The use of an anti-β2-glycoprotein-I assay for discrimination between anticardiolipin antibodies associated with infection and increased risk of thrombosis

Summary. Antiphospholipid antibodies (aPAs), occurring in association with infection, are not generally associated with an increased risk of thrombosis. Anticardiolipin antibodies (aCL) from patients with infection, unlike those from patients with SLE, do not have the β₂GPI cofactor requirements. Antibodies to β₂GPI (αβ₂GPI) are more closely associated with a previous history of thrombosis than aCL in patients with SLE. In the present study we have investigated the reactivity of the αβ₂GPI assay for aPAs associated with infection. Serum from 114 patients with infections including syphilis ( n = 11), tuberculosis ( n = 63) and Klebsiella ( n =42) were assayed for αβ₂GPI and aCL antibodies. The incidence of aCL in serum of patients with tuberculosis, Klebsiella infection and syphilis was 6.0%. 5.0% and 64.0%. respectively, but all patients were negative for αβ₂GPI. These results indicate that the αβ₂GPI assay is negative in patients with transiently positive aCL assays associated with infection.     

β2 glycoprotein-I antigen is increased in primary hyperlipidaemia

Summary. In order to determine whether elevated levels of β₂ glycoprotein-I (β₂GPI) are associated with increased plasma lipids, we measured plasma β₂ GPI antigen levels in 47 patients with primary hyperlipidaemia (20 severe hypercholesterolaemia, nine severe hypertriglyceridaemia, and 18 mixed hyperlipidaemia) and 34 normal healthy subjects. Mean β₂GPI levels were significantly increased in each patient group (302.3, 272.9 and 299.1 mg/1. respectively) compared to controls (199.6 mg/1) (p<0.01). Significant correlations were demonstrated between β₂GPI levels and triglyceride and total cholesterol levels in the control group (r = 0.387, r =0.559: P<0.5), but were not observed in all patient groups. These results indicate that β₂GPI is increased in hyperlipidaemia and that its distribution between plasma lipid fractions is perturbed. Plasma lipid levels should therfore be considered when interpreting results of β₂GPI antigen assays.     

Prevalence and significance of anticardiolipin, anti-β2 glycoprotein I and anti-prothrombin antibodies in chronic hepatitis C

Antiphospholipid antibodies have been demonstrated in chronic hepatitis C, but their clinical and pathogenetic significance remains elusive. We prospectively studied 115 patients (85 men, mean age 36.9 years) with chronic hepatitis C without cirrhosis and treated by α-interferon (α-IFN). Antiphospholipid determinations comprised anticardiolipin (ACA), anti-β₂-glycoprotein I and anti-prothrombin antibodies of the IgG and IgM classes. At entry, 24 patients (21%) were found to possess low to moderate ACA levels (18 IgG, two IgM and four both isotypes) compared with only 4/115 age- and sex-matched control subjects (3.5% P  = 0.001). ACA positivity rate increased to 31% ( P  = 0.01) after a 6-month course of α-IFN treatment. In contrast, the prevalence of anti-β₂-glycoprotein I and anti-prothrombin antibodies was not significantly different from controls at either time point. The presence of ACA correlated with that of antinuclear antibodies ( P  = 0.0002), but was not associated with parameters such as histological activity, viral burden and response to α-IFN, nor with a history of thrombosis or pregnancy loss. However, a non-significant trend of higher incidence of mild thrombocytopenia among ACA-positive patients was observed. We conclude that low-titre ACA positivity is a common finding in patients with chronic hepatitis C, especially following α-IFN treatment, but does not select a category with different clinical features. These data are in keeping with the absence of associated anti-β₂GPI and anti-prothrombin antibodies, and do not support a role for HCV infection in the pathogenesis of the antiphospholipid syndrome.     

Are immunoglobulins with lupus anticoagulant activity specific for phospholipids?

