Which are the best biological markers of the antiphospholipid syndrome?

The diagnosis of antiphospholipid syndrome (APS) requires the presence of both clinical and biological features. Due to the heterogeneity of anti-phospholipid antibodies (aPL) the laboratory approach for their detection includes clotting-based tests for lupus anticoagulant (LA) as well as solid-phase assays for anticardiolipin antibodies (aCL). In addition, as it has been shown that autoimmune aPL recognize epitopes on phospholipid (PL)-binding plasma proteins, assays detecting antibodies to beta 2-glycoprotein I (beta 2-GPI) or prothrombin have been developed. The association between venous or arterial thrombosis and recurrent fetal loss with the presence of conventional aPL (LA and/or aCL) has been confirmed by many studies. The LA and IgG aCL at moderate/high titre seem to exhibit the strongest association with clinical manifestations of the APS. Several reports indicate that LA is less sensitive but more specific than aCL for the APS. Assays against PLs other than CL as well as the use of mixtures of PLs have been proposed to improve the detection of APS-related aPL. Concerning antibodies to PL-binding proteins (detected in the absence of PLs), there is evidence that anti-beta 2-GPI are closely associated with thrombosis and other clinical features of the APS. Moreover, these antibodies may be more specific in the recognition of the APS and in some cases may be present in the absence of aPL detected by standard tests. Many issues are still under debate and are discussed in this review, such as the problems of standardization of anti-beta 2-GPI assays, detection of the IgA isotype of aCL and anti-beta 2-GPI, the coagulation profiles of LA in the recognition of the thrombotic risk and the association of particular markers with subsets of patients with APS.     

Binding properties of antibodies to prothrombin and beta2-glycoprotein I (beta2-GPI) assayed by ELISA and dot blot

Most anti-phospholipid antibodies (aPL) associated with the anti-phospholipid syndrome are autoantibodies with specificity towards beta2-GPI (anti-beta2-GPI) or prothrombin (anti-II). They are mainly screened by ELISA using polyoxygenated plates. However, some authors have claimed that immunoblotting can also be used. Exposure of cryptic epitopes or increase of antigen density on its binding to either phospholipids or suitable plastic surfaces are the two hypotheses proposed for the interaction of beta2-GPI or prothrombin with their antibodies. Forty-five patients with aPL were studied: 25 with lupus anti-coagulant (LA) and anti-cardiolipin antibodies (aCL), 10 with LA alone and 10 with aCL but negative LA. All patients with LA and aCL were positive for anti-beta2-GPI by ELISA and dot blot, while 15/25 had anti-IIELISA and 14 of them also had anti-II by dot blot assay. No patient with LA alone tested positive for anti-beta2-GPI by ELISA or dot blot, whereas 6/10 had anti-IIELISA (five of them were also positive by dot blot). Four out of 10 aCL-positive patients had anti-beta2-GPI by ELISA and dot blot, while none of this group had anti-II by ELISA or dot blot. Antibody binding to beta2-GPI or prothrombin in both ELISA and dot blot was significantly reduced by phospholipid liposomes mixed together with beta2-GPI or prothrombin, whereas liposomal eluants retained it in both assays. Parallel fluid-phase inhibition experiments using increasing concentrations (up to 200 microg/ml) of beta2-GPI or prothrombin demonstrated that antibody binding reduction was more evident on dot blot than on ELISA. It was almost completely abolished on dot blot, while on ELISA a moderate inhibition was achieved even at the highest protein concentration. However, antibody binding on ELISA was virtually abolished when diluted sera were incubated with high protein concentrations applied to nitrocellulose membranes. We could infer that ELISA and dot blot detect antibodies with some differences in avidity but directed against native epitopes on beta2-GPI and prothrombin.     

Anti-phospholipid antibodies in HIV infection and SLE with or without anti-phospholipid syndrome: comparisons of phospholipid specificity, avidity and reactivity with beta2-GPI.

