Antibodies and diagnostic tests in antiphosholipid syndrome

Lupus anticoagulant

LA is a functional test that detects immunoglobulins inducing an in vitro elongation of PL-dependent clotting time. LA phenomenon is mediated by high-titer antibodies, usually IgG, targeting β2GPI (most commonly, DI) or prothrombin (PT).

According to updated guidelines, LA should be assessed by a three-step strategy envisaging— (1) a screening test (to demonstrate the prolongation of clotting time); (2) a mixing test (to exclude a deficiency in coagulation factor); and (3) a confirming test (to identify the inhibitor of coagulation as PL-dependent). Several standardization issues and the interference with concomitant anticoagulation may affect LA reproducibility and interpretation, with obvious clinical implications in APS. Very recently, an Italian study showed a 45% discrepancy rate for LA results, with an even higher inconsistency figure among patients on vitamin K antagonists. Nevertheless, LA has consistently been identified as the strongest thrombotic risk-factor among the three aPL assays. In a systematic literature review, LA emerged as an independent predictor of thrombosis; noteworthy, this association held significance irrespectively of thrombotic site and of an associated diagnosis of systemic lupus erythematosus (SLE). Similarly, LA was the strongest predictor of recurrent pregnancy loss (RPL) and placenta-mediated pregnancy complications.

A single study has to date addressed the clinical significance of isolated, β2GPI-independent LA: patients with a mere LA positivity presented a thrombotic risk similar to the general population. Therefore, an isolated LA conveys a very different clinical risk compared to LA associated with aCL/anti-β2GPI antibody positivity. Another recent study from the same group showed that LA positive patients negative for anti-β2GPI antibodies display mainly IgM anti-phosphatidylserine (PS)/PT antibodies associated with LA and a lower thrombotic risk in comparison with LA patients positive for anti-β2GPI antibodies.

Anticardiolipin antibodies

aCL are usually detected by solid-phase assays employing cardiolipin (CL)-coated matrix in the presence of bovine serum, thus detecting antibodies binding to CL alone (β2GPI-independent aCL) and those that bind to CL bound to bovine β2GPI (β2GPI-dependent aCL). Some assays employ human β2GPI, in order to avoid missing aCL that recognize CL when bound to human—but not bovine- β2GPI. IgG and IgM aCL are expressed in GPL and MPL units, established using calibrators (affinity-purified polyclonal IgG and IgM aCL). Medium/high titers, required to diagnose APS, are defined as above 40 GPL/MPL or the 99th percentile.

In a systematic literature review, aCL IgG were associated with thrombosis, either venous or arterial; at further analysis, aCL correlated with cerebral stroke and myocardial infarction, but not deep vein thrombosis. With regards to pregnancy morbidity, aCL IgG have been related to early and late fetal losses, while aCL IgM were associated with late losses only. When considering placenta-mediated adverse outcomes, an association of aCL was reported with preeclampsia, the association being stronger for severe preeclampsia. Unfortunately, data interpretation is affected by the wide heterogeneity in aCL testing, which partially prevents result comparability.

Anti-β2 glycoprotein I antibodies

Anti-β2GPI antibodies are usually detected by solid-phase assays using matrix coated with purified β2GPI; current classification criteria consider an antibody titer greater than the 99th percentile. Even though inter-laboratory agreement in anti-β2GPI assay appeared to be higher than aCL ELISA, the lack of accepted calibrators raises harmonization issues. International efforts have been recently focused to identify a suitable reference material—monoclonal and affinity-purified polyclonal anti-β2GPI IgG have been evaluated. The latter has been identified to produce stable and reproducible results, allowing to start establishing international units.

In a 2003 meta-analysis, anti-β2GPI antibodies were associated with thrombosis in 57% of evaluated studies, the rate increased to 61% when considering IgG isotype and decreased to 47% for IgM. With regards to pregnancy morbidity, anti-β2GPI antibody positivity was not related to RPL but with placenta-mediated complications. Unfortunately, anti-β2GPI antibodies have to date been assessed in few studies; such paucity of data prevents the drawing of definite conclusions about their clinical significance, despite their widely accepted pivotal pathogenic role in experimental models.

Every aPL test conveys a different clinical significance, implying that each aPL profile carries a characteristic hazard for related events. On one hand of the spectrum, there are patients with double/triple aPL positivity, likely driven by reactivity against β2GPI. On the other hand, there are subjects with single aPL positivity, whose clinical significance is still under investigation. Another critical issue concerns low-titer antibody positivity—aPL are widely accepted to confer prothrombotic susceptibility only at medium/high titers, while it is increasingly recognized that low-titer aPL might be implicated in pregnancy morbidity. Attempts to better characterize the clinical risk upon aPL profile culminated in the development of two independent scoring systems, aPL-S (aPL score, comprising five clotting assays and six ELISA) and GAPSS (Global APS score, which includes also independent risk-factors for thrombosis and pregnancy morbidity), whose utility in clinical practice is currently being evaluated.

