aPL Tests Research Articles

Low-titer antiphospholipid antibodies is associated with increased risk of late preterm stillbirth

Antiphospholipid (aPL) antibodies are present in up to 42% of patients with pregnancy loss. Patients with low-titer aPL antibodies who do not meet Sapporo criteria for antiphospholipid syndrome (APLS) often receive conflicting counseling in regards to pregnancy loss and perinatal outcomes by both hematologists and perinatologists. We performed a retrospective cohort study of pregnant patients who had aPL testing and compared pregnancy outcomes between subjects with low-titer aPL and those with negative aPL. Low-titer aPL was defined as positive anticardiolipin or anti-B2 glycoprotein IgG or IgM < 40 GPL or MPL or < 99th percentile. The primary outcome consisted of early pregnancy loss (< 13 weeks), spontaneous abortion (≥13, < 20 weeks), and fetal demise/stillbirth (≥20 weeks). Bivariable and multivariable analyses were performed. There were 2,898 patients with a pregnancy episode and aPL testing. Compared to those with negative aPL, low-titer aPL individuals were more likely to be younger, identify as Black or African-American, have hematologic or autoimmune disorders, have a history of substance use disorder or psychiatric disorder. Notably, there was increased use of aspirin but no difference in anticoagulation use in this group. (Table 1). After multivariable analysis, patients with low-titer aPL were not at higher risk for early pregnancy loss or spontaneous abortion when compared to those with negative aPL. However, they were at higher risk for stillbirth between 28-37 weeks compared with their negative aPL counterparts with an OR 1.81 (1.01-3.23), once adjusted for high risk comorbidities. (Table 2). Patients with low-titer aPL had higher rates of expected autoimmune and hematologic disorders compared to those with negative aPL. While there was no increased risk of spontaneous abortion, these patients were more likely to have a late preterm fetal demise, even when adjusted for medical and social comorbidities and aspirin use. While these patients do not carry a formal APLS diagnosis, they have a higher risk of stillbirth than patients with negative aPL.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

An Overview of Laboratory Testing for Antiphospholipid Antibodies.

Antiphospholipid antibodies (aPL) represent a group of autoantibodies directed against phospholipids. These antibodies may arise in a number of autoimmune conditions, of which the antiphospholipid (antibody) syndrome (APS) is best recognized. aPL can be detected by various laboratory assays, essentially comprising both solid-phase (immunological) assays and "liquid-phase" clotting assays identifying so-called lupus anticoagulants (LA). aPL are associated with various adverse pathologies, including thrombosis and placental/fetal morbidity and mortality. The type of aPL present, as well as the pattern of reactivity, is variously associated with the severity of the pathology. Thus, laboratory testing for aPL is indicated to help assess the future risk of such events, as well as representing certain "classification" criteria for APS, also used as surrogates for diagnostic criteria. The current chapter overviews the laboratory tests available to measure aPL and their potential clinical utility.

Neural Network Diagnoses the Antiphospholipid Syndrome without Interrupting Anticoagulant Therapy

Background The antiphospholipid syndrome (APS) is characterized by the presence of antiphospholipid antibodies (aPL) and is associated with an increased risk of thrombosis and pregnancy morbidity. The Laboratory diagnosis of APS is difficult because most patients already use anticoagulants due to a thrombotic event, resulting in interference with the clotting assays used for lupus anticoagulant (LAC) testing. Nevertheless, the aPL profile defines patient management, making aPL testing warranted during anticoagulant therapy. Sometimes, cessation of anticoagulant treatment is necessary to perform the lupus anticoagulant (LAC) test, which is unfavorable. The thrombin generation (TG) test is a functional and global hemostatic test. Clinical studies have shown that increased TG is associated with thrombosis, also in thrombotic APS patients. We have previously shown that the artificial intelligence method neural networks can be used to diagnose untreated APS patients from a group of healthy, auto-immune disease, and thrombosis controls with high accuracy. At the moment of aPL testing, many patients are already on anticoagulants, hampering the detection of LAC. Therefore, we developed a neural net (NN) for diagnoses APS in a cohort of anticoagulated subjects, based on the TG test. Methods Antiphospholipid antibodies positivity was diagnosed according to the Sapporo-Sydney guidelines. TG was measured using the calibrated automated thrombogram assay (CAT) on platelet poor plasma with PPP reagent (intermediate concentration of tissue factor) in 48 APS patients and 64 control non-APS patients, both treated with vitamin K antagonists (VKAs) TG was explored in more depth by quantifying parameters of prothrombin conversion and thrombin inactivation, using thrombin dynamics (TD) analysis, and assessing the sensitivity of the activated protein C pathway through the addition of thrombomodulin (TM). Five NNs were developed and the most accurate NN was selected for clinical validation based on its sensitivity and specificity. Input parameters for the NN were TG parameters lag time, peak, endogenous thrombin potential, time-to-peak, and velocity index, and TD parameters total prothrombin conversion, maximum prothrombin conversion rate, thrombin-antithrombin complex formation, and thrombin-α2-macroglobulin complex formation, and the inhibition of TG and TD by TM. The top performing NN was clinically validated using the TG and TD data acquired in the validation cohort, consisting of 33 APS patients and 62 controls, both on VKA therapy, 49 auto-immune disease controls, 38 thrombosis controls, 92 patients visiting the hospital for other indications and 37 healthy subjects. All control subjects were negative for aPL. Results TG was slightly increased in APS patients compared to controls. VKA therapy abolished the typical lag time prolongation seen in non-anticoagulated APS patients due to the LAC effect. The rate of prothrombin conversion was significantly increased in anticoagulated APS patients compared to controls, indicating a more prothrombotic phenotype. The selected NN classified APS patients with a sensitivity of 92% and a specificity of 95% in the developmental cohort consisting of APS patients and controls on VKA antagonists (ROCAUC = 0.9805; 0.9542-1.000; p<0.0001). On average, controls and APS patients were assigned an APS likelihood score of 12% and 89%, respectively. The NN was clinically validated in a separate validation cohort of 311 individuals, including 33 anticoagulated APS patients, 62 anticoagulated controls, 38 thrombosis patients, 49 auto-immune disease patients, 92 hospital controls, and 37 healthy controls. The sensitivity of the NN for the detection of APS was 85%. The overall specificity of the NN was 93%, and 100%, 96%, 96%, 92% and 76% respectively in healthy controls, auto-immune disease controls, hospital controls, anticoagulated controls, and thrombosis controls. Conclusions We developed a NN that accurately classifies APS under anticoagulant treatment. Therefore, this NN could be an alternative for diagnosing APS patients already treated with VKAs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Assessing the Rate of Compliance with the Revised Sapporo/Sydney Criteria in Diagnosing Antiphospholipid Syndrome: A Multicenter Retrospective Analysis

