Establishment of ELISA-comparable moderate and high thresholds for anticardiolipin and anti-ß2 glycoprotein I chemiluminescent immunoassays according to the 2023 ACR/EULAR APS classification criteria and evaluation of their diagnostic performance.

OBJECTIVES: Recently published 2023 ACR/EULAR APS classification criteria emphasize the importance of quantifying single-, double-, and triple-antiphospholipid antibody positivity, distinguishing between IgG and IgM isotypes, and delineating moderate/high levels of anticardiolipin (aCL) and anti-ß2 glycoprotein I (anti-ß2GPI) antibodies. We aimed to establish clinically important moderate/high thresholds for aCL and anti-ß2GPI IgG/IgM chemiluminescent immunoassays (CLIA), in particular QUANTA Flash, comparable to our in-house ELISAs used for over two decades, and to evaluate their diagnostic performance. METHODS: QUANTA Flash CLIA and in-house ELISAs were used to measure aCL and anti-ß2GPI IgG/IgM. Moderate thresholds for QUANTA Flash CLIA were determined using a non-parametric approach, calculating a 99th percentile on serum samples from 139 blood donors, and by mirroring the diagnostic performance of in-house ELISA on 159 patient samples. RESULTS: Thresholds for QUANTA Flash CLIA achieving diagnostic performance equivalent to in-house ELISAs were 40 CU for moderate and 80 CU for high levels for aCL and anti-ß2GPI IgG and IgM. The assays showed good qualitative agreement, ranging from 76.10 to 91.19 %. When considering in-house ELISA results, 14 out of 80 (17.5 %) patients did not fulfill the new ACR/EULAR laboratory classification criteria, while 27 out of 80 (33.8 %) did not when considering QUANTA Flash CLIA results. CONCLUSIONS: We determined moderate and high thresholds for aCL and anti-ß2GPI IgG and IgM detected with QUANTA Flash CLIA, aligning with long-established in-house ELISA thresholds. These thresholds are crucial for seamlessly integrating of the new 2023 ACR/EULAR classification criteria into future observational clinical studies and trials.

Antiphospholipid Antibody Syndrome-Associated Increased Surface Expression of VLA4 Integrin on Human Monocytes.

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by thrombosis and/or obstetric complications in the presence of antiphospholipid antibodies (aPL). Catastrophic APS (CAPS) is the most severe form of the disease, in which microvascular thromboses develop rapidly, leading to multiorgan failure. Monocytes, along with endothelial cells, are critical players in the pathogenesis of APS. Recruitment of these cells to the site of injury/inflammation involves a series of events, including capture, rolling, adhesion enhancement, and transmigration, which are controlled by surface adhesion molecules. The aim of our study was to investigate the surface adhesion profile of monocytes from APS patients and monocytes stimulated in vitro with aPL from a CAPS patient. The surface expression of the adhesion molecules LFA1, L-selectin, MAC1, PSGL1, and VLA4 was analyzed by flow cytometry. To our knowledge, this preliminary study was the first to show that VLA4 was significantly increased on the surface of monocytes from APS patients. Moreover, in vitro stimulations mimicking CAPS showed an even greater increase in VLA4. Our data suggest that the surface adhesion profile on monocytes is altered in APS and CAPS and may be involved in the thrombotic pathophysiology of the disease by enhancing monocyte adhesion.

The Predictive Value of the aCL and Anti-ß2GPI at the Time of Acute Deep Vein Thrombosis-A Two-Year Prospective Study.

Antiphospholipid syndrome (APS) is an important cause of deep vein thrombosis (DVT). According to current APS classification criteria, APS cannot be confirmed until 24 weeks after DVT. This time frame results in frequent discontinuation of anticoagulant treatment before APS is diagnosed. Therefore, the aim of our study was to evaluate the potential predictive value of anticardiolipin (aCL) and anti-ß2glycoprotein I (anti-ß2GPI) before discontinuation of anticoagulation therapy. Patients with newly diagnosed DVT were included into a 24-month prospective study. All patients received anticoagulant therapy. aCL and anti-ß2GPI were determined at inclusion and every four weeks for the first 24 weeks and then one and two years after inclusion. APS was confirmed in 24/221 (10.9%) patients. At the time of acute DVT 20/24 (83.3%), APS patients had positive aCL and/or anti-ß2GPI. Two patients had low aCL levels and two were negative at the time of acute DVT but later met APS criteria due to lupus anticoagulant (LA). Our data indicate that negative aCL and/or anti-ß2GPI at the time of acute DVT make further aPL testing unnecessary; however, LA should be determined after discontinuation of anticoagulant therapy. Positive aCL and/or anti-ß2GPI at the time of acute DVT have a strong positive predictive value for APS and may support therapeutic decisions.

