Noncriteria Antiphospholipid Antibodies in the Pathogenesis of Obstetric Pathology

We aimed to analyse the prevalence of non-criteria anti-phospholipid (aPL) antibodies and their role in the diagnosis, treatment and prognosis in a cohort of patients with clinical features consistent with a diagnosis of antiphospholipid syndrome (APS), but persistently negative for criteria aPL - anti-cardiolipin antibodies (aCL), anti-β2-glycoprotein I antibodies (aβ2-GPI) and lupus anticoagulant (LA) - named seronegative APS (SN-APS). Sera from SN-APS patients were tested for aCL by TLC-immunostaining, anti-vimentin/cardiolipin (aVim/CL) and anti-phosphatidylserine/prothrombin (anti-PS/PT) by ELISA. Control groups of our study were APS patients and healthy controls. We enrolled 114 consecutive SN-APS patients, 69 (60.5%) resulted positive for at least one non-criteria test in two occasions 12 weeks apart. Among the persistently positive patients to these tests, 97% resulted positive for aCL by TLC-immunostaining, 52.3% for aVim/CL and 17.4% for aPS/PT. SN-APS patients with double positivity (aCL by TLC-immunostaining and aVim/CL) showed a likelihood positive ratio of 8 to present mixed thrombotic and obstetrical features. Among SN-APS patients tested positive, after the therapeutic changes, three cases of recurrent thrombosis were observed [median follow-up 41 months (IQR 39.5)]. Twenty pregnancies were recorded in 17 SN-APS patients after the detection of unconventional aPL and 12 of them (60%) experienced a good outcome under conventional treatment for APS. This is the largest monocentric study demonstrating that aCL tested by TLC-immunostaining and aVim/CL can detect aPL positivity in SN-APS. It may encourage clinicians to monitor and provide adequate targeted therapy, which improve SN-APS prognosis.

Backrground Laboratory diagnosis of the antiphospholipid antibody syndrome (APS) requires evidence ofpersistently positive lupus anticoagulant (LAC), detected using clot-based assays and/or thepresence of high-titer serum anti-cardiolipin (aCL) and/or anti-beta-2 glycoprotein-1 (aB2GPI)antibodies, measured by semi-quantitative solid-phase immunoassays. Several non-criteria anti-phospholipid antibodies (NC-APLA) have been proposed as candidates for APS diagnosis:phosphatidyl-serine/ prothrombin (aPS/PT; IgM and IgG isotypes), anti-phosphatidyl serine (aPS;IgM and IgG), anti-prothrombin (aPT-IgG). However, their diagnostic relevance in routine APSevaluation is still unclear. The objectives of this study were 1) to determine the prevalence andassociation of NC-APLA in relation to LAC, aCL and aB2GPI antibodies’ positivity,independently and in combination and, 2) to assess the ability of NC-APLA to predict LACactivity, as an indirect measure of their function. Methods Results from 492 patients submitted for antiphospholipid antibody (APLA) panel or standaloneserology testing between January 2016 to January 2023 were retrieved in accordance withinstitutional guidelines. Patients were grouped according to LAC status and serology positivity(using manufacturer-suggested cutoff) into three groups: Single-positives (SP) for LAC, aCL oraB2GPI; Double-positives (DP) for aCL and aB2GPI; Triple-positives (TP) for LAC, aCL andaB2GPI. NC-ALPA titers were compared between LAC-positive and negative patients. Statisticalsignificance was established using unpaired Student’s t-test. Receiver operating curve (ROC)analysis was employed to assess the ability of NC-APLA to predict LAC status. Results Out of the total 492 patients, 1.8% (9/492) were TP, 2.4% (12/492) were DP and 24.7% (122/492)were SP. All NC-APLA titers were significantly higher in TP than in SP patients (P < 0.001).aPS/PT IgG (Mean: 76.3 vs 12.8 Units) and aPS IgG (Mean: 64.8 vs 5.5 GPS) antibodies weresignificantly higher in TP than DP patients (P = 0.008 and <0.0001, respectively). aPS/PT IgM(Mean: 62.5 vs 19.4 Units) and aPS IgM titers (Mean 31.9 vs 7.7 MPS) were significantly higherin DP compared to SP patients (P < 0.0001). When classified by LAC status, 40 patients werepositive and 452 were negative. Levels of all NC-APLA antibody titers (P < 0.0001) followed byaCL (Mean IgG 21.9 vs 7.8 GPL; IgM 20 vs 12.6 MPL; P < 0.01) and aB2GPI (Mean IgG 20.1 vs 4.9 SGU; IgM 13.8 vs 7.6 SMU, P < 0.001) antibodies were significantly higher in LAC-positivepatients compared to the LAC-negative individuals. ROC analyses based on the LAC statusresulted in the highest area under the curve (AUC, 95% CI) for aPS/PT IgM (0.72, 0.62 to 0.81)Private Information and aPS/PT IgG (0.74, 0.65 to 0.83) antibodies relative to aCL (IgM:0.53, 0.43 to 0.64; IgG:0.66, 0.56 to 0.77) or aB2GPI (IgM:0.5, 0.51 to 0.72 and IgG:0.6, 0.51 to 0.72) antibodies; aPS IgMand aPT IgG displayed similar performances (0.65, 0.54 to 0.75 and 0.66, 0.57 to 0.74,respectively). Whereas aPS IgG was of lower AUC (0.58, 0.48 to 0.68). Conclusion Our data demonstrated that at high titers, NC-APLA, display increased association with LACpositivity in comparison to aCL or aB2GPI antibody levels. Particularly, aPS/PT antibodiesdisplayed the superior ability to determine the LAC presence.Private Information