Increased prevalence of anti-phospholipid antibodies (aPL) and increased levels of lipid peroxidation have been described in patients with HIV infection. To assess the binding specificity and avidity of aPL antibodies in HIV infection, sera from 44 HIV-1 infected patients were evaluated for antibodies to cardiolipin (aCL), phosphatidyl serine (aPS), phosphatidyl inositol (aPI) and phosphatidyl choline (aPC) using enzyme linked immunosorbent assay (ELISA) methods. Sera from 30 patients with systemic lupus erythematosus (SLE), but without features of anti-phospholipid syndrome (APS) (SLE/non APS), six with SLE and secondary APS, (SLE/APS) and 11 with primary APS (PAPS) were also evaluated as controls. The resistance of the aPL antibody binding to dissociating agents was evaluated by treating the ELISA wells, after serum incubation with 2 M urea or 0.6 M NaCl for 10 min. An anti-beta2-glycoprotein-I (beta2-GPI) ELISA was used to assess serum reactivity against beta2-GPI, a plasma protein considered as the true antigen of aCL antibodies occurring in APS and SLE patients. The prevalence of aCL, aPS, aPI and aPC antibodies in HIV-1 infection was 36%, 56%, 34% and 43% respectively, which was comparable to that found in SLE/APS and PAPS patients and significantly higher than that observed in SLE/non-APS patients. Anti-beta2-GPI antibodies occurred in 5% of HIV-1 infected vs. 17% in SLE/non-APS (P=0.11), 50% in SLE/APS (P=0.009) and 70% in PAPS patients (P=0.0014). A significant decrease of aPL binding after urea and NaCl treatment was observed in the sera of HIV-1-infected, compared to that of APS patients, indicating that aPL antibodies from HIV-1 infected individuals have low resistance to dissociating agents. In conclusion, aPL antibodies (1) occur in HIV-1 infection; (2) tend to recognize various phospholipids but not beta2-GPI; and (3) are of low resistance to dissociating agents-a finding probably reflecting low antibody avidity. Finally, these, like the autoimmune-type aCL antibodies, tend to recognize the oxidized CL-a finding probably indicating autoantibody generation as a result of neoepitope formation by oxidized PLs.     

Detection of antiphospholipid antibodies by automated chemiluminescence assay

The laboratory diagnosis of antiphospholipid antibody syndrome (APS) requires the demonstration of antiphospholipid antibodies (aPL) by lupus anticoagulant (LAC) measured through coagulation assays, anticardiolipin IgG or IgM antibodies (aCL) and/or anti-β2-glycoprotein I IgG or IgM antibodies (anti-β2-GPI), usually detected by ELISA. In this study we tested aCL by a new automated system using the chemiluminescence principle. Our results showed that, while almost all the sera from APS patients, positive for IgG aCL and anti-β2-GPI by ELISA, were also positive for IgG aCl by chemiluminescence, only 30.13% of patients without clinical manifestations of APS, but positive for aCL and persistently negative for anti-β2-GPI (by ELISA) and LA, confirmed the positive test by chemiluminescence. This difference was highly significant (p<0.0001). Interestingly, this test also prompted to identify 20% of patients positive for LA, but persistently negative for both aCL and anti-β2-GPI IgG (ELISA). Thus, the new technology of automated chemiluminescence assay for measuring aPL may represent an useful tool to identify "true" APS patients.     

Inhibitor(s) of natural anti-cardiolipin autoantibodies.

IgG fractions were purified on Sepharose anti-human IgG column from eight sera of healthy donors, having no anti-cardiolipin (aCL) activity as measured by anti-cardiolipin ELISA assay (aCL-ELISA). All the IgG fractions, after elution with 4.9 M MgCl2, reacted with CL. The antigen-binding characteristics of the IgG fractions purified from normal human serum (NHS) were similar to those of IgG fractions purified from sera of four patients with the anti-phospholipid syndrome (APLS). Competition assay confirmed the specificity of the binding of the purified IgG fractions to CL. The same results have been achieved with IgG fractions purified on Sepharose Protein-A column. The binding to CL was completely inhibited by either whole NHS and sera from various animal species, or by beta 2-glycoprotein I (beta 2-GPI). Our results support the notion of the existence of both natural anti-CL antibodies and serum inhibitor(s) in sera of healthy individuals. It is conceivable that in part the pathogenesis of APLS entails defects in the natural inhibitors of aCL antibodies.     