Background: Antiphospholipid syndrome (APS) is a systemic autoimmune disease defined by thrombotic or obstetrical events in patients with persistent elevation of antiphospholipid antibodies (aPL) (Lim, 2013). The revised Sapporo/Sydney criteria has been adopted by most professional societies as a guidance to APS diagnosis. To be diagnosed with APS, a patient has to meet at least one clinical criteria and one laboratory criteria. Clinical criteria includes either vascular thrombosis or an adverse outcome during pregnancy. The lab criteria consist of the presence of one or more specified aPL, identified on two or more occasions, at least 12 weeks apart (Miyakis et al., 2006). Antibody testing in patients with suspected APS involves immunoassays for IgG and IgM antibodies to cardiolipin (aCL), and beta2-glycoprotein I (anti-β2GPI), as well as a functional assay for the lupus anticoagulant (LA) phenomenon (Miyakis et al., 2006). Testing asymptomatic individuals is not recommended and carries a significant rate of false positivity, given that up to 10% of healthy individuals may have transient aPL elevation (Vila et al., 1994). Furthermore, certain medications, infections, malignancies, autoimmune diseases, and rheumatologic diseases can also induce transient elevation of aPL antibodies without associated thrombotic complications, emphasizing the importance of repeated aPL measurements in the diagnosis (Lim, 2013; Rand & Wolgast, 2012). Weakly positive levels of aCL and anti-β2GPI antibodies should not be considered significant. Most authorities require Ab levels to be at or above the 99th percentile to be considered clinically significant (Rand & Wolgast, 2012). We aim to assess the compliance with revised Sapporo/Sydney criteria in diagnosing APS in our community hospital system. Methods: We conducted a multi-center, retrospective study among adult patients who were diagnosed with APS in a community healthcare system in southeast Michigan. Demographic and clinical characteristics were collected along with history of venous thrombosis, arterial thrombosis, pregnancy complications, aPL testing results and use of anticoagulants. We used SAS for exploratory data analysis and descriptive statistics. Results: A total of 781 patients were diagnosed with APS between August 2016 to December 2021 (table 1). The median age at diagnosis was 51.3 years (interquartile range 18-93 years). 639 (81.8%) patients were females and 142 (18.2%) were males. Majority of patients were white (n=563, 72.1%). 570 (73.0%) patients had history of venous or arterial thrombosis, 309 (49.9%) had pregnancy complications included in the revised Sapporo/Sydney criteria, and 211 (27.0%) had neither history of thrombosis nor pregnancy complications, not meeting the clinical criteria for APS diagnosis. 528 (67.6%) patients did not meet the laboratory criteria for the diagnosis (figure 1). Of these patients, 439 (83.1%) did not have a repeated testing of aPL antibodies.88 (16.7%) had weakly positive levels of aCL and anti-β2GPI antibodies, below the 99th percentile, and 1 (0.2%) had repeated antibodies testing in less than12 weeks. Out of 528 patients who did not meet the laboratory criteria, 321 (60.8%) were started on anticoagulants. Of these 321 patients, 135 (42%) did not have any history of thrombosis or other appropriate indication of anticoagulation, such as atrial fibrillation. Of 321 patients who were treated with anticoagulation, warfarin was the most prescribed accounting in 120 patients (37.4%). The remainder of patients (62%) were treated with apixaban, rivaroxaban or enoxaparin. Conclusion: Our results showed poor compliance with the current guidelines in both selecting the appropriate candidate for testing, and interpreting aPL testing results, leading to an alarmingly high rate of false diagnosis of APS. Obtaining a positive aPL test result in an otherwise disease-free individual can contribute to unnecessary anticoagulation and side effects including bleeding and increased cost burden to patients and families. Therefore, caution must be exercised while ordering aPL tests and adherence to current guidelines should be encouraged in the routine clinical settings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Thrombophilia Testing Patterns, Result Interpretation, and Clinical Outcomes in a Retrospective Cohort of Patients with Retinal Vascular Occlusion

