Xavier Bossuyt

Background: In the American College of Rheumatology/European Alliance of Rheumatology Associations 2010 classification criteria for rheumatoid arthritis (RA), rheumatoid factor (RF) and anti-citrullinated protein/peptide antibodies (ACPA) are assigned the same weight, although ACPA have proven more specific than RF. We aimed to refine the serological weight factors for RA classification. Materials and methods: Diagnostic samples from 398 patients with RA and 448 diseased controls (DC) were evaluated with RF and ACPA assays from five different manufacturers. A grid search was conducted across all possible integer score pairs for RF and ACPA to identify the weighting combination that optimised the specificity of RA classification. This combination was applied in a refined scoring system (RSS) for three confirmatory cohorts comprising, respectively, 67, 157 and 132 patients with RA and 441, 153 and 193 DC. Findings: In the investigative cohort, the optimal combination of weighting scores was characterised by higher scores for ACPA, higher antibody levels and double RF/ACPA positivity. Applying the optimal combination of weighting scores decreased misclassification compared with the 2010 RA classification criteria, minimally affecting sensitivity. For seropositive patients, RSS significantly lowered misclassification from 38.3–55.8% to 15.0–26.9%. The improved specificity of RSS (compared with the 2010 criteria) was validated in the confirmatory cohorts. Considering only seropositive patients, the RSS significantly decreased RA misclassification (8.6–36.8% vs 20.0–78.9%), minimally affecting the sensitivity (86.7–97.3% vs 86.7–96.0%). Interpretation: Serological weight factors for RA classification can be improved by taking into account the antibody type, the antibody levels and single or combined positivity. The application of an RSS reduced the number of RA misclassifications.

Objectives: Autoantibodies to extractable nuclear antigens (anti-ENA) play a crucial role in the diagnosis of systemic autoimmune rheumatic diseases (SARD). Analytical methods differ in antigen composition and diagnostic performance. We aimed to document the extent of variability amongst laboratories in how anti-ENA testing is conducted, controlled and verified. Methods: A survey was developed to collect information on anti-ENA methodology and quality assurance practices. The questionnaire was distributed amongst laboratories by Sciensano (Belgium), national EASI groups (Italy, Croatia, Portugal, Estonia, Greece) and ICAP (worldwide). Results: A total of 427 laboratories participated (61 % European, 21 % American, 14 % Asian). More than half (52 %) use two or more anti-ENA methods. Turnaround time varied, but 71 % perform the analysis within 72 h. Urgent testing was offered in 42 % of laboratories. Internal quality control (IQC) was performed in 91 % of laboratories, predominantly with commercial materials. External quality assessment (EQA) participation was also high (87 %), with nearly half participating in more than three distributions annually. Reagent lot evaluation was less widely implemented (36 %). Forty-three percent tried to limit variability by limiting lot changes. Method verification was performed by 81 % of the laboratories, though sample type and numbers, as well as reproducibility replicates varied considerably. Conclusions: This survey highlights substantial heterogeneity in anti-ENA laboratory practice. While most laboratories apply IQC and EQA, significant variation remains in test method(s), turnaround times, lot variability evaluation and verification strategies. Development of more comprehensive recommendations on these topics are essential to improve reliability and comparability of results across laboratories.

Objective: In a fraction of patients with rheumatoid arthritis (RA), antinuclear antibodies (ANA) are present. However, in most ANA-positive samples from patients with RA, no autoantigens can be identified. We aimed to characterise the ANA landscape in RA and to identify known and novel autoantigens using an unbiased immunoprecipitation-mass spectrometry (IP-MS) approach. Methods: Sera from 51 ANA-positive, DMARD-naïve patients with RA were analysed by IP-MS using HeLa nuclear or cytoplasmic extracts (depending on the ANA staining pattern). Immunoprecipitated proteins were digested and identified using liquid chromatography coupled with MS. The prevalence of new or unfamiliar autoantigens was assessed in 43 healthy individuals and 88 ANA-positive individuals with a systemic rheumatic disease (systemic lupus erythematosus, Sjögren’s disease, systemic sclerosis and idiopathic inflammatory myopathy). Results: Autoantigens were detected in 10 of 51 RA samples (in 2/29 with titre 1/160, in 4/12 with titre 1/320 and in 4/10 with titre ≥1/640). Autoantigens identified included SSA-Ro60, SSB-La, Ku, fibrillarin, components of the mitochondrial multienzyme complex, protein argonaute 1, 2, 3 and T-cell restricted intracellular antigen 1 and TIA-1-related protein. Two novel autoantigens were discovered: the N-terminal acetyltransferase C (NatC) complex and mitochondrial chaperone BCS1. Neither NatC nor BCS1 was detected in healthy individuals or individuals with other systemic rheumatic diseases. Conclusion: IP-MS revealed a diverse spectrum of autoantigens in patients with ANA-positive RA, including previously unreported targets. These findings expand our knowledge of RA-associated autoimmunity and highlight antibodies to NatC and BCS1 as potential novel biomarkers. Further studies are needed to assess their clinical relevance.

