and lecture fees. Xavier Bossuyt reports a relationship with Werfen that includes: consulting or advisory and speaking and lecture fees. Kevin Deane reports a relationship with Werfen that includes: consulting or advisory. Kevin Deane reports a relationship with Thermo Fisher Scientific Inc. that includes: consulting or advisory. Lieve Van Hoovels reports a relationship with Thermo Fisher Scientific Inc. that includes: speaking and lecture fees. Veena Joy reports a relationship with Thermo Fisher Scientific Inc. that includes: employment. Nina Olschowka reports a relationship with Thermo Fisher Scientific Inc. that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Current classification criteria for rheumatoid arthritis (RA) encompass clinical and immunological items and are capable of correctly identifying the majority of symptomatic RA patients. The presence of positive rheumatoid factor (RF) and/or and anti-cyclic citrullinated protein/peptide antibodies (ACPA) gaining increasing importance according to their serological titer eases the recognition of RA, yet the debate is open on whether this scoring system ought to be optimized by hierarchizing ACPA or the combination of ACPA and RF over single positivity, prioritizing specificity over sensitivity. The risk of misdiagnosis and misclassification are often entangled, yet they are not the same. In fact, while ideal diagnosis requires 100% sensitivity and specificity, classification criteria are conceived to gather a homogeneous patient population, favoring specificity over sensitivity. Nevertheless, as they are frequently summoned to support the diagnostic process in clinical practice, issues arise on how comprehensive those should be and on how frequently they should be updated in light of novel acquisitions regarding measurable RA-related abnormalities. In this viewpoint two different views on the topic are confronted, discussing the performance of available criteria and the potentiality and pitfalls of their refinement according to novel data on ACPA and RF contribution and emergence of newly discovered specificities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Aim: Calprotectin (CLP) is a heterodimeric complex formed by two S100 proteins (S100A8/A9), which plays a pivotal role in innate immunity. Due to its intrinsic cytotoxic and proinflammatory properties, CLP controls cell differentiation, proliferation and NETosis and has been associated with a wide range of rheumatic diseases. Our review summarizes the widespread interest in circulating CLP (cCLP) as a biomarker of neutrophil-related inflammation, in autoimmune rheumatic disease (ARD) and non-ARD., Methods: A thorough literature review was performed using PubMed and EMBASE databases searching for circulating calprotectin and synonyms S100A8/A9, myeloid-related protein 8/14 (MRP8/MRP14), calgranulin A/B and L1 protein in addition to specific ARDs and autoimmune non-rheumatic diseases. We selected only English-language articles and excluded abstracts without the main text., Results: High cCLP serum levels are associated with worse structural outcomes in rheumatoid arthritis and to a lesser extent, in spondyloarthritis. In addition, cCLP can predict disease relapse in some autoimmune diseases including systemic lupus erythematosus (SLE), anti-neutrophil cytoplasmic antibodies-associated vasculitis (AAV) and some severe manifestations of connective tissue diseases, such as glomerulonephritis in SLE, AAV, juvenile idiopathic arthritis, adult-onset Still's disease and lung fibrosis in systemic sclerosis. Therefore, cCLP levels enable the identification of patients who need an accurate and tight follow-up. The clinical usefulness of cCLP as an inflammatory marker has been suggested for inflammatory/autoimmune non-rheumatic diseases, and especially for the monitoring of the inflammatory bowel diseases patients. Currently, there are only a few studies that evaluated the cCLP efficacy as a clinical biomarker in inflammatory/autoimmune non-rheumatic diseases with controversial results. Future studies are warranted to better clarify the role of cCLP in relation to the disease severity in myasthenia gravis, multiple sclerosis, chronic inflammatory demyelinating polyneuropathy, Graves' orbitopathy, autoimmune bullous diseases and uveitis., Conclusion: Our literature review supports a relevant role of cCLP as potential prognostic biomarker mirroring local or systemic inflammation, especially in chronic inflammatory rheumatic diseases., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interests, (Copyright © 2023 Elsevier B.V. All rights reserved.)

