Anti-Phospholipid Antibodies in COVID-19 Are Different From Those Detectable in the Anti-Phospholipid Syndrome.
ABSTRACT: Background:Critically ill patients with coronavirus disease 2019 (COVID-19) have a profound hypercoagulable state and often develop coagulopathy which leads to organ failure and death. Because of a prolonged activated partial-thromboplastin time (aPTT), a relationship with anti-phospholipid antibodies (aPLs) has been proposed, but results are controversial. Functional assays for aPL (i.e., lupus anticoagulant) can be influenced by concomitant anticoagulation and/or high levels of C reactive protein. The presence of anti-cardiolipin (aCL), anti-beta2-glycoprotein I (anti-?2GPI), and anti-phosphatidylserine/prothrombin (aPS/PT) antibodies was not investigated systematically. Epitope specificity of anti-?2GPI antibodies was not reported. Objective:To evaluate the prevalence and the clinical association of aPL in a large cohort of COVID-19 patients, and to characterize the epitope specificity of anti-?2GPI antibodies. Methods:ELISA and chemiluminescence assays were used to test 122 sera of patients suffering from severe COVID-19. Of them, 16 displayed major thrombotic events. Results:Anti-?2GPI IgG/IgA/IgM was the most frequent in 15.6/6.6/9.0% of patients, while aCL IgG/IgM was detected in 5.7/6.6% by ELISA. Comparable values were found by chemiluminescence. aPS/PT IgG/IgM were detectable in 2.5 and 9.8% by ELISA. No association between thrombosis and aPL was found. Reactivity against domain 1 and 4-5 of ?2GPI was limited to 3/58 (5.2%) tested sera for each domain and did not correlate with aCL/anti-?2GPI nor with thrombosis. Conclusions:aPL show a low prevalence in COVID-19 patients and are not associated with major thrombotic events. aPL in COVID-19 patients are mainly directed against ?2GPI but display an epitope specificity different from antibodies in antiphospholipid syndrome.
Project description:BACKGROUND:The aim of this study was to determine the frequency of anti-cardiolipin antibodies (aCL) and anti-?2 glycoprotein I antibodies (a?2GPI) among Tunisian patients with rheumatoid arthritis (RA). METHODS:Ninety RA patients with positive anti-cyclic citrullinated antibodies (anti-CCP) and 90 healthy blood donors (HBD) were studied. aCL and a?2GPI of isotype IgG, IgA and IgM were detected by ELISA. RESULT:The frequency of antiphopholipid antibodies (aPL) (aCL and/or a?2GPI) was significantly higher in patients with RA than in HBD (35.5% vs 11.1%, P = .0001). The frequencies of aCL and a?2GPI were significantly higher in patients than in healthy subjects (15.5% vs 5.5%, P = .04 and 32.2% vs 11.1%, P = .0005 respectively). a?2GPI-IgA were significantly more frequent in patients than in the control group (26.7% vs 7.8%, P = .0007). In patients, a?2GPI-IgA were significantly more frequent than a?2GPI-IgG (26.7% vs. 6.7%, P = .0003) and a?2GPI-IgM (26.7% vs 5.6%, P = .0001). In RA patients, the frequency of a?2GPI was significantly higher than that of aCL (32.2% vs 15.5%, P = .008). a?2GPI-IgA was significantly more frequent than aCL-IgA (26.7% vs 4.4%, P = .00005). The average titer of anti-CCP in aPL positive patients was significantly higher than in aPL negative patients (170.6 ± 50 RU/mL vs 147.7 ± 51 RU/mL, P = .04). Significant correlation was found between a?2GPI-IgA and anti-CCP (r = .235, P = .026). CONCLUSIONS:aPL and particularly a?2GPI-IgA are frequent in RA and are correlated with anti-CCP.