Background Low quality evidence exists to support an association between retinal vascular occlusion and thrombophilia. However, testing may be complicated by the impact of anticoagulants, hormone use and other factors on the results. Even when test results are interpretable, the implications for clinical decision-making are not established. We performed a retrospective chart review to assess patterns of test selection, result interpretation, and clinical outcomes in a cohort of patients who received thrombophilia testing after presenting to a tertiary care center for retinal vascular occlusion. Methods Eligible patients were >18 years with retinal vein occlusion (RVO) or retinal artery occlusion (RAO) and referred for thrombophilia testing by an ophthalmologist between 3/16/2011 and 3/22/2021 at the University of Washington. Electronic medical records were extracted for relevant clinical characteristics, test results, use of antithrombotic therapy, and recurrent arterial or venous events. Continuous variables are summarized by means and standard deviations or medians and range, as applicable. Categorical variables are described by proportions. Differences between groups were assessed using Student's t-tests and Fisher's exact tests for continuous and categorical variables, respectively. Analyses were conducted in R. Results A cohort of 57 patients were identified, with median age of 44 years at diagnosis (Table 1). 38 (66.7%) presented with RVO and 23 (40.4%) presented with RAO, including 4 patients with concurrent RVO and RAO at diagnosis. Baseline characteristics were not significantly different between patients presenting RAO versus RVO, apart from a trend toward higher prevalence of stroke history in the RAO group (4/23 vs 1/38, p = 0.06). Thrombophilia test selection varied: protein C activity was most commonly tested (80.7%) while activated protein C resistance (40.4%) was least commonly ordered (Figure 1). Among the 50 patients who received some combination of antiphospholipid antibody (aPL) testing, only 44% had all Sydney criteria aPL assays performed. The underlying prevalence of thrombophilia was 3.5% (2/57). One patient was found to be homozygous for the factor V Leiden mutation (FVL) after presenting with a central RVO and 1 was diagnosed with antiphospholipid syndrome (APS) in the context of a branch RAO plus persistent positivity of anti-beta-2 glycoprotein I (IgM) and anticardiolipin (IgM) antibodies. Another 5 patients had abnormal test results that were not diagnostic of an underlying thrombophilia (1 heterozygous FVL, 1 heterozygous prothrombin gene mutation, and 3 positive aPL). In total, 15 patients were initiated on low-dose aspirin following diagnosis, including the patient diagnosed with APS. Four patients were treated with anticoagulation, including the heterozygous FVL and heterozygous prothrombin gene mutation patients, who were placed on indefinite anticoagulation. The patient found to be homozygous for FVL briefly received prophylactic dosing of rivaroxaban before switching to low-dose aspirin due to side effects. After a median follow-up of 3.1 years, 3 patients had recurrent events (1 RVO, 2 strokes). One developed recurrent stroke while taking rivaroxaban for atrial fibrillation, while the other 2 were not receiving antithrombotic therapy at the time of thrombosis recurrence. Conclusion A low prevalence of thrombophilia was observed in a young cohort of patients presenting with retinal vascular occlusion. Incomplete testing was common, including for antiphospholipid syndrome, which is associated with 2-fold increased risk of retinal vascular occlusion. Thrombophilia testing did not clearly aid in clinical decision making; the tests ordered and the actions taken based on the test result were inconsistent. Both patients identified to have underlying thrombophilia lacked other history of venous or arterial thromboembolism and ultimately opted against long-term anticoagulation. An additional 14 patients opted for antithrombotic therapy with low-dose aspirin after an entirely negative thrombophilia workup. Misinterpretation of test results likely led to inappropriate recommendations for indefinite anticoagulation in at least 2 patients. The utility of thrombophilia testing in retinal vascular occlusion is poorly studied. In this cohort, we found no evidence that such testing should influence clinical decision-making. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Predictors and Clinical Complications Associated with Antiphospholipid Antibodies in Sickle Cell Disease