Non-criteria antiphospholipid antibodies (aPLs) increase the diagnostic value for antiphospholipid syndrome (APS) and contribute to better recognition of seronegative APS (SNAPS). However, the clinical utility and the diagnostic value of non-criteria aPLs are inconsistent. This study aimed to investigate the prevalence and clinical significance of 7 non-criteria aPLs in a large APS cohort. Seven non-criteria aPLs, including anti-phosphatidylserine/prothrombin (aPS/PT) antibodies IgG/IgA/IgM, anti-phosphatidylethanolamine antibodies (aPE) IgG/IgA/IgM, anti-Annexin V antibodies (aAnnexinV) IgG/IgA/IgM, anti-phosphatidylserine antibodies (aPS) IgM, aPS IgG, antibodies directed againsta mixture of phospholipids(APhL) IgG, and APhL IgM were tested among 175 patients with APS, 122 patients with other autoimmune diseases (as disease controls), and 50 healthy controls. In the present study, the highest prevalence of non-criteria aPLs was seen in aAnnexinV (58.86%). APhL IgG and aPS IgM showed the highest specificity (95.35%) and aPS/PT showed the highest Youden index (0.3991) for the diagnostic value of APS. The aAnnexinV also showed the highest prevalence in SNAPS (43.3%), followed by APhL IgM (21.7%), aPE (16.7%) and aPS/PT (16.7%). APhL IgG, aPS/PT, and aPS IgG showed positive association with thrombotic events in APS patients [APhL IgG: odds ratio (OR) = 2.26, 95% confidence interval (CI) 1.18-4.34, p = 0.013; aPS/PT: OR = 2.48, 95% CI: 1.32-4.69, p = 0.004; aPS IgG: OR = 1.90, 95% CI 1.01-3.60, p = 0.046; respectively). The inclusion of the non-criteria aPLs increased the accuracy of APS diagnosis from 65.7% to 87.4%. Our data provide evidence that adding the non-criteria aPLs can improve the diagnostic accuracy in APS. APhL IgG, aPS/PT, and aPS IgG may be potential biomarkers to predict the risk of thrombosis in APS.