Summary. Recent studies have suggested that the lupus anticoagulant (LA) may be specific for prothrombin. prothrombin-phospholipid complexes, or β₂ glycoprotein 1 (β₂, GP1) rather than phospholipids. We performed a series of experiments to determine whether LA is indeed phospholipid specific. IgG was purified from sera of six patients with the antiphospholipid syndrome (APS) and 10 healthy controls. The six IgG-APS preparations had both LA and anticardiolipin (aCL) activity. Incubation of the six IgG-APS preparations with cardiolipin (CL), phosphatidylserine (PS), phosphatidyl-choline (PC), or PS/PC (20:80) liposomes in Tris-buffered saline, resulted in loss of LA activity from the supernatant. We postulated that loss of activity might have resulted from absorption of IgG LA antibodies by phospholipids, a dilutional effect, or the presence of phospholipids in the supernatant causing 'by-pass' of IgG LA inhibitory activity. To distinguish between these possibilities, we re-isolated IgG from the supernatants and re-tested them for LA activity. IgG re-isolated from the PS, CL and PS/PC supernatants had no LA activity, but LA activity remained in the PC supernatant. This suggested that IgG with LA activity was absorbed by negatively charged but not by zwitterionic phospholipids. In like manner, PS, CL and PS/PC, but not PC liposomes, absorbed IgG with aCL activity. Mixtures of the phospholipid liposomes with β₂GP1 did not modify the absorption of IgG with LA or aCL activity. Finally, we demonstrated that IgG eluted from immunoglobulin-cardiolipin liposome complexes had LA activity. Based on these findings. we conclude that at least one population of antibodies with LA activity is phospholipid specific.     

Factors affecting cardiolipin antibody assays: modification with polyethylene glycol compound

Anti-cardiolipin antibody (aCL) measurement is only semi-reproducible, and current assays detect irrelevant as well as clinically significant antibodies. Factors found to influence results included the source of the enzyme-linked immunoabsorbent assay (ELISA) plate, and its pre-treatment with solvents; the nature of the blocking solution; and the composition of the diluent used for reagents. Fetal calf serum (FCS) in the diluent appeared to reduce non-specific (clinically irrelevant) binding and was not simply a source of β₂-glycoprotein-1 (β₂GP1). A polyethylene glycol compound was found to be an effective blocking agent, and its use enhanced the discrimination between positive and negative samples. A high level of variation may be inherent in aCL-ELISA methodology, but the use of polyethylene glycol compound appeared to be a helpful modification.     

Genetic risk factors of thrombosis in the antiphospholipid syndrome

The possibility of a genetic predisposition to develop antiphospholipid syndrome (APS) and to produce anticardiolipin antibodies and lupus anticoagulant has been addressed by family studies and population studies. Various studies suggest a familial occurrence of anticardiolipin antibodies and lupus anticoagulant, with or without clinical evidence of APS. This familial tendency could be genetically determined. Multiple human leucocyte antigen-DR or -DQ associations with antiphospholipid antibodies have been described. Genetic studies of a representative antigen, beta2-glycoprotein-I (β₂GPI), have been carried-out and a particular valine²⁴⁷/leucine polymorphism could be a genetic risk for presenting anti-β₂GPI antibodies and APS. Many other thrombosis-related genetic factors have been investigated in APS, but no additional risk for thrombosis has been indicated in affected patients. Although the mechanisms and pathophysiology of thrombosis in APS are highly heterogeneous and multifactorial, different genes and acquired factors seem to be involved. In this review, we will focus on those genetic variants that could contribute to the development of thrombosis in APS.     