Antibodies to dsDNA are an important laboratory parameter for diagnosis, monitoring and classification of systemic lupus erythematosus (SLE). In clinical laboratories, several techniques are used to detect and quantify anti-dsDNA antibodies. Each technique has its advantages and disadvantages regarding sensitivity, specificity, avidity and assay procedure. Assays differ with respect to the antigen source (native versus synthetic versus molecular biological) used and the way the antigen is presented (e.g. in solution, covalently linked to a solid phase,…). Consequently, correlation between assays can be poor and standardization of anti-dsDNA antibody tests is challenging. We here provide an overview of the currently available anti-dsDNA tests frequently used in clinical laboratories [Crithidia luciliae immunofluorescence test (CLIFT), Enzyme linked immune sorbent assay (ELISA), fluoroenzyme immunoassay (FEIA), chemiluminisence immunoassay (CIA), multiplexed bead-based assays and Farr-RIA] and their performance characteristics. From this literature study, we concluded that performance characteristics differ between assays. Often, a combination of techniques is necessary for the best result interpretation., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Introduction: During the recent SARS-CoV-2 pandemic, circulating calprotectin (cCLP) gained interest as biomarker to predict the severity of COVID-19. We aimed to investigate the prognostic value of cCLP measured in serum, heparin, EDTA and citrate plasma., Materials and Methods: COVID-19 patients were prospectively included, in parallel with two SARS-CoV-2 negative control populations. The prognostic value of cCLP was compared with IL-6, CRP, LDH, procalcitonin, and the 4C-mortality score by AUROC analysis., Results: For the 136 COVID-19 patients, cCLP levels were higher compared to the respective control populations, with significantly higher cCLP levels in serum and heparin than in EDTA or citrate. Higher cCLP levels were obtained for COVID-19 patients with i) severe/critical illness (n = 70), ii) ICU admission (n = 66) and iii) need for mechanical ventilation/ECMO (n = 25), but iv) not in patients who deceased within 30 days (n = 41). The highest discriminatory power (AUC [95% CI]) for each defined outcome was i) CRP (0.835 [0.755-0.914]); ii) EDTA cCLP (0.780 [0.688-0.873]); iii) EDTA cCLP (0.842 [0.758-0.925]) and iv) the 4C-mortality score (0.713 [0.608-0.818])., Conclusion: Measuring cCLP in COVID-19 patients helps the clinician to predict the clinical course of COVID-19. The discriminatory power of EDTA and citrate plasma cCLP levels often outperforms heparin plasma cCLP levels., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Calprotectin (CLP) is a promising biomarker for the evaluation of neutrophil-related inflammation. Our aim was to establish reference values for circulating CLP in different sample types and to study the effect of pre-analytical variables., Methods: Reference values were determined in 100 healthy individuals. Pre-analytical variables were evaluated in 10 healthy controls and four rheumatoid arthritis patients with active disease and covered sample type (serum with/without gel separator, heparin, EDTA and citrate plasma), pre-centrifugation time (<2 h, 6 h, 24 h), storage condition (2-8 °C, 18-25 °C, 30 °C) and storage time (24 h, 72 h, 7 days). CLP measurements were performed with the EliA™Calprotectin 2 assay on Phadia™200 (Thermo Fisher Scientific)., Results: In healthy controls, baseline CLP concentrations in serum were more than double the concentration in EDTA and citrate plasma (0.909 µg/mL versus 0.259 µg/mL and 0.261 µg/mL respectively). Heparin, EDTA and citrate stabilized CLP concentrations for up to 6 h before centrifugation, whereas significant increases in CLP levels were observed when serum was left untreated during that time period., Conclusion: Clinical studies on circulating CLP need to apply sample type-specific reference values and decision limits. To obtain reproducible CLP results in serum, more stringent pre-analytical sample handling instructions are needed., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Objective: Analytical validation of newly released SARS-CoV-2 antibody assays in the clinical laboratory is crucial to ensure sufficient performance in respect to its intended use. We aimed to assess analytical and diagnostic performance of 8 (semi-)quantitative assays detecting anti-nucleocapsid IgG (Euroimmun, Id-Vet) or total Ig (Roche), anti-spike protein IgG (Euroimmun, Theradiag, DiaSorin, Thermo Fisher) or both (Theradiag) and 2 rapid lateral flow assays (LFA) (AAZ-LMB and Theradiag)., Methods: Specificity was evaluated using a cross-reactivity panel of 85 pre-pandemic serum samples. Sensitivity was determined at both the manufacturer's and a 95% specificity cut-off level, using 81 serum samples of patients with a positive rRT-PCR. Sensitivity was determined in function of time post symptoms onset., Results: Specificity for all assays ranged from 92.9% to 100% (Roche and Thermo Fisher) with the exception of the Theradiag IgM LFA (82.4%). Sensitivity in asymptomatic patients ranged between 41.7% and 58.3%. Sensitivity on samples taken <10 days since symptom onset was low (23.3%-66.7%) and increased on samples taken between 10 and 20 days and > 20 days since symptom onset (80%-96% and 92.9%-100%, respectively). From 20 days after symptom onset, the Roche, Id-vet and Thermo Fisher assays all met the sensitivity (>95%) and specificity (>97%) targets determined by the