To reveal the clinical significance of criteria and non-criteria antiphospholipid antibodies detected by line immunoassay in comparison with ELISA, systemic lupus erythematosus patients with and without thrombotic events were investigated. Thus, 107 systemic lupus erythematosus patients (48% with deep vein thrombosis or/and arterial thrombosis) and 120 healthy donors were enrolled. Serum antiphospholipid antibodies were detected by ELISA (Orgentec Diagnostika, Germany) and line immunoassay (GA Generic Assays, Germany). Lupus anticoagulant and IgG to cardiolipin and β2GPI but not IgM as well as triple positivity by ELISA and line immunoassay were linked with thrombosis in systemic lupus erythematosus. IgG to phosphatidylinositol and phosphatidylserine by line immunoassay showed significantly higher levels in systemic lupus erythematosus with deep vein thrombosis/arterial thrombosis than without and were independent risk factors for deep vein thrombosis (odds ratio 3.9, 95% confidence interval 1.1, 13.2) and arterial thrombosis (odds ratio 5.1, 95% confidence interval 1.3, 19.8) as well as thrombosis (odds ratio 3.6, 95% confidence interval 1.1, 11.3) and recurrence thereof (odds ratio 6.9, 95% confidence interval 2.1, 22.6), respectively. The occurrence of >4 IgG antiphospholipid antibodies by line immunoassay was an independent risk factor for thrombosis (odds ratio 10.9, 95% confidence interval 1.2, 101.5), arterial thrombosis (odds ratio 14.6, 95% confidence interval 2.5, 86.3), deep vein thrombosis (odds ratio 5.8, 95% confidence interval 1.0, 32.4) and recurrence of thrombosis (odds ratio 35.9, 95% confidence interval 3.8, 342.8). Line immunoassay is a promising multiplex test for the simultaneous detection of criteria and non-criteria antiphospholipid antibodies. Profiling of antiphospholipid antibodies by line immunoassay can differentiate systemic lupus erythematosus patients with thrombosis from systemic lupus erythematosus patients without and assess the risk for thrombosis and recurrence thereof.

This report describes the development of recurrent cutaneous microthrombosis in a patient with the superposition of Factor V Leiden heterozygosity on a noncriteria IgM antibody to phosphatidylserine/prothrombin complex. The patient was treated with prednisone, apixaban, and rituximab and was stable off of prednisone at her last outpatient visit 22 months after the initial event. This report illustrates the challenges of dealing with multifactor thrombophilia especially when one of those factors is a noncriteria antiphospholipid antibody and reaffirms the value of testing for noncriteria antibodies when clinical findings suggest the presence of antiphospholipid antibodies but the criteria antibodies are negative. This report further shows, in this patient, the benefit of the addition of rituximab-pvv to apixaban in normalizing the level of antiphosphatidylserine/prothrombin complex antibodies with the cessation of cutaneous microthrombotic events, normalization of inflammatory markers, and allowing the discontinuation of prednisone. Because of the relatively high frequency of Factor V Leiden heterozygosity in Caucasian populations, this report suggests that dual-factor thromobophilia due to its combination with criteria or noncriteria antiphospholipid antibodies may be more common than is recognized.

BackgroundIncreasing interest has focussed upon assays which are not currently included in the Antiphospholipid syndrome (APS) classification criteria to detect antibodies directed against other phospholipids (PL), PL binding proteins and/or...