Anti-endothelial cell antibodies to the endothelial hybridoma cell line (EAhy926) in systemic lupus erythematosus patients with antiphospholipid antibodies

The endothelial hybridoma (EAhy926) cell line was employed to clarify whether antiphospholipid antibodies (aPA) [lupus anticoagulant (LA), antiprothrombin antibody (aPT) and/or anticardiolipin antibody (aCL)] and anti-endothelial cell antibodies (AECA) are identical, and establish whether β₂-glycoprotein I (β₂-GPI) is needed for reactivity of aPA to endothelial cells. Ig-G AECA was positive in 9/30 SLE patients with aPA (30.0%) and 10/22 SLE patients negative for aPA (45.5%). Ig-M AECA was positive in one SLE patient with aPA and one SLE patient without aPA. AECA-positivity was not significantly different among unfixed, TNF-stimulated and fixed EAhy926. The influence of β₂-GPI on the reactivity of serum to EAhy926 was minimal, and absorption experiments of serum with cardiolipin-liposome/β₂-GPI or phosphatidylserine-liposome/prothrombin gave little evidence of cross-reactivity of aPA and AECA. The results of our study suggest that aPA and AECA may have partially cross-reacted, but were different antibodies. However, further study is needed to clarify the clinico-pathological significance of AECA.     

Antiphospholipid antibodies in adults with immune thrombocytopenic purpura

To determine the clinical significance of antiphospholipid antibodies (aPL) in patients with immune thrombocytopenic purpura (ITP), anticardiolipin (aCL) (IgG and IgM) and lupus anticoagulant (LA) were sought at diagnosis in 215 ITP adults with platelets <50 × 10⁹/l. aPL (aCL and/or LA) were detected in 55 patients (26%): aCL alone in 39 (18%), aCL and LA in 15 (7%) and LA alone in one (0·5%). LA was significantly associated with high IgG-aCL levels ( P  =   0·001). Among age, sex, initial platelet count, bleeding score, acute or chronic ITP outcome, only younger age was significantly associated with LA-positivity (mean age 29 ± 14 years vs. 45 ± 20 years, P  =   0·002). After a median follow-up of 31 months, 14/215 (7%) patients developed thrombosis (four arterial, 10 venous and/or pulmonary embolism); four of them (29%) had high aCL levels and LA. Multivariate analysis significantly associated thrombosis events only with age [hazard ratio (HR)   =   1·6; 95% confidence interval (CI): 1·2–2·4], LA (HR: 9·9; 95% CI: 2·3–43·4) or high IgG-aCL level (HR: 7·5; 95% CI; 1·8–31·5). Although the thrombosis rate was low, the significant associations between thrombosis and LA or high aCL level suggest that aPL should be tested at ITP diagnosis.     

Clinical significance of antiphospholipid antibodies in Indian scleroderma patients

In patients with systemic sclerosis (SSc), antiphospholipid antibodies (aPL) have been reported to be associated with more severe manifestations including digital infarct, gangrene and pulmonary hypertension. But these findings are not consistent in all studies; moreover, there are no data available from Indian subcontinent. The objective of this study is to assess the prevalence of antiphospholipid antibodies in Indian SSc patients and correlate them with clinical and immunological features. Seventy-two patients were recruited prospectively from rheumatology clinic from 2002 to 2006. Their medical records were reviewed. Anticardiolipin antibodies (IgG, IgM) by ELISA and lupus anticoagulant (LA) were tested in standardized pattern and repeated after 6 weeks. Anti-β2 glycoprotein-I antibodies were done in patients who had aPL antibodies. Nineteen patients had diffuse cutaneous SSc and 53 had limited disease. Seven patients (9.7%) were positive for aPL antibodies in their sera. Only one patient had clinical features of antiphospholipid antibody syndrome and manifested with recurrent abortions and deep vein thrombosis. She was positive for aCL, LA and anti-β2 glycoprotein-I antibodies. Four patients were only aCL (IgG) positive in moderate titers and one each had only aCL (IgM) and LAC positivity. None of the clinical parameters showed an association with aPL antibody.     