Introduction. The antiphospholipid syndrome is characterized by the presence of anti-phospholipid autoantibodies (aPL) leading to a hypercoagulable state as well as an arterial, venous, and microcirculatory vasculopathy. Under several physiological and pathological situations, including the sickling of red blood cells (RBC), negatively charged phospholipids translocate from the inner to the outer surface of cell membranes leading to the potential generation of the antigenic target of aPL. We tested the following two-part hypothesis: 1) repeated exposure of the immune system of patients with sickle cell disease (SCD) to the antigenic target of aPL leads to an increased formation of aPL; and 2) the presence of aPL is associated with pregnancy and SCD-related complications. Methods. We conducted a cross-sectional study in 63 adults with SCD who had aPL testing performed during a hospital admission between August 1999 & June 2017. We compared individual aPL levels with categorical and linear variables using the Kruskal-Wallis test and linear regression analysis, respectively. The anticardiolipin (aCL) IgG and lupus anticoagulant (LA) levels were log-transformed for these analyses. An abnormal aPL was defined as any of the following: IgM or IgG anti-b2GPI >20 IU/mL, IgM or IgG aCL > 20 IU/mL, LA > 10 seconds. Clinical complications were compared by abnormal aPL status using the chi-square test. Results. The median age of the SCD cohort was 32 years (IQR, 21 - 39 years), 49 (78%) were female, 62 (98%) were African American, 5 (8%) had a diagnosis of systemic lupus erythematosus (SLE), 43 (68%) were Hgb SS genotype, 17 (27%) were on hydroxyurea, and 16 (25%) were on chronic anticoagulation therapy. SCD patients with SLE had higher median aCL IgM (6 vs. 1 IU/mL, respectively; P=0.02) and anti-b2GPI IgG (3.5 vs. 0 IU/mL, respectively; P=0.008) levels compared to those without SLE, while lower hemoglobin concentrations were associated with higher aCL IgG levels (β -0.12; P=0.07) and longer LA times (β -0.18; P=0.02). Fifteen of 63 (24%) patients with SCD had an abnormal aPL. Patients with an abnormal aPL had a lower hemoglobin concentration (Figure 1A), a higher white blood cell (WBC; 13.5 vs. 11.8 x109/L, respectively; P=0.06) count, and a lower platelet (268 vs. 340 x109/L, respectively; P=0.05) count at the time of hospitalization compared to those with a normal aPL. A lower hemoglobin concentration (odds ratio [OR], 1.9; P=0.008), a higher WBC count (OR 1.2; P=0.03), and a lower platelet count (10 platelets/µL increment: OR 1.05, P=0.06) were independently associated with the presence of an abnormal aPL by logistic regression analysis, adjusting for age, sex, and prior SLE diagnosis. Consistent with the second part of our hypothesis, several adverse outcomes trended higher in SCD patients with an abnormal aPL. An abnormal aPL was more prevalent among women with ≥ 3 miscarriages, preterm deliveries, or pre-eclampsia as well as during a hospitalization complicated by acute chest syndrome or multiorgan failure (Figure 1B). Conclusions. These data support the hypothesis that patients with SCD have a high predilection to form aPL, and that the presence of an abnormal aPL is associated with pregnancy complications and the severity of acute SCD-related complications during hospitalization. Our study is limited by its small number of patients. Further larger studies are needed to assess the mechanisms of aPL generation as well as to determine the prevalence and associated risk of abnormal aPL status in patients with SCD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Non-criteria antiphospholipid antibodies and pediatric rheumatic disease: a case series

BackgroundNon-criteria antiphospholipid antibodies (NC-aPL) are a relatively undefined subgroup of antiphospholipid antibodies (aPL). Knowledge about NC-aPL in adults is limited and even less is known in pediatric patients. Routine tests for antiphospholipid syndrome (APS)—a clinical state marked by the presence of aPL in association with vascular thrombosis—usually include lupus anticoagulant (LAC), anti-cardiolipin (aCL) and -beta-2 glycoprotein I (aβ2GPI). LAC is a functional screen for prothrombotic aPL, while the latter tests identify specific autoantibodies. Specific targets of NC-aPL include, but are not limited to, phosphatidylethanolamine, phosphatidylserine, and prothrombin.Presentation of casesWe present single-center data from eight pediatric patients with NC-aPL identified during a three-year period. All patients had presenting features raising suspicion for APS. Most patients were female with a primary rheumatic disease. One patient had a stroke. Another patient had alveolar hemorrhage and pulmonary hypertension. Raynaud’s phenomenon, rashes involving distal extremities, and headaches were common. Most patients had a positive LAC, yet their routine aPL tests were negative, prompting testing for NC-aPL.ConclusionsOur findings suggest NC-aPL are associated with typical signs and symptoms of APS in pediatric patients. Pediatricians and pediatric subspecialists should consider NC-aPL when clinical suspicion is high and routine aPL tests are negative, particularly when LAC is positive. While guidelines for NC-aPL do not yet exist for children or adults, these autoantibodies have pathogenic potential. Actionable items could include evaluation for the presence of other (primary) rheumatic diseases, and consultation with hematologists and/or obstetricians regarding anticoagulation/platelet inhibition and thrombosis education. Future guidelines regarding NC-aPL will only be generated by gathering more data, ideally prospectively.