Adrenal failure and antiphospholipid syndrome.

We herein report two cases of primary adrenal insufficiency (AI) associated with antiphospholipid syndrome (APS). In both patients, the main finding that led to the diagnosis was hyponatraemia. The major difference between the two cases was the time at which AI evolved during the course of APS. In the first patient, AI developed acutely along with other presenting features of APS. In the second patient, the AI was unmasked during a stressful situation induced by severe inflammation that occurred 7 years after the first APS manifestation and had probably evolved slowly during the previous few years. These cases emphasise the importance of considering AI in patients with either suspected or newly diagnosed APS as well as in patients who have long been known to have APS. The symptoms and signs alerting the clinician to possible AI are general abdominal complaints, fever, hypotension, and hyponatraemia. Conversely, patients with primary AI should be questioned about the signs and symptoms of APS.

Characterization of Plasma-Derived Small Extracellular Vesicles Indicates Ongoing Endothelial and Platelet Activation in Patients with Thrombotic Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is a systemic autoimmune disease, characterized by thrombosis, obstetric complications and the presence of antiphospholipid antibodies (aPL), which drive endothelial injury and thrombophilia. Extracellular vesicles (EVs) have been implicated in endothelial and thrombotic pathologies. Here, we characterized the quantity, cellular origin and the surface expression of biologically active molecules in small EVs (sEVs) isolated from the plasma of thrombotic APS patients (n = 14), aPL-negative patients with idiopathic thrombosis (aPL-neg IT, n = 5) and healthy blood donors (HBD, n = 7). Nanoparticle tracking analysis showed similar sEV sizes (110-170 nm) between the groups, with an increased quantity of sEVs in patients with APS and aPL-neg IT compared to HBD. MACSPlex analysis of 37 different sEV surface markers showed endothelial (CD31), platelet (CD41b and CD42a), leukocyte (CD45), CD8 lymphocyte and APC (HLA-ABC) cell-derived sEVs. Except for CD8, these molecules were comparably expressed in all study groups. sEVs from APS patients were specifically enriched in surface expression of CD62P, suggesting endothelial and platelet activation in APS. Additionally, APS patients exhibited increased CD133/1 expression compared to aPL-neg IT, suggesting endothelial damage in APS patients. These findings demonstrate enhanced shedding, and distinct biological properties of sEVs in thrombotic APS.

Added value of non-criteria antiphospholipid antibodies for antiphospholipid syndrome: lessons learned from year-long routine measurements.

The international classification criteria for definite antiphospholipid syndrome (APS) include three laboratory measurements: lupus anticoagulant (LA), IgG and IgM isotypes of anti-cardiolipin (aCL) and anti-ß2glycoprotein I antibodies (anti-ß2GPI). When persistently elevated, they are specific for APS; however, many patients that fulfil clinical criteria may exhibit negative serological results. These "seronegative" APS (SN-APS) are exposed to an increased thrombotic risk. The aims of our cross-sectional, retrospective study of consecutive autoimmune patients' samples were to evaluate the association of non-criteria antiphospholipid antibodies (aPL) with thrombosis and obstetric events, to calculate the risk score for adverse events and to assess the specific contribution of single aPL positivity in SN-APS. LA, aCL, anti-ß2GPI and anti-phosphatidylserine/prothrombin antibodies (aPS/PT) of IgG, IgM, and IgA isotypes were determined in sera of 323 patients with autoimmune disorders. Medical records of all patients were carefully analyzed. aCL, anti-ß2GPI and aPS/PT of IgG and IgA isotypes were significantly associated with thrombosis while none of the IgM aPL showed such association. aPS/PT of all isotypes, aCL and anti-ß2GPI of IgG and IgA isotype showed significant correlation to obstetric events. When considering results of aPS/PT ELISA, we could additionally identify 3% of thrombotic patients and 2% of obstetric patients. Thrombotic and obstetric risk scores were calculated showing significantly higher association to clinical events, as compared to evaluating individual risk factors. aPS/PT could represent an additional biomarker in SN-APS patients. IgA aPL are associated with thrombosis and obstetric complications. Risk scores accounting different aPL and conventional risk factors, better assesses risk for adverse event, as compared to evaluating individual factors alone.