Testing for antiphospholipid antibodies: Advances and best practices

The diagnosis of antiphospholipid syndrome (APS) relies on the detection of circulating antiphospholipid antibodies (aPL). Currently, lupus anticoagulant (LAC), anticardiolipin (aCL), and antibeta2‐glycoprotein I antibodies (aβ2GPI) IgG or IgM are included as laboratory criteria if persistently present. Progress has been made on the standardization of tests as guidelines on LAC testing and immunological assays for aCL and aβ2GPI are published. However, LAC measurement remains a complicated procedure with many pitfalls and interfered by anticoagulant therapy. Solid‐phase assays for aCL and aβ2GPI still show interassay differences. These methodological issues make the laboratory diagnosis of APS challenging. In the interpretation of aPL results, antibody profiles help in identifying patients at risk. Noncriteria aPL, such as antibodies against the domain I of beta2‐glycoprotein (aDI) and antiphosphatidylserine‐prothrombin (aPS/PT) antibodies have been studied in the last years and may be useful in risk stratification of APS patients. But, aDI and aPS/PT are not included in the current diagnostic criteria and testing in daily practice is not recommended as these antibodies have no added value in the diagnosis of APS. This review will focus on the technical aspects of the laboratory methods, the clinical relevance of assays and interpretation of aPL results in the diagnosis of APS.     

Anti-prothrombin antibodies combined with lupus anti-coagulant activity is an essential risk factor for venous thromboembolism in patients with systemic lupus erythematosus

Anti-prothrombin antibodies (anti-prothrombin) and anti-beta2-glycoprotein I antibodies (anti-beta2-GP I) are the most common and characterized anti-phospholipid antibodies (aPL) detected using specific enzyme-linked immunosorbent assay (ELISA) systems. Recently, lupus anti-coagulant (LA) activity detected by a phospholipid-dependent coagulation assay was reported to be associated with anti-prothrombin and/or anti-beta2-GP I. Here we show that the co-existence of IgG anti-prothrombin and LA activity might be an essential risk factor for venous thromboembolism (VTE) in patients with systemic lupus erythematosus (SLE). We examined not only the levels of antibodies to prothrombin and anti-beta2-GP I (both IgG and IgM isotypes) using an ELISA system, but also LA activity detected using both diluted Russell's viper venom time (dRVVT) and STACLOT LA test in 124 patients with SLE. The SLE patients were divided into four groups according to the results of ELISA and LA assay results for each aPL: group A, ELISA+ and LA+ group B, ELISA+ and LA-; group C, ELISA- and LA+ group D, ELISA- and LA-. Regarding IgG anti-prothrombin, the prevalence of VTE was significantly higher in group A (16/35 cases, 45.7%, P < 0.001, Fisher's exact probability test) than in the other groups (B, 2/30, 6.7%; C, 1/22, 4.5%; D, 1/37, 2.7%). With respect to IgM anti-prothrombin and IgG or IgM anti-beta2-GP I, the prevalence of VTE was higher in both groups A and C than in group D, but no statistical difference in prevalence was found between groups A and C. Multivariate logistic regression analysis of risk factors for VTE confirmed that the co-existence of IgG anti-prothrombin and LA activity was the only significant risk factor for VTE (odds ratio, 19.13; 95% confidence intervals, 4.74-77.18).     

Prevalence and isotype distribution of antiphospholipid antibodies in unselected Chilean patients with venous and arterial thrombosis