Detection of 'antiphospholipid' antibodies: a single chromogenic assay of thrombin generation sensitively detects lupus anticoagulants, anticardiolipin antibodies, plus antibodies binding beta(2)-glycoprotein I and prothrombin

The diagnosis of the antiphospholipid syndrome (APS) requires both a typical clinical event plus a persistently positive test in an assay for either anticardiolipin (aCL) antibodies or a lupus anticoagulant (LA). Enzyme linked immunosorbent assays (ELISA) specific for autoantibodies against beta(2)-glycoprotein I (beta(2)GPI) or prothrombin are also used, but none of the tests are adequately sensitive or specific. A chromogenic assay was developed that measures the effect of test antibody or plasma samples on in vitro thrombin formation. It is able to detect both LA and beta(2)GPI-dependent aCL antibodies and may have greater specificity for APS than currently available tests. Using this method various monoclonal antibodies (MoAbs) were examined, from mice immunized with beta(2)GPI, mice with a spontaneous animal model of APS, and from three humans with APS. Plasma and affinity purified antibodies from patients with APS and control groups were also examined. Thrombin inhibition was more sensitive to perturbation by MoAbs than a combination of tests for LA (P < 0.05) and at lower antibody concentrations (12.5 microg/ml versus 100 microg/ml). There was a significant correlation between inhibition of thrombin generation and the level of MoAb reactivity to beta(2)GPI (r = 0.90; P < 0.001) but not to CL (r = 0.06; P = 0.76). Plasma and affinity purified antibodies from patients with APS also inhibited thrombin generation, and significantly more so than patients with aPL from causes other than APS. APS patient samples showed thrombin inhibition in the presence of anti-beta(2)GPI or antiprothrombin antibodies. All MoAbs binding beta(2)GPI showed inhibition of thrombin generation, while MoAbs binding domain I of beta(2)GPI had more LA effect.     

Anti-lysobisphosphatidic acid antibodies in patients with antiphospholipid syndrome and systemic lupus erythematosus

Lyso(bis)phosphatidic acid (LBPA) is a novel antigenic target in anti-phospholipid syndrome (APS) and antibodies directed against LBPA (aLBPA) have been detected in sera from APS patients. In this study we first evaluated aLBPA in comparison with the most widely used methods (i.e. anticardiolipin [(aCL)-enzyme-linked immunosorbent assay (ELISA)] and antibeta-2-glycoprotein-I antibodies (abeta(2)-GPI-ELISA) utilized to detect antiphospholipid antibodies in patients with primary or secondary APS, systemic lupus erythematosus, chronic HCV infection and healthy subjects. We then assessed the relationship between aLBPA, lupus anticoagulant (LAC) and the main clinical manifestations of APS. Finally, we evaluated the presence of 'pure' (i.e. beta(2)-GPI-independent) aLBPA in patients with APS and controls. The results indicate that aLBPA as well as abeta(2)-GPI display higher specificity but lower sensitivity for APS compared to aCL. Moreover, serum aLBPA correlate closely with aCL and abeta(2)-GPI in APS patients and are strictly associated with LAC positivity. We demonstrate that beta(2)-GPI binds to LBPA with affinity similar to CL, and antibodies able to react with phosholipid-protein complex exist; however, 'pure' aLBPA can also be detected in sera of APS patients. Altogether these data confirm that LBPA may be an antigenic target in APS and that aLBPA are serological markers of APS with similar sensitivity and specificity compared to abeta(2)-GPI. However, the clinical utility of aLBPA detection alone or in combination with aCL and/or abeta(2)-GPI remains to be elucidated in larger and longitudinal studies.     

Patients with atherosclerotic syndrome, negative in anti-cardiolipin assays, make IgA autoantibodies that preferentially target domain 4 of beta2-GPI

Autoantibodies targeting beta2-glycoprotein l (beta2-GPI), a component of the atherosclerotic plaque, are commonly found in patients with acute ischemic syndromes. Serum samples from APS (antiphospholipid syndrome) patients and from cardiovascular patients exhibiting acute atherosclerotic syndromes were analyzed for IgG and IgA antibodies in both anti-beta2-GPI and anticardiolipin (aCL) ELISA assays. All of the APS samples used here were positive in both assays. Serum samples from 382 atherosclerosis patients were also analyzed for IgG and IgA antibodies in the same assays. In sharp contrast to the APS samples, we found that only 1% of the samples from atherosclerosis patients were positive for IgA aCL, and 1.6% positive for IgG aCL, whereas 35.6% were positive for IgA anti-beta2-GPI and only 1.6% for IgG anti-beta2-GPI. The antigenic specificity of 29 serum samples from atherosclerosis patients was evaluated. Six different recombinant domain-deleted mutants (DM) of human beta2-GPI and full-length human beta2-GPI (wild-type) were used in competitive inhibition assays to inhibit the autoantibodies from binding in the anti-beta2-GPI ELISA assays. Domain-deleted mutants D--345 and D--45 inhibited the binding in the IgA anti-beta2-GPI assay, suggesting that these autoantibodies recognize domain 4 of the beta2-GPI molecule. These results clearly show that IgA anti-beta2-GPI autoantibodies from atherosclerotic patients are distinct from IgA autoantibodies found in APS samples.     