Stroke and antiphospholipid syndrome-antiphospholipid antibodies are a risk factor for an ischemic cerebrovascular event.

Testing for antiphospholipid antibodies could be an important part in determining the cause of a cerebrovascular event (CVE). Currently, it is also unknown whether antiphospholipid antibodies represent a risk factor for the development of a CVE and whether the selected therapy options are efficacious. So, this study aimed at (1) determining the frequency of patients experiencing a CVE and fulfilling the laboratory criterion for an antiphospholipid syndrome (APS), (2) investigating whether the persistent presence of antiphospholipid antibodies represented a risk factor for a CVE, and (3) focusing on the efficacy of the selected treatment strategy in the first year after the CVE. Eighty-nine patients with an acute CVE were prospectively followed for 1 year. At least two sera from each were tested for lupus anticoagulants, anticardiolipin, anti-ß2-glycoprotein I, anti-phosphatidylserine/prothrombin and anti-annexin V antibodies. Twenty out of eighty-nine (22%) of CVE patients fulfilled the criteria for APS (17/20 for definitive and 3 for probable APS). There was a significant association between persistently present antiphospholipid antibodies and the CVE (OR, 4.62). No statistically significant difference was found in the CVE recurrence rate between APS-CVE and non-APS-CVE patients being treated mainly with acetyl salicylic acid. Antiphospholipid antibodies represent an independent risk factor for a CVE. In the first year after the CVE, antiplatelet therapy seemed to be sufficient in secondary CVE thromboprophylaxis in most APS patients.

Anti-Phosphatidylserine/Prothrombin Antibodies Are Associated with Adverse Pregnancy Outcomes.

Objective. To determine the prevalence and clinical association of anti-phosphatidylserine/prothrombin antibodies (aPS/PT) in patients with a history of pregnancy complications relevant to antiphospholipid syndrome (APS). Materials and Methods. Two hundred and eleven patients with a history of (a) three or more consecutive miscarriages before 10th week of gestation (WG) (n = 64), (b) death of a morphologically normal fetus beyond 10th WG (n = 72), (c) premature birth of a morphologically normal neonate before 34th WG due to eclampsia, preeclamsia and placental insufficiency (n = 33), and (d) less than three unexplained consecutive miscarriages before 10th WG (n = 42). Subjects sera were analyzed for lupus anticoagulant (LA), anti-cardiolipin (aCL), anti-ß 2-glycoprotein I (anti-ß 2GPI), and aPS/PT antibodies. Results. 41/169 (24.3%) of patients were positive for at least one measured aPL. The highest prevalence was found for aPS/PT and aCL (13.0% and 12.4%, resp.) followed by LA (7.7%) and anti-ß 2GPI (7.1%). 11/169 with APS-related obstetric manifestations had only aPS/PT. 17.8% of patients were positive for LA or aCL and/or anti-ß 2GPI; however when adding the aPS/PT results, an additional 7% of patients could be evaluated for APS. Conclusion. aPS/PT are associated with recurrent early or late abortions and with premature delivery irrespective of other aPL.

Development of patient-centred standards of care for osteoarthritis in Europe: the eumusc.net-project.