Antiphospholipid antibodies (aPL) are a heterogeneous family of antibodies associated with thrombotic events and other complications. The objective of this study was to investigate the prevalence of aPL in a group of Chilean patients with thrombosis. Two hundred and twenty-six patients with venous and arterial thrombosis and 95 healthy controls were studied. Anticardiolipin (aCL), anti-₂ glycoprotein I (anti-₂GPI), and antiprothrombin (aPT) antibodies were determined. Eighty-eight out of 226 (38.9%) patients with thrombosis had some type of aPL. Fifty-seven patients (25.2%) were positive for aCL, 31 (13.7%) for aPT, and 14 (6.2%) for anti-₂GPI antibodies. Twelve patients (5.3%) were positive for more than one aPL. IgG, IgM and IgA isotypes were observed in aCL, anti-₂GPI, and aPT antibodies. Twenty-six out of 92 (28.3%) patients with venous thrombosis and 31/134 (23.1%) patients with arterial thrombosis were positive for aCL antibodies. With regard to the control group (4/95=4.2%), the odd ratios (OR) were 5.2 (1.3–19.8; p0.01) and 5.7 (1.6–22.3; p0.01), respectively. Additionally, we observed statistically significant OR with aPT and anti-₂GPI antibodies; in the first, with venous and arterial thrombosis, and in the second, only with arterial thrombosis. Our results show a significant prevalence of aPL, predominantly aCL and aPT antibodies, in patients with thrombosis. Additionally, aCL and aPT antibodies appear to be a risk factor for venous and arterial thrombosis, and anti-₂GPI antibodies appear to be a risk factor for arterial thrombosis.Abbreviations aCL Anticardiolipin antibodies - aPL Antiphospholipid antibodies - AMI Acute myocardial infarction - BSA Bovine serum albumin - DVT Deep venous thrombosis - FBS Fetal bovine serum - IS Ischemic stroke - LA Lupus anticoagulant - RT Room temperature - RVT Retinal vein thrombosis - SLE Systemic lupus erythematosus     

Is the determination of anti‐beta2 glycoprotein I antibodies useful in patients with venous thromboembolism without the antiphospholipid syndrome?

Anti-beta2-glycoprotein I (beta2GPI) antibodies are frequently found in patients with lupus anticoagulant (LA). To investigate the prevalence of antibeta2GPI antibodies and their clinical impact in patients with a history of venous thromboembolism (VTE) without LA/anticardiolipin antibodies (ACA), we studied 503 patients [128 (36.2%) men, median age 41 years (interquartile range, IQR 28-54 years)] with previous thrombosis. A group of 113 individuals without VTE [43 (38.1%) men, age 46.7 years (IQR 38-52 years)] served as a control group. Among 418 patients without LA/ACA, anti-beta2GPI-IgG levels were elevated in seven (1.7%), -IgM in 15 (3.6%) and -IgA in 14 (3.3%) cases; in 58 patients with ACA, anti-beta2GPI-IgG levels were elevated in two (3.4%), six (10.3%) and three (5.2%), and in 27 with LA, they were elevated in 18 (66.7%), 19 (70.4%) and 10 (37%) respectively. Thus, the prevalence of elevated anti-beta2GPI antibodies was not increased in patients without LA/ACA but was strongly associated with LA. Patients without ACA/LA who had a recurrent event did not have higher prevalence of elevated anti-beta2GPI-IgG, -IgM or -IgA antibodies than those without a recurrent event. Thus, elevated antibeta2GPI antibodies are not likely to be a predictor of recurrent events in patients without LA. We conclude that determination of anti-beta2GPI antibodies does not improve the clinical management of patients with a history of VTE without LA/ACA.     

Characterization of IgG monoclonal anti-cardiolipin/anti-beta2GP1 antibodies from two patients with antiphospholipid syndrome reveals three species of antibodies

Antiphospholipid antibodies (aPL), including antibodies detected in anti-cardiolipin (aCL) enzyme-linked immunosorbent assays and in lupus anticoagulant (LA) tests, are strongly associated with recurrent thrombosis and recurrent fetal loss, i.e. the antiphospholipid syndrome (APS). Although recent studies suggest that most APS-associated aCL are directed against the phospholipid (PL)-binding plasma protein beta2-glycoprotein 1 (beta2GP1), the precise nature of aCL binding specificities remains controversial. To address the issue of aCL specificity we generated five new monoclonal IgG aCL from two patients with APS. Characterization of these five aCL, as well as two previously published IgG aCL, revealed three patterns of reactivity: (1) four antibodies reacted strongly with human beta2GP1-cardiolipin (CL) complexes and weakly with human beta2GP1 alone; (2) two antibodies recognized bovine beta2GP1, but not human beta2GP1; (3) one antibody reacted with complexes of human beta2GP1 and CL, but not with human beta2GP1 alone. Only one monoclonal displayed weak LA activity. These patient-derived IgG monoclonal antibodies, and additional ones to be generated, may help define varying species of antibodies detected in aCL assays and identify the specific antibodies that may be pathogenic.     