Isotype distribution and clinical relevance of anti-beta2-glycoprotein I (beta2-GPI) antibodies: importance of IgA isotype.

The aim of this study was to evaluate the prevalence of IgG, IgA and IgM anti-beta2-GPI antibodies in anti-phospholipid syndrome (APS), and to establish the clinical significance of IgA type antibodies compared with the other isotypes. Anti-beta2-GPI antibodies were measured in the sera of 70 patients by solid-phase enzyme immunoassay in gamma-irradiated polystyrene plates coated with human purified beta2-GPI. Thirty-three out of the 70 patients were classified as having APS: three of them had primary, and 30 had secondary APS related to systemic lupus erythematosus (SLE). The remaining 37 patients had SLE without APS. Anti-beta2-GPI antibodies of IgG, IgA and IgM isotypes were present in 84.8%, 59.3% and 51.5% of patients with APS. Both the frequency and the level of each isotype were significantly higher in patients with APS. This association was very strong for IgA (P = 0.0004 for the antibody frequency and P < 0.0001 for the antibody level), as well as for IgG type antibodies (P < 0.0001 and P < 0.0001), whereas it was weaker for IgM (P = 0.01 and P = 0.04). A strong relationship was demonstrated between increased IgA anti-beta2-GPI antibody levels and a history of venous thrombosis, thrombocytopenia, heart valve disease, livedo reticularis and epilepsy. IgG anti-beta2-GPI antibodies were associated with the presence of lupus anticoagulant (LA) in addition to the main features of APS. However, antibodies of IgM isotype were related only to thrombocytopenia and heart valve disease. We recommend the evaluation of anti-beta2-GPI antibodies of IgA isotype in addition to IgG in patients with clinical suspicion of APS.     

Antiphospholipid antibodies bind ATP: a putative mechanism for the pathogenesis of neuronal dysfunction

Antiphospholipid antibodies (aPL) generated in experimental animals cross-react with ATP. We therefore examined the possibility that aPL IgG from human subjects bind to ATP by affinity column and an enzyme linked immunosorbent assay (ELISA). Sera with high levels of aPL IgG were collected from 12 patients with the antiphospholipid syndrome (APS). IgG fractions from 10 of 12 APS patients contained aPL that could be affinity-bound to an ATP column and completely eluted with NaCl 0.5 M. A significant (> 50%) inhibition of aPL IgG binding by ATP 5 mM was found in the majority. Similar inhibition was obtained with ADP but not with AMP or cAMP. All the affinity purified anti-ATP antibodies also bound beta2-glycoprotein-I (beta2-GPI, also known as apolipoprotein H) suggesting that, similar to most pathogenic aPL, their binding depends on this serum cofactor. We further investigated this possibility and found that the binding of beta2-GPI to the ATP column was similar to that of aPL IgG in that most was reversed by NaCl 0.5 M. Furthermore, addition of beta2-GPI to aPL IgG significantly increased the amount of aPL binding to an ATP column. We conclude that aPL IgG bind ATP, probably through beta32-GPI. This binding could interfere with the normal extracellular function of ATP and similar neurotransmitters.     

Assay principles of antiphospholipid antibodies and heterogeneity of the antibodies

The antiphospholipid syndrome(APS) is characterized by predominant clinical features of venous and arterial thrombosis and recurrent pregnancy loss accompanied by antiphospholipid antibodies(aPL) such as anticardiolipin antibodies(aCL) and lupus anticoagulant(LA). In 1990, three individual research groups, including us, first reported that a 50 kD plasma cofactor is required for the binding of aCL to cardiolipin(CL) and now, beta 2-glycoprotein I(beta 2-GPI), which binds to anionic phospholipids(PLs), is widely believed to be the major antigen for aCL. It was also reported that epitopes for such aCL are cryptic and that they appear only when beta 2-GPI interacts with lipid membranes containing anionic PLs, such as CL and phosphatidylserine, or with a polyoxygenated polystyrene surface. In contrast, prothrombin was recently identified as the "true" antigen for LA. In this review paper, we would like to describe on specificity of aPL and also on a possible mechanism on autoantibody-dependent development of atherosclerosis.     