OBJECTIVE: The eumusc.net project is an initiative founded by the European Community and the European League Against Rheumatism. One aim of the project was to facilitate equal standards for musculoskeletal health across Europe. The aim of this work-package was to develop patient-centred and consensus based standards of care (SOC) for osteoarthritis (OA), which should be available in a professional and a patient version. METHODS: A systematic review concerning guidelines dealing with OA was conducted. Furthermore, experts in musculoskeletal diseases were contacted to ensure that 'grey' literature was not excluded. Documents that fulfilled predefined inclusion/exclusion criteria were included and all interventions for OA were extracted and categorised. Based on this list of interventions, a three round Delphi exercise with an international and multidisciplinary expert panel, including patient research partners, was performed to achieve expert consensus. RESULTS: Six documents were included and used for further analysis. Out of them, 46 interventions have been extracted and 10 consensus based SOC were formulated. In addition, a patient version, written in a lay-understandable wording and in the format of checklist questions was developed. An example is SOC 5: "People with OA should achieve optimal pain control using pharmacological and non-pharmacological means." The matching patient-centred checklist question reads: "Do I know how to control pain associated with OA?" CONCLUSIONS: The SOC for OA will be available in the 23 languages of the European Union to enhance unified information to patients and professionals and to further harmonise the treatment/care of OA within Europe.

Obstetrical APS: is there a place for hydroxychloroquine to improve the pregnancy outcome?

The use of the conventional APS treatment (the combination of low-dose aspirin and LMWH) dramatically improved the obstetrical prognosis in primary obstetrical APS (OAPS). The persistence of adverse pregnancy outcome raises the need to find other drugs to improve obstetrical outcome. Hydroxychloroquine is widely used in patients with various autoimmune diseases, particularly SLE. Antimalarials have many anti-inflammatory, anti-aggregant and immune-regulatory properties: they inhibit phospholipase activity, stabilize lysosomal membranes, block the production of several pro-inflammatory cytokines and, in addition, impair complement-dependent antigen-antibody reactions. There is ample evidence of protective effects of hydroxychloroquine in OAPS similar to the situation in SLE arising from in vitro studies of pathophysiological working mechanism of hydroxychloroquine. However, the clinical data on the use of hydroxychloroquine in primary APS are lacking and prospective studies are necessary.

Optimization of unnicked ß2-glycoprotein I and high avidity anti-ß2-glycoprotein I antibodies isolation.

Patient biological material for isolation of ß2-glycoprotein I (ß2GPI) and high avidity IgG anti-ß2-glycoprotein I antibodies (HAv anti-ß2GPI) dictates its full utilization. The aim of our study was to evaluate/improve procedures for isolation of unnicked ß2GPI and HAv aß2GPI to gain unmodified proteins in higher yields/purity. Isolation of ß2GPI from plasma was a stepwise procedure combining nonspecific and specific methods. For isolation of polyclonal HAv aß2GPI affinity chromatographies with immobilized protein G and human ß2GPI were used. The unknown protein found during isolation was identified by liquid chromatography electrospray ionization mass spectrometry and the nonredundant National Center for Biotechnology Information database. The average mass of the isolated unnicked purified ß2GPI increased from 6.56 mg to 9.94 mg. In the optimized isolation procedure the high molecular weight protein (proteoglycan 4) was successfully separated from ß2GPI in the 1st peaks with size exclusion chromatography. The average efficiency of the isolation procedure for polyclonal HAv anti-ß2GPI from different matrixes was 13.8%, as determined by our in-house anti-ß2GPI ELISA. We modified the in-house isolation and purification procedures of unnicked ß2GPI and HAv anti-ß2GPI, improving the purity of antigen and antibodies as well as increasing the number of tests routinely performed with the in-house ELISA by ~50%.

Comparison of the performance of the different classification criteria for primary Sjögren's syndrome: a prospective cohort study.

The objective of the study is to compare the longitudinal performance of different classification criteria for primary Sjögren's syndrome (SS) in a cohort of patients previously diagnosed with primary SS. In each patient, we repeated diagnostic tests as required by the Copenhagen, European, Californian, and American-European Consensus Group (AECG) or the new American College of Rheumatology (ACR) classification criteria. Sixty-three out of 90 eligible patients (70 %) consented to participate. During the follow-up (mean (standard deviation, SD) 7.6 years (0.5)), we observed evolution from primary SS to SS with another systemic autoimmune disease (SAD) in 9/63 (14 %) patients, on average after 4.0 years (SD 0.9). The evolution from primary SS to SS-SADs was significantly more common if the diagnosis of primary SS was initially made using AECG (17 %, p = 0.008) or ACR (16 %, p = 0.016) criteria. In the 34 patients who underwent a full diagnostic reassessment, the diagnosis retention rate was statistically significant for all the criteria, except the European criteria. At reassessment, 3/32 (9 %) patients initially diagnosed as having primary SS using the European criteria could not be classified as having primary SS by any of the criteria. The differences in classification when using the AECG and the new ACR criteria were not statistically significant. The longitudinal diagnosis retention rate was highest for the Californian and AECG criteria. Regardless of the classification criteria, some patients eventually develop another SAD.