WHO. Antibody signal response was significantly higher in the critically ill patient group., Conclusion: Antibody detection can complement rRT-PCR for the diagnosis of COVID-19, especially in the later stage, or in asymptomatic patients for epidemiological purposes. Addition of IgM in LFAs did not improve sensitivity., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The clinical presentation of celiac disease (CD) varies between children. The objective of this study was to document the pre-test probability for CD based on symptoms and routine laboratory test and to evaluate the performance of two IgA anti-tissue transglutaminase (tTG) assays. We critically reviewed the concept of using multiples of the manufacturer's upper limit of normal (ULN), as proposed in the ESPGHAN guidelines (if IgA tTG is >10 times ULN, no biopsy is needed)., Methods: The retrospective study included 91 children with newly diagnosed CD and 605 controls (<16 years). All underwent upper endoscopy with small bowel biopsies. Four laboratory parameters and 16 symptoms were registered. All patients were tested for IgA anti-tTG antibodies with assays from Inova Diagnostics and Thermo Fisher Scientific., Results: Some combinations of clinical symptoms and laboratory parameters had a high pre-test probability for CD, such as (combinations of) anorexia, failure to thrive, low ferritin level and elevated AST. The diagnostic performance of both IgA anti-tTG assays was excellent and comparable (no difference in ROC curve area under the curve). At a threshold that corresponds to a specificity of 100% (5 times ULN for Inova Diagnostics and 2 times ULN for Thermo Fisher), the sensitivity was 82% for both assays. At the 10 times ULN threshold, the sensitivity differed between the assays (77% vs. 57%), indicating that such threshold does not completely align interpretation across companies., Conclusions: Our study showed that some combinations of symptoms and aberrant laboratory parameters had a high pre-test probability. The use of the ESPGHAN non-biopsy approach could reduce small bowel biopsies, but thresholds for IgA-tTG levels are not aligned across assays and should be based on predefined likelihood ratios or specificity., Competing Interests: Declaration of Competing Interest PV is a senior clinical investigator of the Fund for Scientific Research – Flanders. XB has been consultant for Inova Diagnostics and Thermo Fisher., (Copyright © 2020. Published by Elsevier B.V.)

Herpes simplex virus (HSV) has increasingly been associated with pulmonary disease in critically ill patients. However, the clinical relevance of HSV is still a topic of debate. Monitoring of HSV in a quantitative way could potentially give relevant information on its role in the pathogenesis of lower respiratory tract infection. A fast and reliable quantitative real-time PCR (Q-PCR) for the quantitative detection of HSV-1 and HSV-2 DNA was developed. A prospective observational study was performed in an intensive-care unit (ICU) to monitor the HSV viral load in lower respiratory tract aspirates of long-term mechanically ventilated patients. HSV was common in the lower respiratory tract (LRT) of critically ill patients with mechanical ventilation for at least 48 h (62%, n = 65/105). Detection of HSV was significantly associated with prolonged mechanical ventilation (p <0.01), prolonged ICU stay (p <0.01), and development of ventilator-associated pneumonia (p = 0.02). Corticosteroid administration (p <0.01) in the ICU and anti-HSV IgG seropositivity (p <0.01) were risk factors for the occurrence of HSV in the LRT. The fact that no HSV-seronegative patient became positive suggests that all HSV DNA-positive patients had HSV reactivations. Monitoring the HSV viral load in the LRT of critically ill patients showed a typical homogeneous pattern of HSV kinetics. HSV emerged in tracheal and bronchial aspirates after a median of 7 days of intubation (5-11 days), and this was followed by an exponential increase (c. 1 log copies/mL/day) to reach very high HSV peaks (10(6)-10(10) copies/mL) in 78% of the HSV DNA-positive patients.

Background: Anti-neutrophil cytoplasmic antibodies (ANCA) are associated with primary small vessel vasculitis (SVV). Proteinase-3 (PR3)-ANCA are primarily associated with Wegener granulomatosis, whereas myeloperoxidase (MPO)-ANCA are primarily associated with microscopic polyangiitis (MPA) and vasculitic Churg-Strauss syndrome. We evaluated whether a strategy that is based on screening with ELISA or fluoroenzymeimmunoassay (FEIA) is an accurate alternative to screening with indirect immunofluorescence (IIF)., Methods: C-ANCA and P-ANCA were determined by IIF and PR3-ANCA and MPO-ANCA were determined by ELISA (Inova) or FEIA (Phadia) on 326 patients (38 with newly diagnosed SVV and 288 diseased controls)., Results: Specificity and positive likelihood ratios were higher for ELISA and FEIA than for IIF. Post-test probability for SVV of a positive test result was higher for ELISA and FEIA than for IIF. Decision tree analysis in which several testing strategies were compared revealed that a testing strategy that is based on screening with ELISA or FEIA had an expected clinical utility that was comparable to screening with IIF and confirming with ELISA or FEIA. The highest expected clinical utility was found when both IIF and ELISA or FEIA were performed on all samples., Conclusions: A strategy based on screening for ANCA with ELISA or FEIA (without prior IIF) is a valuable alternative to screening with IIF and confirming with ELISA or FEIA.