Antiphospholipid antibodies are persistently positive at high titers. Additive value of platelet-bound C4d.

BackgroundClassification criteria for antiphospholipid syndrome (APS) require that antiphospholipid antibody (aPL) positivity is confirmed after at least 12 weeks. We tested the hypothesis that aPL at high titers remain positive while low titers fluctuate over time. As both platelet-bound C4d (PC4d) and aPL are associated with thrombosis in systemic lupus erythematosus (SLE), we also evaluated whether PC4d can aid in APS diagnosis.MethodsData from serum or plasma sent to Exagen’s laboratory for routine aPL testing were analyzed. Anti-cardiolipin (aCL) and anti-beta2 glycoprotein-1 antibodies (aB2GP1) were measured by chemiluminescence or ELiA fluorescence enzyme immunoassay; anti-phosphatidylserine/prothrombin complex antibodies (aPS/PT) by ELISA; PC4d by flow cytometry. Statistical analysis included descriptive statistics, logistic regression, and Pearson correlation.ResultsMore than 80% of positive samples with aCL and aB2GP1 at high titers - but not low titers - were positive at a retest. Non-criteria aPL (aPS/PT) followed a similar trend. aCL and aB2GP1 measured with two different technologies were highly correlated. PC4d and IgG of the three aPL were at best moderately correlated even when only positive aPL samples were analyzed (coefficient: 0.1917 to 0.2649).ConclusionsHigh titers aPL are often persistently positive, allowing an earlier diagnosis and risk assessment at the time of the initial screening. Conversely, a retest may be necessary for low titers. The high correlation between two methodologies suggests that these findings are independent of assay platform. The low to moderate correlation between PC4d and aPL might suggest a possible additive value to evaluate association with thrombosis in autoimmune diseases.

Neuroimaging features of antiphospholipid antibody-related stroke compared with atrial fibrillation-related stroke

Recognizing the lesion pattern of antiphospholipid antibody-related stroke (aPL-stroke) may contribute to establishing the cause in patients with cryptogenic stroke. We aimed to describe the neuroimaging features of aPL-stroke compared with atrial fibrillation-related stroke (AF-stroke), a major hidden cause of cryptogenic stroke. Using a prospective stroke registry, we identified consecutive aPL- and AF-stroke patients without other potential causes of stroke. Neuroimaging features based on diffusion-weighted imaging and angiographic findings at admission were compared. A total of 56 and 333 patients were included in the aPL- and AF-stroke groups, respectively. aPL-stroke patients more often presented with single small lesions (aPL-stroke, 30.4% vs. AF-stroke, 7.5%, p < 0.001), while the predominant pattern in AF-stroke patients was large territorial lesions (26.8% vs. 56.5%, p < 0.001). aPL-stroke patients had smaller infarct volume (1.58 mL [0.45; 9.41] vs. 11.32 mL [2.82; 33.08], p < 0.001) and less experience of relevant artery occlusion (17.9% vs. 54.7%, p < 0.001). The proportion of multi-territory lesions, an embolic pattern, was similar between the two groups (28.6% vs. 22.8%, p = 0.44). In comparison only including patients with multi-territory lesions as well, aPL-stroke patients showed small lesion dominance and smaller infarct volume. Multivariate analyses showed independent associations between mild neuroimaging features (small lesion prevalence, smaller infarct volume, and absence of relevant artery occlusion) and aPL-stroke. Patterns of small lesion prevalence, small infarct volume, and absence of relevant artery occlusion were suggestive of aPL-stroke rather than AF-stroke. Cryptogenic stroke patients with such neuroimaging features may benefit from aPL testing for a precise diagnosis.

Venous Thromboembolic Events in African American Lupus Patients in Association With Antiphospholipid Antibodies Compared to White Patients.

The risk of thrombotic events is elevated in patients with systemic lupus erythematosus (SLE) compared to the general population and has been attributed to both systemic inflammation and to the presence of antiphospholipid antibodies (aPLs). Our objective was to examine differences in aPL prevalence in White and African American patients with SLE and venous thromboembolic (VTE) events, and to compare inflammatory markers at the time of a VTE event. Records of White and African American patients with SLE and VTE events were retrieved from a rheumatology practice based at an academic hospital. A clinically significant aPL profile was defined as anti-cardiolipin IgG/IgM and/or anti-β2 -glycoprotein I IgG/IgM ≥40 units, and/or positive lupus anticoagulant ≥1.3. Logistic regression was used to determine predictors of a clinically significant aPL profile. Ninety-seven patients fulfilled American College of Rheumatology and/or 2012 Systemic Lupus Erythematosus International Collaborating Clinics classification criteria for SLE, had a history of VTE events, and had available aPL tests (59 White and 38 African American patients). African American patients were 66% less likely (95% confidence interval 0.12-0.96; P = 0.04) to have a clinically significant aPL profile compared to White patients in multivariable regression. Triple positivity was most frequent among White patients, while 7 of 8 African American patients had a positive lupus anticoagulant test. At the time of a VTE event, African American patients had significantly higher levels of anti-double-stranded DNA (P = 0.02), lower hemoglobin (P = 0.01), and higher erythrocyte sedimentation rate (P = 0.008). Among patients with SLE and VTE events, African American patients were less likely to have a clinically significant aPL profile compared to White patients, indicating that a negative aPL profile in African American patients does not decrease VTE risk.