Introduction. Patients with antiphospholipid syndrome (APS) may have a large spectrum of thrombotic clinical events (arterial or venous), but the factors that determine the occurrence of specific clinical manifestation has not been clearly established. Objective. The aim of the study was to determine the factors associated with a history of deep vein thrombosis (DVT) in patients with APS. We were especially interested to fi nd an association between the criteria and non-criteria antiphospholipid antibodies (APLAs) and the DVT. Methods. We realized a cross-sectional study, with consecutive enrollment of all patients presented in our department with the diagnosis of APS in the period 2008-2011. From the total of 106 patients for which the demographic, clinical and biological parameters were collected, the assessment of the criteria and non-criteria APLAs was performed in 73 patients. Results. The mean age at inclusion was 44.7 years, the female-to-male ratio 6.6, and the mean APS disease duration 6.7 years. The majority of the patients included presented with secondary APS (70 patients). Lupus anticoagulant was the most frequent immunological marker used to sustain the diagnosis of APS, found positive in 102 patients (96.2%). The recurrent DVT was more frequently observed in patients with primary APS than in those with secondary APS (p = 0.02). No significant association with DVT was found for the clinical parameters traditionally associated with the risk of thrombosis that we have taken into study: smoking, body mass index (BMI), waist circumference and waist-to-hip ratio. We found a positive association between the DVT history and the positivity for the IgM anti-β2 glycoprotein I antibodies [OR (95%CI) = 6.95 (1.36-35.58), p = 0.01]. The titer of IgG antiprothrombin antibodies and IgG antiphosphatidylethanolamine antibodies was higher in patients with previous DVT [3.0 (0.0-151.0) versus 2.0 (0.0-2.0), p = 0.01, respectively 5.0 (1.0-33.0) versus 3.0 (1.0-7.0), p = 0.01]. In the subgroup of patients with secondary APS, the previous DVT was associated only with the positivity for IgM anti-β2 glycoprotein I antibodies [OR (95%) = 13.63 (1.46-127.15), p = 0.02]. In the same subgroup, the levels of IgG anticardiolipin and the IgG antiphosphatidylethanolamine antibodies were higher in patients with previous DVT [2.0 (0.0-64.0) versus 1.0 (0.0-20.0), p = 0.02, respectively 3.0 (0.0-151.0) versus 2.0 (0.0-7.0), p = 0.03]. In patients with primary APS, the titer of IgG antiprothrombin antibodies was significantly higher when DVT history were present [5.0 (1.0-3.0) versus 3.0 (1.0-7.0), p = 0.01. Only the titer of IgG antiprothrombin antibodies was associated with the history of recurrent thrombosis [5.0 (1.0-3.0) versus 3.0 (1.0-7.0), p = 0.01]. Conclusions. In patients with APS, the DVT history was related with the prevalence of the anti-β2 glycoprotein I antibodies, but also with the levels of some non-criteria antibodies, IgG antiprothrombin antibodies and IgG antiphosphatidylethanolamine antibodies. In patients with primary APS, previous DVT is associated only with the titer of IgG antiprothrombin antibodies, whereas in patients with secondary APS, the DVT history were related with the presence of anti-β2 glycoprotein I antibodies and with the titers of both criteria antibodies (IgG anticardiolipin antibodies) and non-criteria antibodies (IgG antiphosphatidylethanolamine antibodies). The risk of recurrent thrombosis was higher in patients with primary APS when compared with those with secondary APS. Only the titers of IgG antiprothrombin antibodies were correlated with recurrent thrombosis.

The 2006 Sydney classification criteria, which is most commonly used in the dignosis of antiphospholipid syndrome (APS) by far, improves the sensitivity of the diagnosis but suffers a lack of specificity. Extra-criteria manifestations are associated with antiphospholipid antibody (aPL) and thrombosis, thus fully recognizing them could help make treament decision and improve the prognosis of part of the patients with probable APS, in whom serological examination suggests APS but clinical criterias are not met. On the other hand, with the development of basic researches, new serum antibodies have been discovered to be related to arteriovenous thrombosis and pregnancy morbidities. Those new biomarkers could help physicians enhance risk assessment and treatment decision for patients with adequate clinical menifestations for the classification criteria but negative laboratory tests, also known as seronegative APS (ANAPS). This article would spread out extra-criteria manifestations and non-criteria antiphospholipid antibodies associated with APS. Key words: Antiphospholipid syndrome (APS); Extra-criteria manifestations; Non-criteria antiphospholipid antibodies; Seronegative APS (SNAPS)

BackgroundThe study of other non-criteria antiphospholipid antibodies (aPL) may improve the stratification of patients with antiphospholipid syndrome (APS) and assist in the interpretation of conflicting results on aPL testing.ObjectivesTo determine...

Antiphospholipid syndrome (APS) is an autoimmune, thromboinflammatory disease, which affects children and adults. There are particular features of the disease and nuances to diagnosis and management in a pediatric population, which must be appreciated to improve clinical care. Pediatric-specific epidemiological studies highlight that pediatric APS is quite rare with incidence in some populations of 0.2 per 100 000. There are new classification criteria in APS, which include a wider range of clinical features increasingly identified in registry data and case series of pediatric APS, though validation in pediatric APS is still needed. There is a particularly high proportion of pediatric APS patients with noncriteria antiphospholipid antibodies (aPL). Recurrent thrombosis is especially common in pediatric APS, highlighting the difficulty of management of this disease with high morbidity in children. Recent research has enhanced understanding of pediatric-specific APS epidemiology, laboratory findings, the wide variety of clinical features, and challenges in successful treatment. Future directions could include evaluation of potentially unique features in pediatric pathophysiology, an evaluation of the new APS classification criteria in children, broader prospective data on clinical and laboratory features, and a continued search for treatment beyond committing young patients to lifelong anticoagulation.

Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by thrombotic symptoms (venous, arterial, or small vessels) and/or gestational morbidity in patients carrying antiphospholipid antibodies (aPLs). Criteria aPLs include anti-cardiolipin antibodies, anti-beta 2 glycoprotein I (aB2GPI) antibodies of the IgG or IgM isotypes, and lupus anticoagulant (LA). However, there are aPLs that are associated with APS events but are not included in the criteria (extra-criteria). The aim of this study is to evaluate the prevalence and association of criteria and extra-criteria aPLs with APS clinical events. A total of 838 patients with clinical manifestations of APS were studied. In total, 715 presented with vascular manifestations, and 130 presented with obstetric morbidity. We measured levels of criteria aPLs, and the extra-criteria aPLs determined were anti-phosphatidylserine/prothrombin (aPS/PT) of IgG and IgM isotypes and aB2GPI IgA. Classic aPL, aPS/PT, and aB2GPI IgA positivity showed a significant and independent association with thrombosis (OR: 2.40, 2.36, and 2.53 respectively). IgA aB2GP1 was the only aPL significantly associated with the five types of thrombotic events (venous thrombosis, pulmonary embolism, stroke, acute myocardial infarction, and arterial thrombosis). Regarding obstetric APS, the most relevant antibodies were classic aPL of IgM isotype (OR: 36.04) and aPS/PT of both isotypes (OR: 4.4). The other aPL studied did not show association in multivariate analysis. The degree of clinical association for each group of aPLs was different depending on the form of presentation (vascular or obstetric) and the presence or absence of autoimmune diseases. Moreover, a fair level of agreement between LA and aPS/PT positivity was found; therefore, aPS/PT should not be referred to as a surrogate marker of LA.

To identify the osteoprotegerin (OPG) correlates with antiphospholipid syndrome (APS) parameters. Our cohort included 40 patients with primary APS disease associated with systemic lupus erythematosus (SLE) (mean age, 43.7 years; 87% female). Data on cardiovascular risk factors and specific clinical events in APS were collected. Then we tested OPG and 10 criteria and noncriteria antiphospholipid antibodies (aPLs) on preserved specimens in all cases. A total of 26 patients (65%) had high serum OPG levels. Patients with high OPG were mostly overweight. In patients with SLE, the OPG levels were associated with anti-double-stranded DNA (anti-dsDNA) and anti-Sm titers. However, we did not find significant correlations of the OPG with any of the 10 aPLs tested. Also, we found no relationship regarding venous APS events. In APS, high OPG levels are not linked to serum aPL expression.

The place of non-criteria antiphospholipid antibodies (aPLs) in the diagnosis of antiphospholipid syndrome (APS) is still debatable. The aim of this research was to evaluate the correlations between the titres of non-criteria aPLs (anti-phosphatidylethanolamine (aPE), anti-phosphatidylserine (aPS), and anti-prothrombin (aPT) antibodies), and the ones of the already studied criteria aPLs (anti-cardiolipin (aCL) and anti-β2 glycoprotein I-aβ2GPI antibodies). Altogether, 72 APS (30 primary and 42 secondary) patients were included in our study. High correlation coefficients (rs) were found between aPS IgM and aCL IgM, overall (0.77, p < 0.01), as well as in the primary (0.81, p < 0.01), and secondary (0.75, p < 0.01) APS subgroups. Low or statistically insignificant correlations were observed between IgG/IgM isotypes of aPT and aCL, or aβ2GPI, in the entire study population, and when evaluating the subgroups. Therefore, moderate correlations were mainly identified between the tested non-criteria antibodies and the criteria ones, suggesting little added value for the use of the tested non-criteria aPLs, with the exception of aPT, which seems to have different kinetics and might be a promising APS diagnostic tool.