Development of patient-centred standards of care for rheumatoid arthritis in Europe: the eumusc.net project.

OBJECTIVE: The eumusc.net project is a European Union (EU) commission and European League Against Rheumatism (EULAR)funded project that aims to facilitate equal standards for musculoskeletal health in all EU countries. One work-package was to develop evidence-based and patient-centred standards of care (SOC), for rheumatoid arthritis (RA) understandable for patients and professionals across Europe. METHOD: A review of documents covering clinical practice 'guidelines' and SOC for RA was conducted. The obtained documents were evaluated using the Appraisal of Guidelines for Research and Evaluation II (AGREE II) criteria, and all recommended methods to treat RA were extracted. Based on this information, a three-round Delphi exercise was performed including a consensus group meeting of 21 researchers and patient representatives. RESULTS: 16 patient-centred SOC were formulated including a lay version in the format of a checklist. An example is SOC 3: 'People with RA should receive a treatment plan developed individually between them and their clinician at each visit.' The corresponding checklist question reads: 'Have I received a treatment plan which includes an explanation of my management, expected goals and outcomes and important contact details?' CONCLUSIONS: The SOC for RA will be available in all 23 official European languages and contribute to more unified treatment approaches in Europe.

Detection of antiphosphatidylserine/prothrombin antibodies and their potential diagnostic value.

Antiprothrombin antibodies, measured with phosphatidylserine/prothrombin complex (aPS/PT) ELISA, have been reported to be associated with antiphospholipid syndrome (APS). They are currently being evaluated as a potential classification criterion for this autoimmune disease, characterized by thromboses and obstetric complications. Given the present lack of clinically useful tests for the accurate diagnosis of APS, we aimed to evaluate in-house and commercial assays for determination of aPS/PT as a potential serological marker for APS. We screened 156 patients with systemic autoimmune diseases for antibodies against PS/PT, ß2-glycoprotein I, cardiolipin and for lupus anticoagulant activity. We demonstrated a high degree of concordance between the concentrations of aPS/PT measured with the in-house and commercial assays. Both assays performed comparably relating to the clinical manifestations of APS, such as arterial and venous thromboses and obstetric complications. IgG aPS/PT represented the strongest independent risk factor for the presence of obstetric complications, among all tested aPL. Both IgG and IgM aPS/PT were associated with venous thrombosis, but not with arterial thrombosis. Most importantly, the association between the presence of IgG/IgM aPS/PT and lupus anticoagulant activity was highly significant. Taken together, aPS/PT antibodies detected with the in-house or commercial ELISA represent a promising serological marker for APS and its subsets.

European registry of babies born to mothers with antiphospholipid syndrome.

OBJECTIVES: This study aimed to describe the long-term outcome and immunological status of children born to mothers with antiphospholipid syndrome, to determine the factors responsible for childhood abnormalities, and to correlate the child's immunological profile with their mothers. METHODS: A prospective follow-up of a European multicentre cohort was conducted. The follow-up consisted of clinical examination, growth data, neurodevelopmental milestones and antiphospholipid antibodies (APL) screening. Children were examined at 3, 9, 24 months and 5 years. RESULTS: 134 children were analysed (female sex in 65 cases, birth weight 3000±500 g, height 48±3 cm). Sixteen per cent had a preterm birth (<37 weeks; n=22), and 14% weighted less than 2500 g at birth (n=19). Neonatal complications were noted in 18 cases (13%), with five infections (4%). During the 5-year follow-up, no thrombosis or systemic lupus erythematosus (SLE) was noted. Four children displayed behavioural abnormalities, which consisted of autism, hyperactive behaviour, feeding disorder with language delay and axial hypotony with psychomotor delay. At birth lupus anticoagulant was present in four (4%), anticardiolipin antibodies (ACL) IgG in 18 (16%), anti-ß(2) glycoprotein-I (anti-ß2GPI) IgG/M in 16 (15%) and three (3%), respectively. ACL IgG and anti-ß2GPI disappeared at 6 months in nine (17%) and nine (18%), whereas APL persisted in 10% of children. ACL and anti-ß2GPI IgG were correlated with the same mother's antibodies before 6 months of age (p<0.05). CONCLUSION: Despite the presence of APL in children, thrombosis or SLE were not observed. The presence of neurodevelopmental abnormalities seems to be more important in these children, and could justify long-term follow-up.