Carbamylation of β2-glycoprotein I generates new autoantigens for antiphospholipid syndrome: a new tool for diagnosis of 'seronegative' patients.

Antiphospholipid syndrome (APS) is a prothrombotic condition defined by recurrent thrombosis, pregnancy complications and circulating antiphospholipid antibodies (aPL), including anti-β2-glycoprotein I (β2-GPI). In clinical practice it is possible to find patients with APS persistently negative for the aPL tests according to Sydney criteria ('seronegative APS', SN-APS). Recently, several autoimmune responses have been described as a consequence of post-translational modifications of their target autoantigens. This study was undertaken to test carbamylated-β2-GPI (Carb-β2-GPI) as a new autoantigen of APS. β2-GPI was carbamylated by potassium cyanate and used to investigate its effect on monocyte-derived dendritic cell (moDC) phenotype and function. Sera from 114 SN-APS patients, 60 APS, 20 patients with RA, 20 non-APS thrombosis and 50 healthy donors were analysed for anti-Carb-β2-GPI by ELISA. Carb-β2-GPI is able to activate moDCs, inducing upregulation of CD80, CD86 and CD40, activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase and nuclear factor-κB, and IL-12p70 release. Serological results showed that both 37/114 SN-APS (32.46%) and 23/60 APS (38.33%) patients resulted positive for anti-Carb-β2-GPI. Interestingly, SN-APS patients who tested positive for anti-Carb-β2-GPI showed a higher prevalence of thrombocytopenia (P = 0.04, likelihood positive ratio of 3.9). Data obtained from both functional tests on moDCs and immunological approaches prompted identification of Carb-β2-GPI as a 'new' antigenic target in APS. In particular, anti-Carb-β2-GPI revealed a potential usefulness in identification of a significant proportion of SN-APS patients. Moreover, since patients who tested positive for anti-Carb-β2-GPI reported a high risk of thrombocytopenia, this test may be considered a suitable approach in the clinical evaluation of SN-APS.

Clinical Management of Thrombotic Antiphospholipid Syndrome.

Thrombotic manifestations of antiphospholipid syndrome are often a therapeutic dilemma and challenge. Despite our increasing knowledge of this relatively new disease, many issues remain widely unknown and controversial. In this review, we summarise the latest literature and guidelines on the management of thrombotic antiphospholipid syndrome. These include the laboratory assays involved in antiphospholipid antibodies (aPL) testing, the use of direct oral anticoagulants in secondary prevention, management of recurrent thrombosis, individuals with isolated aPL, and catastrophic antiphospholipid syndrome. Treatment aims to prevent the potentially fatal and often disabling complications of APS with antithrombotic and cardiovascular risks prevention strategies. Some insights and updates on topical issues in APS are provided. We also include our current practice, which we believe is the pragmatic approach based on the currently available evidence.

Characteristics of Patients With Antiphospholipid Antibody Positivity in the APS ACTION International Clinical Database and Repository.

To describe the baseline characteristics of patients with positivity for antiphospholipid antibodies (aPLs) who were enrolled in an international registry, the Antiphospholipid Syndrome (APS) Alliance for Clinical Trials and International Networking (APS ACTION) clinical database and repository, overall and by clinical and laboratory subtypes. The APS ACTION registry includes adults who persistently had positivity for aPLs. We evaluated baseline sociodemographic and aPL-related (APS classification criteria and "non-criteria") characteristics of patients overall and in subgroups (aPL-positive without APS, APS overall, thrombotic APS only, obstetric APS only, and both thrombotic APS/obstetric APS). We assessed baseline characteristics of patients tested for the presence of three aPLs (lupus anticoagulant [LAC] test, anticardiolipin antibody [aCL], and anti-β2 -glycoprotein I [anti-β2 GPI]) antibodies by aPL profiles (LAC only, single, double, and triple aPL positivity). The 804 aPL-positive patients assessed in the present study had a mean age of 45 ± 13 years, were 74% female, and 68% White; additionally, 36% had other systemic autoimmune diseases. Of these 804 aPL-positive patients, 80% were classified as having APS (with 55% having thrombotic APS, 9% obstetric APS, and 15% thrombotic APS/obstetric APS). In the overall cohort, 71% had vascular thrombosis, 50% with a history of pregnancy had obstetric morbidity, and 56% had experienced at least one non-criteria manifestation. Among those with three aPLs tested (n=660), 42% were triple aPL-positive. While single-, double-, and triple aPL-positive subgroups had similar frequencies of vascular, obstetric, and non-criteria events, these events were lowest in the single aPL subgroup, which consisted of aCLs or anti-β2 GPI only. Our study demonstrates the heterogeneity of aPL-related clinical manifestations and laboratory profiles in a multicenter international cohort. Within single aPL positivity, LAC may be a major contributor to clinical events. Future prospective analyses, using standardized core laboratory aPL tests, will help clarify aPL risk profiles and improve risk stratification.