Project description:BACKGROUND:Despite expansion in the 2006 Sydney antiphospholipid syndrome (APS) classification criteria to include IgG/IgM anti-?2-glycoprotein (a?2GPI) 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. METHODS:One 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, a?2GPI IgA, and anti-?2GPI Domain 1 (a?2GPI-D1) IgG. RESULTS:Up 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 a?2GPI Domain 1 IgG seem to be the most significant risk factors for thrombotic events and pregnancy morbidity, respectively. aPS/PT IgG/IgM and a?2GPI-D1 IgG were detected in some SNAPS patients, while IgA isotypes of aCL/a?2GPI tended to appear together with other biomarkers. The combined analysis showed enhanced diagnostic performance with the inclusion of non-criteria aPLs. CONCLUSIONS:Recognition 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.
Project description:Background: Anti-phospholipid syndrome (APS) and systemic lupus erythematous (SLE) are autoimmune disorders that often co-occur. Anti-phospholipid antibodies (aPL) are typical of both conditions and may be associated with vascular events and pregnancy-related morbidities. Whereas, aPL-screening is mandatory for individuals with suspected SLE, the clinical value of longitudinal aPL analyses in established SLE is unclear. Methods: We investigated the occurrence and variation of IgG/IgA/IgM anti-cardiolipin (aCL) and anti-?2-glycoprotein-I (anti-?2GPI) antibodies, using both the manufacturer's cut-off and a cut-off based on the 99th percentile of 400 apparently healthy donors, in recent-onset SLE. Furthermore, we evaluated the relationships between aPL levels and SLE/APS manifestations, as well as the pharmacotherapy. Patients with SLE who met validated classification criteria were included in this prospective study (N = 54). Samples were obtained at 0, 6, 12, 24, 36, 48, 60, 72, 84, and 96 months after SLE diagnosis. Results: Depending on the cut-off applied, 61.1 or 44.4% showed a positive result for at least one aPL isotype or the lupus anticoagulant test over time. Median values for all six aPL isotypes numerically decreased from inclusion to last follow-up, but none of the isotypes met statistical significance. Seroconversion (from positive to negative, or the opposite direction) was occasionally seen for both aCL and anti-?2GPI. IgA and IgM anti-?2GPI were the most common isotypes, followed by IgM aCL. Presence of IgG aCL associated significantly with myocardial infarction and miscarriage, and IgG/IgA anti-?2GPI with miscarriage. Conclusion: aPL were common during the first years of SLE. Even though the levels fluctuated over time, the patients tended to remain aPL positive or negative. Repeated aPL testing in the absence of new symptoms seems to be of uncertain value in patients with recent-onset SLE.
Project description:PURPOSE:To explore the role of plasmatic platelet-activating factor acetylhydrolase (PAF-AH), a marker of cardiovascular risk, in patients with anti-phospholipid antibodies (aPL). METHODS:PAF-AH activity was assessed in a series of 167 unselected patients screened for aPL in a context of thrombotic events, risk of thrombosis or obstetric complications and in 77 blood donors. RESULTS:116/167 patients showed positive results for at least one aPL among IgG/IgM anti-prothrombin/phosphatidylserine (aPS/PT), anti-cardiolipin (aCL), anti-beta2-glycoprotein I (a?2GPI) or lupus anticoagulant (LAC), while 51/167 patients resulted aPL-negative. LAC+ patients disclosed higher PAF-AH than LAC-negative (22.1 ± 6.4 nmol/min/ml vs. 19.5 ± 4.1 nmol/min/ml; p = 0.0032), and aPL-negative patients (p = 0.03). Patients presenting positive IgG a?2GPI disclosed higher PAF-AH than patients with only IgM a?2GPI-positive antibodies (23.1 ± 7.2 nmol/min/ml vs. 20.1 ± 5.3 nmol/min/ml; p = 0.035), as well as than patients showing only isolated LAC, aCL or aPS/PT (16.9 ± 3.8 nmol/min/ml; p = 0.003). CONCLUSIONS:PAF-AH plasmatic activity is particularly up-regulated in LAC+ and in a?2GPI IgG+ patients, possibly representing an alternative prognostic biomarker for the therapeutic management of APS patients.