International cohort study of 73 anti-Ku-positive patients: association of p70/p80 anti-Ku antibodies with joint/bone features and differentiation of disease populations by using principal-components analysis.

INTRODUCTION: An international cohort study of 73 anti-Ku-positive patients with different connective tissue diseases was conducted to differentiate the anti-Ku-positive populations of patients based on their autoantibody profile and clinical signs/symptoms and to establish possible correlations between antibodies against Ku p70 and Ku p80 with autoimmune diseases. METHODS: Sera of anti-Ku-positive patients were collected from six European centers and were all secondarily tested (in the reference center); 73 were confirmed as positive. Anti-Ku antibodies were detected with counter-immunoelectrophoresis (CIE), line immunoassay (LIA), and immunoblot analyses. All clinical and laboratory data were follow-up cumulative data, except for anti-Ku antibodies. Statistical analyses were performed by using R (V 2.12.1). The Fisher Exact test was used to evaluate the association between anti-Ku antibodies and diagnosis, gender, clinical signs, and other observed antibodies. The P values were adjusted for multiple testing. Separation of disease populations based on the presence of antibodies and clinical signs was investigated by principal-components analysis, which was performed by using thr// R's prcomp function with standard parameters. RESULTS: A 16% higher prevalence of anti-Ku p70 was found over anti-Ku p80 antibodies. In 41 (57%) patients, a combination of both was detected. Five (7%) patients, who were CIE and/or LIA anti-Ku positive, were negative for both subsets, as detected with the immunoblot; 31% of the patients had undifferentiated connective tissue disease (UCTD); 29% had systemic sclerosis (SSc); 18% had systemic lupus erythematosus (SLE); 11% had rheumatoid arthritis; 7% had polymyositis; and 3% had Sjögren syndrome. CONCLUSIONS: A significant positive association was found between female patients with anti-Ku p70 and joint/bone features, and a significant negative association was found between female patients with anti-Ku p80 only and joint/bone features (P = 0.05, respectively). By using the first and the third components of the principal-component analysis (PCA) with 29 parameters evaluated, we observed that the anti-Ku-positive population of UCTD patients had overlapping parameters, especially with SLE, as opposed to SSc, which could be helpful in delineating UCTD patients.

Comparison and evaluation of different methodologies and tests for detection of anti-dsDNA antibodies on 889 Slovenian patients' and blood donors' sera.

AIM: To evaluate four different commercially available assays for anti-double stranded DNA (dsDNA) detection and compare them with the in-house radioimmunoassay according to Farr (FARR-RIA) in order to select the optimal primary method for use in combination with FARR-RIA. METHODS: Sera from 583 consecutive patients sent to our laboratory for routine diagnosis, 156 selected patients with autoimmune diseases (76 systemic lupus erythematosus [SLE] patients and 80 patients with other autoimmune diseases), and 150 blood donors were tested for anti-dsDNA antibodies with two enzyme-linked immunoassays (ELISA), two Crithidia luciliae immunofluorescence tests (CLIFT), and FARR-RIA. The specificities and sensitivities of the tests were calculated and compared. RESULTS: FARR-RIA and CLIFT 2 showed the highest specificity for SLE (100%), with CLIFT 2 showing higher sensitivity (33% vs 47%). Both ELISAs showed higher sensitivities (>53%) than FARR-RIA but lower specificities (<93%), whereas CLIFT 1 showed the lowest overall agreement with FARR-RIA. CONCLUSION: CLIFT 2 was selected as the primary test for use in combination with FARR-RIA. The use of CLIFT 2 reduced the number of sera that needed to be tested by FARR-RIA, the time needed to report the results, and environmental toxicity, cancerogenicity, and radioactivity.