The prevalence of antibodies to anionic phospholipids in patients with the primary antiphospholipid syndrome, systemic lupus erythematosus and their relatives and spouses

OBJECTIVES: Antiphospholipid antibodies (aPL) have been associated with syndromes involving thrombosis, fetal loss and thrombocytopenia. Genetic and environmental conditions are among the factors attributed to the cause of autoimmune diseases such as the antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE). The aim of this study was to determine whether these factors determine the prevalence of aPL. METHODS: Three groups of patients were tested for the presence of IgG, IgM and IgA anticardiolipin (aCL), antiphosphatidylinositol (aPI), antiphosphatidylglycerol (aPG) and antiphosphatidylserine (aPS) antibodies: (i) patients with primary APS (PAPS); (ii) patients with SLE and secondary APS; and (iii) patients with SLE without APS. First-degree relatives and spouses of patients with SLE/APS were also tested for circulating aPL. RESULTS: IgG aPL were particularly prevalent in patients with PAPS. IgG aPI and aCL were more prevalent in patients with PAPS than the IgM equivalents (P < 0.0001). Notably, none of the patients with PAPS had IgA aPL. A significantly higher number of relatives of patients with SLE/APS possessed IgG aPL than the normal controls. Except for aPG (P < 0.03), the prevalence of these antibodies in the relatives was not significantly different from patients with SLE/APS. The relatives also had significantly higher prevalence of IgG aPI, aPS and aCL antibodies than IgM aPL antibodies. In contrast, the prevalence of IgG aPL in the spouses was no different than in the healthy controls. CONCLUSIONS: Genetic factors, shared by patients and their relatives, seem to have some effect on the prevalence of aPL in the subjects studied, while environmental factors shared by spouses appear to have no influence.     

Relative value of different antiphospholipid antibodies detected in a department of internal medicine: retrospective study of 124 patients

PURPOSE: The value of antiphospholipid antibodies (aPL) detected in the sera of the patients of an Internal Medicine department is not univocal and is still much debated. To test the contribution of such new markers, we reviewed the records of patients having antiphospholipid antibodies detected between 1996 and 1997. METHODS: One hundred and twenty four patients, having at least one of these two aPL: lupus anticoagulant (LA), anticardiolipin antibodies (aCL), or one of these two anti-proteins: anti-beta 2glycoprotéin I antibodies (anti-beta 2GPI) or anti-prothrombin antibodies (aPT), were studied. LA was detected by a PTT-LA technique and aCL, anti-beta 2GPI and aPT by ELISA-sandwich techniques. For each patient we recorded sex, age, personal and familial history of thrombosis, fetal losses and systemic disease, the reason of aPL detection, the final diagnosis, activated partial thromboplastin time (aPTT), platelets count and type of aPL. RESULTS: The population was composed of 77 women (62%) and 47 men (38%) with a mean age of 54 years [12-92 years]. A thrombocytopenia was strongly correlated to aCL presence (OR = 6.15 et p = 0.03). The reason of aPL detection was venous thrombosis, recurrent fetal losses, systemic disease, infectious disease or fortuitous discovery of a prolonged aPTT. The final diagnosis was a systemic disease in 57% of cases, an infectious disease in 14.5%, a thrombosis in 4.5% and a neoplasia in 3%. LA was detected in 54% of patients, aCL in 39.5%, anti-beta 2GPI in 23% and aPT in 31%. No relationship between the aPTT value and the type of aPL could be established. CONCLUSION: Our study shows that familial histories of venous thrombosis or systemic disease are useful to enhance antiphospholipid antibodies detection; that LA is mostly associated to systemic and infectious diseases; that aCL and anti-beta 2GPI are predominant in case of venous thrombosis and that thrombocytopenia has to enhance aCL detection and the discussion about a possible APS.     