Current status and role of antiphospholipid antibody testing in cryptogenic stroke.

Although it is not recognized as essential to test for antiphospholipid antibody (aPL) in stroke of unknown cause, aPL-related stroke may account for a considerable number of cryptogenic strokes. We aimed to assess the current status and diagnostic value of aPL testing in cryptogenic stroke patients. Consecutive patients admitted with acute ischemic stroke were examined to confirm the factors associated with performing aPL testing and with positive aPL test results in real-world practice. Cryptogenic stroke patients were separately examined in the same manner. The antibody profiles of cryptogenic stroke patients with aPL positivity were compared by age. Among 2947 patients, 606 (20.6%) were tested for aPLs and 129 (21.3%) were positive. Physicians tended to perform aPL testing in patients aged <50years and in cryptogenic stroke patients. Cryptogenic stroke was a strong predictor of positive aPL results (adjusted odds ratio 3.70, 95% confidence interval 2.38-5.76). However, aPL positivity did not differ by age in stroke patients. Among 283 cryptogenic stroke patients, 136 (48.1%) were tested for aPLs and 56 (41.2%) were positive. aPL tests were performed predominantly in patients aged <50years rather than in older patients, even among cryptogenic stroke patients. The two age groups had similar positivity rates of >40% (<50years: 43.2%; ≥50years: 40.4%; p=0.92) and their antibody profiles were similar. A significant number of patients with cryptogenic stroke had positive aPL results regardless of age. aPL testing may offer additional diagnostic opportunities in cryptogenic stroke patients, and thus may reduce the incidence of cryptogenic stroke.

COVID-19 and antiphospholipid antibodies: A position statement and management guidance from AntiPhospholipid Syndrome Alliance for Clinical Trials and InternatiOnal Networking (APS ACTION).

Coronavirus disease 2019 (COVID-19) is associated with a high rate of thrombosis. Prolonged activated partial thromboplastin times (aPTT) and antiphospholipid antibodies (aPL) are reported in COVID-19 patients. The majority of publications have not reported whether patients develop clinically relevant persistent aPL, and the clinical significance of new aPL-positivity in COVID-19 is currently unknown. However, the reports of aPL-positivity in COVID-19 raised the question whether common mechanisms exist in the pathogenesis of COVID-19 and antiphospholipid syndrome (APS). In both conditions, thrombotic microangiopathy resulting in microvascular injury and thrombosis is hypothesized to occur through multiple pathways, including endothelial damage, complement activation, and release of neutrophil extracellular traps (NETosis). APS-ACTION, an international APS research network, created a COVID-19 working group that reviewed common mechanisms, positive aPL tests in COVID-19 patients, and implications of COVID-19 infection for patients with known aPL positivity or APS, with the goals of proposing guidance for clinical management and monitoring of aPL-positive COVID-19 patients. This guidance also serves as a call and focus for clinical and basic scientific research.

A new pro-thrombotic mechanism of neutrophil extracellular traps in antiphospholipid syndrome: impact on activated protein C resistance.

In APS, precise evaluation of thrombotic risk is a major challenge. Different players, such as activated protein C (APC) resistance or neutrophil extracellular traps (NETs) contribute to the risk of thrombosis. Nevertheless, no study has investigated the interaction between these actors. The main objective of this study was to investigate the relation between NETs and APC resistance. We designed a cross-sectional study including APS/antiphospholipid antibodies (aPL) patients and patients with autoimmune diseases (AID). We performed thrombin generation tests without and with APC to determine APC resistance. To evaluate circulating NETs, we measured plasma levels of MPO-DNA complexes and cell-free DNA with ELISA. We recruited 117 patients with definite APS/aPL or AID. We found a positive correlation between NETs and APC resistance, in APS patients and specifically in patients with high thrombotic risk, displaying LA or positivity of all three aPL tests (triple+), or anti-domain I IgG (aDI+). All these patient subgroups had increased NETs concentrations and APC resistance. As the risk profile for thrombosis increased, the relationship between NETs and APC resistance was stronger. We have shown that NETs participate in the hypercoagulable state of APS patients by contributing to APC resistance, in particular in high-risk patients. In these most at-risk patients, a targeted action on NETs could reduce APC resistance and constitute a new therapeutic approach in the treatment of APS patients in addition to antithrombotic therapy.