BackgroundDespite expansion in the 2006 Sydney antiphospholipid syndrome (APS) classification criteria to include IgG/IgM anti-β2-glycoprotein (aβ2GPI) antibodies in addition to IgG/IgM anti-cardiolipin antibodies (aCL) and lupus anticoagulant (LAC), some individuals with clinical features of APS remain seronegative (seronegative APS or SNAPS) and are at risk of recurrent thrombosis and pregnancy morbidities. Our aim was to assess the value of “non-criteria” aPL antibodies to detect these SNAPS patients.MethodsOne hundred ninety-two APS patients, 90 SNAPS patients, 193 autoimmune disease controls, and 120 healthy controls were evaluated. Ten antiphospholipid antibodies (aPLs) were tested using commercial kits, including 5 non-criteria aPLs: anti-phosphatidylserine/prothrombin antibodies (aPS/PT) IgG/IgM, aCL IgA, aβ2GPI IgA, and anti-β2GPI Domain 1 (aβ2GPI-D1) IgG.ResultsUp to 60.9% of the SNAPS and 93.5% of APS patients were detected by at least one non-criteria aPL. aPS/PT IgG had the highest Youden index in classifying APS and SNAPS from controls. aPS/PT IgG and aβ2GPI Domain 1 IgG seem to be the most significant risk factors for thrombotic events and pregnancy morbidity, respectively. aPS/PT IgG/IgM and aβ2GPI-D1 IgG were detected in some SNAPS patients, while IgA isotypes of aCL/aβ2GPI tended to appear together with other biomarkers. The combined analysis showed enhanced diagnostic performance with the inclusion of non-criteria aPLs.ConclusionsRecognition of SNAPS patients is critical for clinical management and prevention of potential thrombotic and obstetric adverse events. The non-criteria antiphospholipid antibodies help to identify a considerable portion (60.9%) of these patients who otherwise may remain untreated and at clinical risk.

Background and Aims: Anti-phosphatidylserine/ prothrombin antibodies (aPS/PT) are non-criteria antiphospholipid antibodies (aPL) strongly associated with thrombosis and pregnancy complications related to antiphospholipid syndrome (APS). The latest ACR/EULAR classification criteria for APS aim to classify APS with an increased specificity of up to 99%, but also recommend exploring the relevance of other aPL tests, such as aPS/PT, to guide future updates. In this study, we aimed to assess the clinical relevance of a single aPS/PT-positivity. Materials and Methods: Sixty-nine patients prospectively followed between January 2011 and December 2024 were enrolled from the University Medical Centre Ljubljana (UMCLJ) and the University of Padua (UniPD). IgG and IgM antibodies against cardiolipin (aCL), β2-glycoprotein I (anti-β2GPI) and aPS/PT antibodies were tested at UMCLJ using in-house ELISAs (1) and at UniPD using commercial assays from Orgentec Diagnostika, Mainz, Germany, and INOVA Diagnostics Inc., San Diego, CA, USA, respectively (2). Lupus anticoagulant (LAC) was determined using a three-step procedure in accordance with the ISTH guidelines. Results: The combined cohort comprised 69 patients (11 men, mean age at diagnosis 45.5±16.1 years, range 10-77 years) who were persistently single aPS/PT positive, confirmed by at least two measurements between 2014 and 2024. Importantly, 27 of 69 (39%) patients achieved a clinical domain score of more than 3 weight points (WP), thus fulfilling the clinical domain of the new classification criteria. Among them, 11 patients were LAC-negative and therefore did not meet the laboratory criteria (&lt;3 WP), whereas 16 patients had persistent LAC positivity and met the new criteria. In the LAC-negative group, the mean clinical domain weight was 4.5 compared to 5.4 in the LAC-positive group, which was not a statistically significant difference (Figure 1). Eight out of eleven (73%) LAC-negative patients and twelve out of sixteen (75%) LAC-positive patients had an underlying autoimmune disease, mostly SLE. Seven LAC-negative patients experienced venous thromboembolism (VTE), one with a high-risk VTE profile, three experienced arterial thrombosis (AT), two with a high-risk CVD profile, one had established microvascular manifestations and three had obstetric manifestations, two of which were PEC, three patients had cardiac valve manifestations and three had thrombocytopenia. Nine LAC-positive patients experienced VTE, one with a high-risk VTE profile, six experienced AT, all without a high-risk CVD profile, two had established and three suspected microvascular manifestations and four had obstetric manifestations; none of them were PEC. Four patients had cardiac valve manifestations, and five had thrombocytopenia. Conclusions: Single aPS/PT positivity is associated with all six clinical domains included in the 2023 ACR/EULAR APS classification criteria, even independently of LAC positivity. The association of aPS/PT with newly added clinical domains, known to be linked to damage accrual, makes aPS/PT a tool for risk stratification in APS. Figure 1. Clinical relevance of single aPS/PT positivity. Created in https://BioRender.com