Project description:Anti-phospholipid antibodies (aPL) analyzed by line immunoassay (LIA) can recognize beta2-glycoprotein I (?2GPI) domain 1 (D1) epitopes depending on ?2GPI binding to distinct phospholipids. The aPL LIA was compared with consensus ELISA to investigate whether both techniques can discriminate anti-phospholipid syndrome (APS) patients from aPL-positive, systemic autoimmune rheumatic diseases (SARD) patients without clinical symptoms of APS and controls.Thirty-four APS patients (14 arterial/venous thrombosis, 16 pregnancy morbidity, and 4 both), 41 patients with SARD lacking clinical APS criteria but demonstrating positivity for anti-?2GPI (a?2GPI) IgG, and 20 healthy subjects (HS) were tested for aPL to cardiolipin (aCL), phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol (aPG), phosphatidylinositol, phosphatidylserine, ?2GPI, prothrombin, and annexin V by LIA. Samples were also tested for aCL, a?2GPI, a?2GPI-domain 1 (aD1), and a?2GPI-domains 4-5 (aD4-5) by ELISA and for lupus anti-coagulant.Comparison of LIA with ELISA revealed a good agreement for the consensus criteria aPL a?2GPI and aCL IgG (kappa?=?0.69, 0.68, respectively) and a moderate agreement for IgM (kappa?=?0.52, 0.49, respectively). Regarding ELISA, aD1/aD4-5 demonstrated the best performance of differentiating APS from asymptomatic SARD [area under the curve (AUC): 0.76]. aPG IgG had the best performance by LIA (AUC: 0.72) not significantly different from aD1/aD4-5. There was a good agreement for aPG IgG with aD1/aD4-5 (kappa?=?0.71).aD1/aD4-5 (ELISA) and aPG IgG (LIA) differentiate APS from SARD patients. PG appears to interact with ?2GPI of APS patients and exposes D1 thereof for disease-specific aPL binding in LIA.
Project description:Objectives:We evaluate the performance characteristics of antiphosphatidylserine (anti-PS), antiphosphatidylinositol (anti-PI), and antiphospholipid mixture (APhL) enzyme-linked immunosorbent assays (ELISAs) compared with anticardiolipin (aCL) and anti-?2 glycoprotein I (anti-?2GPI) in a large group of patients with antiphospholipid (aPL)-related diseases. Methods:Serum samples from 548 patients from the Hopkins and Jamaican systemic lupus erythematosus cohorts, the PROMISSE cohort, and the Antiphospholipid Standardization Laboratory were examined for immunoglobulin G (IgG)/immunoglobulin M (IgM) positivity in aCL, anti-?2GPI, anti-PS, anti-PI, and APhL ELISA assays. Results:All IgG assays were associated with one or more thrombotic and/or obstetric manifestations, with an increased risk associated with higher antibody titers. Analytical performance was similar among assays, but IgG assays performed better than IgM counterparts. Conclusions:Increasing titers of APhL, anti-PS, and anti-PI antibodies could indicate an increased risk of thrombotic and/or obstetric aPL-related manifestations. These assays may be promising biomarkers for particular APS manifestations.
Project description:OBJECTIVES:aPL are present in between 20 and 30% of patients with SLE. They can cause vascular events (VE) or pregnancy morbidity. aCL and anti-beta-2-glycoprotein I (anti-?2GPI) are measured in clinical practice. Domain I (DI) of ?2GPI is the main site for aPL binding. We investigated the prevalence of IgG anti-DI, aCL and anti-?2GPI antibodies in early SLE and their association with mortality and development of VE. METHODS:Samples from 501 patients with SLE that had been obtained and stored early during their disease were tested for IgG anti-DI, aCL and anti-?2GPI antibodies by ELISA. LA status and history of VE were obtained by reviewing medical records. Kaplan-Meier analysis was used to investigate mortality and occurrence of VE, comparing groups with and without aPL in early disease. RESULTS:Of 501 patients, 190 (38%) had at least one of these aPL, of whom 112 had anti-DI alone. Of 276 patients with complete vascular history, 83 had experienced VE. The 39 patients who were double or triple-ELISA-positive for any combination of the three aPL were more likely to have or develop lupus anticoagulant (P<0.0001) than those who were single-ELISA-positive or negative. In Kaplan-Meier analysis, they showed a trend towards developing more VE (P = 0.06). CONCLUSION:IgG anti-DI antibodies were present in early serum samples from 29% of patients and were more common than IgG aCL or anti-?2GPI. There was some evidence suggesting that double or triple-ELISA-positivity for these antibodies identified a group with worse outcomes.