Evaluation of Frequency, Clinical Correlation, and Antibodies Confirmation Profile in Patients with Suspected Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by arterial and venous thrombotic manifestations and/or pregnancy-related complications in patients with persistent antiphospholipid (aPL) antibodies. The introduction of Sapporo's classification criteria allowed uniformity in the classification of this pathology, representing a considerable advance in its diagnosis. However, currently some doubts about the application of these criteria still persist. The aim of this study was to contribute to the better understanding of APS by the assessment of aPL prevalence, the association between clinical and laboratory tests, and evaluation of the aPL confirmatory profile.In this study, 1,179 samples from patients with suspected APS of both genders, without age restrictions, who were advised to test for complete aPL's profile were analyzed. The samples were tested for lupus anticoagulant (LAC), anticardiolipin immunoglobulin (Ig) G/IgM and anti-β-2-glycoprotein I IgG/IgM antibodies. Patient samples with isolated test requests for analysis and samples from patients under the influence of anticoagulants or in an infectious process were excluded.The overall positivity found was 17.9% and the most frequent aPL was LAC. The antibodies were determined in isolation and in association. The prevalence of triple positivity was 0.8% and double positivity was 1.8%. Positivity was higher in inpatient/emergency services compared with outpatient services. There was a higher positivity in individuals over 41 years, males, patients with systemic lupus erythematosus, kidney complications, and deep vein thrombosis/thrombophlebitis. The positivity confirmation with second sample was 39.5% and the confirmation profile shows that 50.6% of samples confirmed with same positivity profile; 17.3% with a different profile and regarding to these, 2.5% of the samples confirmed positivity with a different antibody from the previously detected.This study suggests that the aPL's positivity tends to increase with age, showing that the aPL's testing should be avoided during an acute event and reinforces the need for complete aPL laboratory profile in the second sample and subsequent determinations.

Indication and outcome of lupus anticoagulant and antiphospholipid antibodies testing in routine clinical practice.

ObjectivesLupus anticoagulans (LACs) and aPLs, both further summarized as aPL, are frequently assessed in routine daily clinical practice in diagnostic workups for suspected autoimmune diseases or to test for underlying risk factors in patients with thrombosis or obstetric complications. The aim of this study was to determine the prevalence of aPL positivity in patients with an indication for aPL testing in routine clinical practice.MethodsIn this retrospective single-centre study, indication for aPL testing, aPL test results and clinical data were collected for patients tested between June 2015 and April 2018.ResultsDuring the study period, 16 847 single aPL tests were performed in 2139 patients. In 212 patients one or more positive aPL test was found, confirmed in 43.9% with a second positive test. Indications for aPL testing were diagnostic workup/follow-up of autoimmune diseases (33.6%), thrombosis (21.4%) and obstetric complications (28%). Seventy-four patients (3.5% of all patients) fulfilled the criteria of APS, of whom 51% were newly diagnosed. Second positive aPL titres and titres of APS patients were significantly higher compared with positive aPL titres at the first measurement (P < 0.05). Patients with indications of arterial thrombosis and diagnostic workup/follow-up of autoimmune diseases had significantly higher levels of aCL IgG and anti-β2 glycoprotein I (β2GPI) IgG compared with patients with other indications.ConclusionThe prevalence of one or more positive aPL test was 9.9% and APS was diagnosed in 3.5% of the patients. Patients with arterial thrombosis had significantly higher anti-β2GPI IgG and aCL IgG, which should be confirmed in future studies.

The Predictive Value of the aCL and Anti-β2GPI 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-β2glycoprotein I (anti-β2GPI) 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-β2GPI 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-β2GPI. 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-β2GPI 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-β2GPI at the time of acute DVT have a strong positive predictive value for APS and may support therapeutic decisions.

Assessment of Anti-β2 glycoprotein1 antibody in Systemic Lupus Erythrematosus Patients

Secondary anti-phospolipid syndrome (APS) is diagnosed in many patients with systemic lupus erythrematosus (SLE) especially with thromboembolic events and/or pregnancy loss in the presence of persistent laboratory evidence for anti-phospholipid antibody (aPL). In this work, we aimed to detect the prevalence of IgG and IgA anti-β2 glycoprotein1 (β2GP1) in SLE patients. Serum samples were collected from 50 female patients with SLE (25 had APS and 25 patients who did not have APS), in addition to 22 apparently healthy females with matched age as a control group. All samples from patients and control were tested for lupus anticoagulant (LA), IgG and IgA isotypes of antiβ2GPI, Anti-nuclear antibody (ANA), Anti-double strand antibody (Anti-dsDNA). Number of patients positive for Anti-β2GP1 antibodies were significantly increased in APS patients compared to non-APS patients (P=0.015). Anti-β2GP1 IgA isotype was significantly higher in APS patients than in non-APS patients (P=0.011) and significantly correlated with deep venous thrombosis and pregnancy morbidity (P=0.004). There was no difference in anti-β2GPI IgG isotype between APS patients and non-APS patients. We concluded that although anti-β2GPI IgA is not within Sapparo diagnostic criteria, it seems to contribute to the pathogenesis of thrombotic manifestations of SLE and may represents a useful indicator particularly when standard aPL tests are negative.