Clinical relevance of multiple antibody specificity testing in anti-phospholipid syndrome and recurrent pregnancy loss

We wanted to evaluate whether testing for anti-phosholipid antibodies other than anti-cardiolipin (aCL) and anti-beta-2 glycoprotein I (abeta2GPI) immunoglobulin (Ig)G and IgM identifies patients with recurrent pregnancy loss (RPL) who may be positive for anti-phospholipid syndrome (APS). In a cross-sectional study comprising 62 patients with APS, 66 women with RPL, 50 healthy blood donors and 24 women with a history of successful pregnancies, we tested IgM and IgG antibodies to phosphatidic acid, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl inositol and phosphatidyl serine with and without beta-2 glycoprotein I (beta2GPI) from a single manufacturer as well as aCL and abeta2GPI antibodies. Diagnostic accuracies of individual and combined anti-phospholipid (aPL) assays were assessed by computing sensitivities, specificities, positive predictive values and negative predictive values together with their 95% confidence intervals. There was a general trend for increased sensitivities in the presence of beta2GPI co-factor with significant effect for certain specificities. The overall combined sensitivity of the non-recommended aPL assays was not significantly higher than that of the aCL and aB2GPI tests. Multiple aPL specificities in RPL group is not significantly different from controls and therefore of no clinical significance.     

Anti-beta(2)-glycoprotein I autoantibody expression as a potential biomarker for strokes in patients with anti-phospholipid syndrome

Anti-phospholipid syndrome (APS) is an autoimmune disease. Cerebral ischemia associated with APS occurs at a younger age than typical atherothrombotic cerebrovascular disease, is often recurrent, and is associated with high positive IgG anti-phospholipid (GPL) unit levels. This study sought to determine the frequency rates of anti-cardiolipin (aCL) dependent on the presence of beta(2)-GPI, anti-beta(2)-glycoprotein I (abeta(2)-GPI), and anti-phosphatidyl serine (aPS) IgG autoantibodies among stroke patients, and thus demonstrate the importance of testing for abeta(2)-GPI autoantibodies. For these study, stroke patients and control subjects recruited from Mosul, Erbil, and Dohuk provinces in Northeren Iraq between March 2004 and March 2005 were evaluated. All cases were under 50 years-of-age and had no recognizable risk factors. Using ELISA to evaluate the presence of IgG isotype of aCL, abeta(2)-GPI, and aPS autoantibodies in their blood, the results indicated that the frequency of abeta(2)-GPI was 14/50 (28%), aCL was 11/50 (22%), and aPS was 9/50 (18%) among stroke patients. In contrast, aCL was detected in 2/30 (6.7%) of control subjects; each of the other anti-phospholipid antibodies (APLA) was never observed. Of all the abeta(2)-GPI(+) cases, the incidence of stroke patients having the combined profile of abeta(2)-GPI + aCL was 11/14 (78.6%) and of abeta(2)-GPI + aPS was 9/14 (64.3%). Only 2/14 (14.3%) of these abeta(2)-GPI(+) patients also expressed aCL in the absence of aPS. The frequency of patients expressing all three markers was only 9/14 (64.3 %). In none of the APS/stroke patients were aCL or aPS expressed in the absence of the abeta(2)-GPI. Conversely, abeta(2)-GPI as a sole marker was seen in 3/14 (21.4%) of these patients (i.e., in absence of either other marker). It can be concluded from these studies that the among the three major forms of APLA examined, the presence of abeta(2)-GPI IgG autoantibodies appeared to correlate best with stroke in patients who were concurrently suffering APS.     

Molecular definition of human beta 2-glycoprotein I (beta 2-GPI) by cDNA cloning and inter-species differences of beta 2-GPI in alternation of anticardiolipin binding.