To assess the prevalence of anti-aminoacyl tRNA synthetase antibodies (anti-ARS) in a large cohort of untreated early rheumatoid arthritis (RA) patients compared to healthy controls. The baseline serum samples of patients with early RA (n = 332) included in the multicentre CareRA trial, a completed interventional trial in untreated early RA, and healthy controls (n = 72) were screened for anti-ARS antibodies with a commercial dot immunoassay (DIA). Radiolabelled protein immunoprecipitation (IP) was performed to confirm the presence of suspected anti-ARS antibodies, in addition to HEp-2 indirect immunofluorescence and a commercial line immunoassay (LIA). Anti-ARS antibodies (anti-PL12 n = 2, anti-OJ n = 1) were detected in 0.9% of early RA patients using a DIA, of which one anti-PL12-positive sample was also positive on LIA. Presence of these anti-ARS antibodies could, however, not be confirmed by protein immunoprecipitation. No anti-ARS antibodies were detected in the healthy control group. We report that the presence of anti-ARS antibodies cannot be confirmed by IP in a multicentre cohort of untreated early RA patients. Anti-ARS autoantibodies should not routinely be evaluated in patients with early RA. Sensitivity and specificity issues with DIA and LIA for anti-ARS should be considered in the interpretation of anti-ARS testing.

Extract Screening for autoantibodies is an essential part of the work-up of interstitial lung disease (ILD) as detection of specific autoantibodies can lead to timely diagnosis and treatment of an underlying systemic autoimmune disease (SAD). In practice, screening for autoantibodies is mostly done with the HEp-2 indirect immunofluorescence assay (HEp-2 IIFA), often referred to as the antinuclear antibody (ANA) test, together with solid-phase assays for the most common SAD-associated autoantibodies. A positive HEp-2 IIFA without detection of a specific autoantibody in a patient with a radiographic usual interstitial pneumonia (UIP) pattern on high-resolution computed tomography (HRCT) of the chest is a common clinical scenario. Shareable abstract Immunoprecipitation–mass spectrometry can identify new and rare autoantigens in persons with ANA-positive usual interstitial pneumonia without a diagnosis of a definite systemic autoimmune disease https://bit.ly/4iAIRK5

Objectives: This study aimed to perform an extended analytical verification of the Immunoglobulin Isotypes (GAM) for the EXENT ^® Analyzer (EXENT ^® -GAM) assay, a MALDI-TOF mass spectrometry-based method for detecting and quantifying serum M-proteins in patients with plasma cell dyscrasias, and to compare it with conventional serum protein electrophoresis (SPEP), serum immunofixation electrophoresis (sIFE) and serum free light chains (sFLC) assays. Methods: Imprecision, linearity, limit of quantification (LOQ), quantification comparison with SPEP, isotyping concordance with sIFE and sFLC, interference from therapeutic monoclonal antibodies (t-mAbs), sample stability, and reagent lot consistency were evaluated. Results: EXENT ^® -GAM demonstrated acceptable imprecision (CV ≤20 % for low and ≤15 % for high M-protein levels) and wide linear range (∼0.03–30 g/L). The polyclonal immunoglobulin background negatively influenced the assays LOQ. M-proteins with mass-shifted light chains (i.e., glycosylated light chains) are prone to non-linearity and inferior LOQ. M-protein quantification by EXENT ^® differed systematically and proportionally from quantification by SPEP, highlighting non-interchangeability. EXENT ^® demonstrated 97 % concordance with sIFE for M-protein isotyping and identified numerous additional (low-level) M-proteins. Some proved to be clinically relevant (residual disease in sIFE-negative samples); others lacked correlation with sFLC result or clinical diagnosis. EXENT ^® reliably distinguished endogenous M-proteins from t-mAbs, except talquetamab, which interfered with quantification and was partially misclassified. Conclusions: EXENT ^® -GAM enables sensitive and reproducible quantification and isotyping of M-proteins below the detection limit of SPEP and sIFE. Its ability to resolve analytical challenges posed by SPEP and sIFE represents a significant advancement. Further clinical studies are needed to confirm its potential in residual disease detection.