Prevalence of antibodies against oxidised LDL in a cohort of 163 patients with positive anti-phospholipid antibodies and recent thrombosis

We evaluated the prevalence of anti-β₂-glycoprotein I (a-β₂-GPI), anti-phosphatidylserine/prothrombin (a-PP), anti-prothrombin (a-PT), and anti-oxidised low-density lipoprotein (a-oxLDL) antibodies in a cohort of patients with a recent thrombotic event. Among consecutive sera sent to an autoimmune laboratory for routine testing for anti-cardiolipin antibodies (aCL) 473 were found positive for IgG and/or IgM aCL. The referring physicians were requested for clinical information about thrombo-embolic events occurring within 6 months prior to testing. One hundred and sixty-three individuals of which 82 had suffered from cerebro-vascular infarction and 49 from deep venous thromboses or pulmonary emboli were included in the study. Ninety-four sera were positive for IgG aCL and 69 were negative. There was a strong correlation between the presence of IgG aCL and the three other phospholipid-related antibodies: a-β₂-GPI, a-PP, and a-PT. However, IgG a-oxLDL antibodies were almost equally distributed among sera positive and negative for IgG aCL. The presence of antibodies of all subgroups was most pronounced among patients with venous thrombo-embolic disease. In this cohort antibodies to β₂-GPI, PP, PT seem to coexist with aCL. However, a-oxLDL antibodies appear to define a subset of patients, who were older, had more arterial vascular disease, and with no apparent association to the presence of IgG aCL.     

Heterogeneity of antibodies to beta2-glycoprotein 1 from patients with systemic lupus erythematosus

We studied antibodies to beta2-glycoprotein 1 (anti-beta2GP1) from 72 patients with systemic lupus erythematosus (SLE) with or without antiphospholipid syndrome (APS) or with or without anticardiolipin antibodies (aCL). Fifteen patients had APS and positive antiphospholipid antibodies [clinical APS(+)/aPL(+)], 12 patients had APS, negative serum IgG and IgM aCL, antiphosphatidylethanolamine, anti-phosphatidylserine and no lupus anticoagulant [clinical APS(+)/ aPL(-)]. A third group included 16 patients without APS but high aCL levels [clinical APS(-)/ aPL(+)]. In a fourth group we studied 29 patients without clinical manifestations of APS or aCL [clinical APS(-)/aPL(-)]. One hundred anticardiolipin and VDRL-negative normal sera were studied as controls. IgG antibodies to cardiolipin proper in a bovine beta2GP-free system, to human beta2GP1 immobilized on cardiolipin or to human beta2GP1 alone were detected in all sera by ELISA using irradiated and nonirradiated plates from two manufacturers. Sera from APS(+)/aPL(+) patients showed IgG binding to CL, CL + beta2GP1 and beta2GP1 in irradiated and nonirradiated plates. APS(+)/ aPL(-) sera had more significant IgG binding to beta2GP1 than normal controls when studied in both irradiated or nonirradiated plates (P = 0.001). This binding was inhibited by solid-phase cardiolipin in a dose-dependent manner. Sera from the APS(-)/aPL(+) subgroup had comparable IgG activity in both the CL and CL + beta2GP1 assays, while no anti-beta2GP1 activity was detected in these sera. Sera from the clinical APS(-)/aPL(-) patients were negative in the three ELISA systems. Antibodies to human beta2GP1 from SLE patients recognize various epitopes. Those from APS(+)/ aPL(+) patients appear to react with an epitope boosted by cardiolipin in addition to another one present in the native protein. In contrast, anti-beta2GP1 from patients with APS(+)/aPL(-) are blocked by cardiolipin, suggesting that their epitope is the phospholipid-binding site.     