Human beta 2-glycoprotein I (beta 2-GPI) is involved in cardiolipin (CL) binding of anticardiolipin antibodies (aCL) in systemic lupus erythematosus (SLE). We examined the inter-species differences of beta 2-GPI in alternation of CL binding of aCL. beta 2-GPI preparations were obtained from human, bovine, and rat sera by sequential CL--polyacrylamide affinity, DEAE--cellulose, and anti-human IgG-conjugated Sepharose CL-4B column chromatography, and they had apparent molecular weights of 50, 53, and 55 kDa respectively. Human beta 2-GPI not only enhanced CL binding by aCL in SLE but also depressed it by those in syphilis. Either bovine and rat beta 2-GPI exerted no or quite small inhibition of the binding of syphilitic aCL compared with human beta 2-GPI whereas all three species of beta 2-GPI generated binding of aCL in SLE to a similar degree. Further, a complete cDNA clone, p beta 2-GPI, was isolated from a human hepatoma cell line, HepG2, and its nucleotide sequence was analyzed. The sequences of bovine and rat counterpart molecules (beta 2-GPI) are highly homologous to that of the deduced sequence, and their corresponding regions are 84.0 and 82.5% identical to the complete domain and to the amino acid sequence 53-326 of human beta 2-GPI respectively. One of major differences appears at position 154 in human beta 2-GPI, and might be associated with the inhibitory effect on the binding of syphilitic aCL. The sequencing analysis of these beta 2-GPI proteins might provide leads to functional sites of domains which would be associated with such serological phenomena.     

Patients with atherosclerotic syndrome,negative in anti-cardiolipin assays,make IgA autoantibodies that preferentially target domain 4 of β2-GPI

Autoantibodies targeting β₂-glycoprotein l (β₂-GPI), a component of the atherosclerotic plaque, are commonly found in patients with acute ischemic syndromes. Serum samples from APS (antiphospholipid syndrome) patients and from cardiovascular patients exhibiting acute atherosclerotic syndromes were analyzed for IgG and IgA antibodies in both anti-β₂-GPI and anticardiolipin (aCL) ELISA assays. All of the APS samples used here were positive in both assays. Serum samples from 382 atherosclerosis patients were also analyzed for IgG and IgA antibodies in the same assays. In sharp contrast to the APS samples, we found that only 1% of the samples from atherosclerosis patients were positive for IgA aCL, and 1.6% positive for IgG aCL, whereas 35.6% were positive for IgA anti-β₂-GPI and only 1.6% for IgG anti-β₂-GPI. The antigenic specificity of 29 serum samples from atherosclerosis patients was evaluated. Six different recombinant domain-deleted mutants (DM) of human β₂-GPI and full-length human β₂-GPI (wild-type) were used in competitive inhibition assays to inhibit the autoantibodies from binding in the anti-β₂-GPI ELISA assays. Domain-deleted mutants D—345 and D---45 inhibited the binding in the IgA anti-β₂-GPI assay, suggesting that these autoantibodies recognize domain 4 of the β₂-GPI molecule. These results clearly show that IgA anti-β₂-GPI autoantibodies from atherosclerotic patients are distinct from IgA autoantibodies found in APS samples.     

Prevalence and clinical significance of anti-phospholipid antibodies in multiple sclerosis: a study of 89 patients

The prevalence of serum anti-phospholipid antibodies (aPL) was evaluated in multiple sclerosis (MS) patients to search for a possible association with a distinct form of the disease. Anti-cardiolipin antibodies (Ab) (aCL) and anti-beta 2 glycoprotein I Ab (abeta2GPI) were measured together with antinuclear Ab (ANA), anti-double-stranded DNA Ab (anti-ds DNA) and anti-myelin Ab in 89 patients. Twenty-nine (32.6%) patients had serum aPL, 19xaCL (15 of the IgG and four of the IgM isotype), 14 abeta2GPI (two IgG and 12 IgM) (four of these patients had both Ab). Prevalence of aCL correlated with that of ANA, which were positive in 52 patients (P=0. 005) and with anti-myelin Ab detected in two patients (P=0.046) but not with that of anti-ds DNA (mostly of the IgM class) detected in 28% of case by ELISA. No correlation could be found between aPL and age, sex, duration of the disease from diagnosis, category of MS, clinical course, clinical symptoms, serum IgM levels nor atypical lesions by magnetic resonance imaging. Hence, aCL and abeta2GPI are neither rare in MS nor associated with a specific clinical form of the disease and they cannot be a diagnosis exclusion criteria.     