Introduction: Rheumatoid factor (RF) are autoantibodies that are found in approximately two thirds of patients with rheumatoid arthritis, a chronic autoimmune disease characterized by potentially destructive inflammation of the joints. RF consists of polyclonal antibodies targeting the Fc part of immunoglobulin G. Despite its clinical relevance, RF is not specific for RA, and conventional assays for RF detection, predominantly solid-phase tests detecting IgM RF, suffer from poor harmonization and the disability to test more than one RF isotype.Methods: We studied RF using a mass-spectrometry-based approach in RF(+), RF(–) rheumatoid arthritis patients and in disease controls. This allowed evaluation of RF at the amino acid level, including the variable and hypervariable region part of RF. RF was captured on Fc coated microwell plates, isolated, digested into peptides and analyzed by liquid chromatography tandem mass spectrometry. An initial proof-of-concept analysis was conducted comprising 12 samples, followed by a larger-scale experiment comprising 86 samples.Results: Principal component analysis and sparse partial least squares discriminant analysis demonstrated that RF(+) RA patients displayed peptides that were differentially expressed compared with disease control patients. Framework region-derived peptides, variable region-derived peptides as well as de novo sequenced peptides not present in the human proteome database, were found to be enriched in RF(+) sera compared to disease control sera. Interestingly, some of these peptides were also upregulated in sera from RF(–) RA patients. Furthermore, mass spectrometry analysis revealed different RF isotypes. In addition to IgM, also IgA and IgG isotypes were observed. RF-IgG2 isotype was observed in RF(+) as well as in RF(–) RA patients.Discussion: In summary, our findings highlight that mass spectrometry provides a platform for elucidating the heterogeneity and isotypic diversity of RF autoantibodies in RA, overcoming limitations inherent to current solid-phase RF assays. Upregulated de novo peptides were found, possibly related to the hypervariable regions of RF. Further validation using integrated proteomic and genomic approaches is required to confirm these novel peptides and their localization within the RF hypervariable regions.

We describe the case of a 50-year old woman presenting with hypertension-associated thrombotic microangiopathy (TMA) and nephrotic syndrome as an unusual initial presentation of a cryoglobulinemic glomerulonephritis secondary to primary Sjögren's disease. This case first highlights the importance of a thorough and systemic work-up in patients with TMA, given the broad differential diagnosis and diagnostic complexity. When initial evaluation does not reveal an underlying cause and TMA parameters improve with blood pressure control, a diagnosis of hypertension-associated TMA may be considered. However, hypertension-associated TMA is often secondary to an underlying auto-immune or primary renal disease, warranting further investigation. In this case, kidney biopsy revealed a membranoproliferative glomerulonephritis with hyaline thrombi. Electron microscopy showed subendothelial electron dense deposits, and serum analysis confirmed the presence of mixed cryoglobulins, establishing the diagnosis of cryoglobulinemic glomerulonephritis. Primary Sjögren's disease was suspected based on positive anti-Ro60 and anti-Ro52 antibodies and supported by the presence of sicca symptoms, and was finally confirmed by a positive Schirmer's test. This case illustrates the diagnostic value of a kidney biopsy in identifying the underlying etiology of TMA and it also highlights the importance of considering cryoglobulinemia in the differential diagnosis of membranoproliferative glomerulonephritis.