IgA anti-beta-2 glycoprotein I antibodies in chronic hepatitis C

Background and study aimsAntiphospholipid antibodies (aPL) have been reported not only in various autoimmune conditions but also in other infections, such as chronic hepatitis C (CHC) infection. The aim of this study is to evaluate the frequency of aPL in patients with CHC.Patients and methodsNinety-six CHC patients and 90 healthy blood donors (HBD) were studied. Fifty-three of the patients were under treatment, and 43 had not yet received any treatment. IgG, IgA, and IgM antibodies against cardiolipin (aCL) and beta-2 glycoprotein I (aβ2GPI) were detected by ELISA.ResultsWe found that the frequency of aPL (aCL and/or aβ2GPI) was significantly higher in CHC patients than in controls (51% vs 11.1%, p <10^?6). The frequencies of aCL and aβ2GPI were significantly higher in patients than in HBD (27.1% vs 5.5%, p < 10^?3, and 44.8% vs 11.1%, p < 10^?6, respectively). The isotype distribution of aCL and aβ2GPI demonstrated that aCL-IgG and aβ2GPI-IgA were more frequent in patients than in healthy subjects (21.9% vs 2.2%, p < 10^?3, and 38.5% vs 7.8%, p < 10^?6, respectively). In CHC patients, the frequency of aβ2GPI was significantly higher than that of aCL (44.8% vs 27.1%, p = 0.01). aβ2GPI-IgA was significantly more frequent than aβ2GPI-IgG (38.5% vs 7.3%, p <10^?6), aβ2GPI-IgM (38.5% vs 9.4%, p <10^?3), and aCL-IgG (38.5% vs 21.9%, p = 0.01). No difference in aPL frequency was observed between the treated and untreated patients.ConclusionOn the basis of the findings of this study, aPL, particularly aβ2GPI-IgA and aCL-IgG, are frequent in CHC patients.     

Clinical audit of antiphospholipid antibody testing in tertiary practice: towards improved relevance in thrombophilia investigations

Background: The antiphospholipid syndrome (APS) is an autoimmune condition characterised by vascular thromboses and/or pregnancy morbidity. Diagnosis of APS typically requires laboratory evidence of antiphospholipid antibodies (aPL). Depending on their clinical presentation, affected individuals might be seen by a variety of clinical specialities. Aim: To evaluate clinical ordering patterns for aPL/APS at a tertiary level public facility. Methods: We performed an audit of internal clinical requests for aPL tests at our institution for a 6‐month period. Results: We identified a wide variety of clinical ordering background for aPL, of predominantly obstetric (72/268; 26.9%) or thrombophilic (78/268; 29.1%) patients. Only 11/268 samples (4.1%) were positive for lupus anticoagulant (LA) and 14/268 (5.2%) were positive for anticardiolipin antibody (aCL). The percentage of aCL positivity in the LA‐positive group was 46% (5/11). None of the 72 obstetric patients tested was identified to have aPL. Of the 11 LA‐positive patients, the reasons identified for testing comprised: prolonged Activated Partial Thromboplastin Time (assay) (n= 3), thrombosis (n= 3), APS (n= 2), systemic lupus erythematosus (n= 2), vasculitis (n= 1). Conclusion: We determined a wide variety of clinical ordering background for aPL at a tertiary level institution, with an overall low rate (<10%) of aPL positivity among a hospital population of predominantly obstetric or thrombophilic patients. That no positive obstetric aPL cases were identified suggests local clinical ordering guidelines may need review, as also potentially practised at other institutions. We also observed a moderate rate (46%) of coincidence of aCL and LA, in agreement with guidelines indicating that multiple tests are required to identify APS.