Conformationally altered beta 2-glycoprotein I is the antigen for anti-cardiolipin autoantibodies

Anti-cardiolipin autoantibodies (aCL) induce thrombosis and recurrent fetal death. These antibodies require a 'cofactor', identified as beta 2-glycoprotein I (beta 2-GPI), to bind phospholipids. We show here that aCL can bind beta 2-GPI in the absence of phospholipid. Binding of aCL to beta 2-GPI is dependent upon the beta 2-GPI being immobilized on an appropriate surface including cardiolipin, irradiated polystyrene and nitrocellulose membrane. This effect cannot be explained by increased antigen density of beta 2-GPI immobilized on these surfaces. Rather, conformational changes that occur following the interaction of beta 2-GPI with phospholipid render this protein antigenic to aCL. Liquid-phase beta 2-GPI was not antigenic for aCL. Thus, aCL cannot bind circulating beta 2-GPI. These findings may explain why patients with aCL can remain healthy for many years but then undergo episodes of thrombosis or fetal loss without changes in their circulating aCL profile, as the triggering event for these pathologies can be predicted to be one that renders beta 2-GPI antigenic for aCL.     

Immunoglobulin A anti‐phospholipid antibodies in Swedish cases of systemic lupus erythematosus: associations with disease phenotypes,vascular events and damage accrual

Immunoglobulin (Ig) G‐ and IgM‐class anti‐cardiolipin antibodies (aCL) and lupus anti‐coagulant (LA) are included in the 1997 update of the American College of Rheumatology (ACR‐97) systemic lupus erythematosus (SLE) criteria. Despite limited evidence, IgA‐aCL and IgA anti‐β₂‐glycoprotein‐I (anti‐β₂GPI) were included in the 2012 Systemic Lupus International Collaborating Clinics criteria. The present study aimed to evaluate IgG‐/IgA‐/IgM‐aCL and anti‐β₂GPI occurrence in relation to disease phenotype, smoking habits, pharmacotherapy, anti‐phospholipid syndrome (APS) and organ damage among 526 Swedish SLE patients meeting ACR‐97. Patients with rheumatoid arthritis (n = 100), primary Sjögren’s syndrome (n = 50) and blood donors (n = 507) served as controls. Anti‐phospholipid antibodies (aPL) were analysed by fluoroenzyme‐immunoassays detecting aCL/anti‐β₂GPI. Seventy‐six (14%) SLE cases fulfilled the Sydney APS‐criteria, and ≥ 1 aCL/anti‐β₂GPI isotype (IgG/IgA/IgM) occurred in 138 SLE patients (26%). Forty‐five (9%) of the SLE cases had IgA‐aCL, 20 of whom (4%) lacked IgG‐/IgM‐aCL. Seventy‐four (14%) tested positive for IgA anti‐β₂GPI, 34 (6%) being seronegative regarding IgG/IgM anti‐β₂GPI. Six (1%) had APS manifestations but were seropositive regarding IgA‐aCL and/or IgA anti‐β₂GPI in the absence of IgG/IgM‐aPL and LA. Positive LA and IgG‐aPL tests were associated with most APS‐related events and organ damage. Exclusive IgA anti‐β₂GPI occurrence associated inversely with Caucasian ethnicity [odds ratio (OR) = 0·21, 95% confidence interval (CI) = 0·06–0·72) and photosensitivity (OR = 0·19, 95% CI = 0·05–0·72). Nephritis, smoking, LA‐positivity and statin/corticosteroid‐medication associated strongly with organ damage, whereas hydroxychloroquine‐medication was protective. In conclusion, IgA‐aPL is not rare in SLE (16%) and IgA‐aPL analysis may have additional value among SLE cases with suspected APS testing negative for other isotypes of aPL and LA.     

Does seronegative antiphospholipid syndrome really exist?

The diagnosis of seronegative (SN-) antiphospholipid syndrome (APS) has been suggested for patients with clinical manifestations indicative of APS but with persistently negative results in the commonly used assays to detect anti-cardiolipin (aCL) antibodies, anti-β2 Glycoprotein I antibodies (aβ2GPI), and lupus anticoagulant (LA). To date the best management of these patients is still unclear. New emerging anti-phospholipid (aPL) assays could improve our ability in diagnosing APS. However, the availability of aPL assays in routine laboratory practice is limited. In fact, even aβ2GPI is routinely tested in only a small number of laboratories, and other aPL, such as anti-prothrombin or anti-annexin antibodies, in only a few research laboratories. On the other hand transient or false negative aPL assay and other genetic or acquired pro-thrombotic conditions can further complicate this issue. This paper is focused on the arguments for and against the diagnosis of SN-APS and is aimed to help the clinician when approaching a patient with clinical manifestations consistent with APS diagnosis but with negative aPL using the commonly available tests.