Project description:BACKGROUND:?2-Glycoprotein I (?2GPI) represents the major antigenic target for antiphospholipid antibodies (aPL), with domain 1 (D1) being identified as a risk factor for thrombosis and pregnancy complications in APS. We aimed to analyse the ability of aPL, and particularly anti-D1 ?2GPI, to stimulate prothrombotic and proinflammatory activity of immune cells in vitro. METHODS:Peripheral blood mononuclear cells (PBMCs) from 11 healthy individuals were incubated with: (1) "anti-D1(+)"-pooled plasma derived from patients suspected of having APS contained anticardiolipin antibodies (aCL), lupus anticoagulant (LA), anti-?2GPI and anti-D1 ?2GPI; (2) "anti-D1(-)"-pooled plasma from patients suspected of having APS contained aCL, LA, anti-?2GPI, and negative for anti-D1 ?2GPI; (3) "seronegative"-negative for aPL. RESULTS:The presence of anti-D1(+) and anti-D1(-) plasma resulted in increased HLA-DR and CD11b on monocytes. While only anti-D1(+) plasma markedly increased the percentage and median fluorescence intensity (MFI) of CD142 (tissue factor, TF) on monocytes in comparison with those cultured with anti-D1(-) and seronegative plasma. Anti-D1(+) plasma resulted in increased percentage and MFI of activation marker CD69 on NK and T cytotoxic cells. Expression of IgG receptor Fc?RIII(CD16) on monocytes and NK cells was down-regulated by the anti-D1(+) plasma. CONCLUSIONS:Taking together, our study shows the ability of patient-derived aPL to induce immune cell activation and TF expression on monocytes. For the first time, we demonstrated the influence of anti-D1 ?2GPI on the activation status of monocytes, NK and cytotoxic T cells. Our findings further support a crucial role of D1 epitope in the promotion of thrombosis and obstetrical complications in APS.
Project description: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-?(2) glycoprotein-I (anti-?2GPI) IgG/M in 16 (15%) and three (3%), respectively. ACL IgG and anti-?2GPI disappeared at 6 months in nine (17%) and nine (18%), whereas APL persisted in 10% of children. ACL and anti-?2GPI 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.
Project description:Patients with COVID-19 are at high risk for thrombotic arterial and venous occlusions. Lung histopathology often reveals fibrin-based blockages in the small blood vessels of patients who succumb to the disease. Antiphospholipid syndrome is an acquired and potentially life-threatening thrombophilia in which patients develop pathogenic autoantibodies targeting phospholipids and phospholipid-binding proteins (aPL antibodies). Case series have recently detected aPL antibodies in patients with COVID-19. Here, we measured eight types of aPL antibodies in serum samples from 172 patients hospitalized with COVID-19. These aPL antibodies included anticardiolipin IgG, IgM, and IgA; anti-?<sub>2</sub> glycoprotein I IgG, IgM, and IgA; and anti-phosphatidylserine/prothrombin (aPS/PT) IgG and IgM. We detected aPS/PT IgG in 24% of serum samples, anticardiolipin IgM in 23% of samples, and aPS/PT IgM in 18% of samples. Antiphospholipid autoantibodies were present in 52% of serum samples using the manufacturer's threshold and in 30% using a more stringent cutoff (?40 ELISA-specific units). Higher titers of aPL antibodies were associated with neutrophil hyperactivity, including the release of neutrophil extracellular traps (NETs), higher platelet counts, more severe respiratory disease, and lower clinical estimated glomerular filtration rate. Similar to IgG from patients with antiphospholipid syndrome, IgG fractions isolated from patients with COVID-19 promoted NET release from neutrophils isolated from healthy individuals. Furthermore, injection of IgG purified from COVID-19 patient serum into mice accelerated venous thrombosis in two mouse models. These findings suggest that half of patients hospitalized with COVID-19 become at least transiently positive for aPL antibodies and that these autoantibodies are potentially pathogenic.