Antiβ2 glycoprotein I domain I antibodies

A positively-charged discontinuous epitope in β2GPI-DI has been identified as the most relevant antigenic target involved in β2GPI/antiβ2GPI antibody binding. The pathogenicity of anti-DI antibodies has been progressively characterized—passive infusion of a synthetic peptide targeting DI partially protected naïve mice from thrombogenic effects of human polyclonal aPL IgG fractions; more recently, a human monoclonal anti-DI IgG-induced clotting and fetal loss in animals. Several methodologies can be employed to detect antiDI antibodies—a two-step solid-phase assay using hydrophilic-hydrophobic plates, several one-step solid-phase assays and chemiluminescence employing different DI sources. Antibodies against DI are more specific for APS compared to antibodies targeting the whole molecule; they are detectable in most APS patients, providing the prevalent β2GPI antibody specificity not only in primary APS but also among aPL-positive patients with associated autoimmune condition. Furthermore, anti-β2GPI-DI IgG antibodies have been found to be associated with LA, vascular thrombosis and -to a lesser extent- with pregnancy complications. Interestingly, patients at higher clinical risk for both vascular and obstetric manifestations, namely those with double/triple aPL positivity, display greater prevalence and titers of antiDI antibodies. A recent meta-analysis considering 11 studies for a total of 1585 aPL positive subjects including 1218 APS patients revealed that antiβ2GPI-DI antibodies may represent a promising tool to assess thrombotic risk in patients with APS, conferring an odds ratio for thrombotic events of about 2. Lower evidence is available on the association between anti-D1 antibodies and miscarriages; recently, antiDI positivity has been shown to predict late pregnancy loss and premature birth but not early pregnancy wastage potentially offering an insight into pathogenic mechanisms. It is still not clear whether antiDI antibodies offer additional prognostic value over double/triple positivity, or simply reflect the fact that APS patients with 2–3 positive tests are more likely to carry anti-DI antibodies. On the other hand, a small but consistent proportion of full-blown APS patients display autoantibodies reacting with β2GPI epitopes other than DI, implying that the assay for the whole molecule cannot yet be replaced.

In addition, antibodies reacting only against epitopes located in domains 4 and 5 of the β2GPI molecule have been recently described. These antibodies are not pathogenic in a thrombosis animal model, are not associated with clinical manifestations of the syndrome and more frequently detectable in the so called asymptomatic aPL-positive carriers or in non-APS patients. Their use in the clinical practice is still matter of research.

Anti-cardiolipin and antiβ2 glycoprotein I antibody IgA

In vivo findings are supportive for a pathogenic role of IgA: aCL and antiβ2GPI IgA purified from APS patients induced a larger thrombus compared with control IgA. Literature on aCL IgA is highly heterogeneous, with a prevalence rate ranging from 0% to 78%. Similarly, data on the association with clinical events are inconsistent—some studies reported a significant association with thrombotic events, pregnancy morbidity and non-criteria manifestations, whereas most failed to observe any relationship. Antiβ2GPI IgA is more promising, particularly in SLE. Anti-β2GPI IgA positivity has been correlated with vascular events and with noncriteria manifestations, even though in most cases it was associated with other APS laboratory criteria. However, in a cohort of 873 lupus patients, isolated antiβ2GPI IgA was reported in 5.7% of subjects, of which 60% presented APS-like clinical manifestations. Based on these data, aPL IgA were included in the recent SLICC classification. A different scenario has been described in primary APS, where controversy still reigns as available studies have provided conflicting results. Indeed, some investigators have reported isolated anti-β2GPI IgA positivity associated with vascular events or RPL, with IgA carrying an hazard ratio for APS even higher than IgM. In particular, in a study on 244 patients with isolated anti-β2GPI IgA, the incidence rate of APS-events was 3.1% per year in anti-β2GPI IgA positive patients as compared to 0.6% per year in the control group. It has been proposed that testing for β2GPI-IgA circulating immune complexes may further help identifying IgA-positive patients at risk of developing a thrombotic event. Conversely, other authors claims that positivity for IgA anti-β2GPI is highly coincidental with other aPL, neglecting any association with APS manifestations. Moreover, another bias affecting IgA testing relies in the poor comparability between kits, prompting the use of more standardized assays. Currently, IgA testing might be reasonable when APS is clinically suspected but conventional markers are negative, even though the faction in the APS community advocating the incorporation of IgA antibodies among APS criteria is progressively growing.

Antiprothrombin antibodies

Antibodies against PT (aPT) exert in vitro thrombogenic effects interfering with fluid-phase coagulation components and activating endothelial cells (EC). Owing to this finding, aPT were thought to represent promising diagnostic/prognostic aPL. However because of the lack of cross-reactivity of human aPT with animal PT, in vivo evidence for their pathogenic effect is not sound and is supported by a recent model only.

ELISA detecting antibodies against PT employs as antigenic target the mere PT or the PS/PT complex; the latter identifies antibodies against PS/PT (aPS/PT), a separate autoantibody population recognizing a conformational epitope(s) different from those present on plain PT. The clinical significance of aPT positivity in APS is still debated—conflicting results emerged about the association with thrombosis. Conversely, most studies highlighted a significant association of aPS/PT with aPL-associated thrombotic manifestations, particularly venous thrombosis. Consistently, a systematic review found both aPT and aPS/PT to increase the thrombotic hazard, with aPS/PT representing a stronger risk-factor for thrombosis than aPT. Positivity for anti-PS/PT has been shown to identify patients at highest thrombotic risk, in particular in case of concomitant positivity for antiDI antibodies. . In addition, antiPS/PT antibodies were shown to predict obstetric complications, in particular intra uterine growth restriction (IUGR) and preeclampsia in women positive for aCL and antiβ2GPI in most of the cases. Noteworthy, aPS/PT, together with antiβ2GPI antibodies and LA, displayed the best diagnostic accuracy for both vascular and obstetric APS among several combinations of six aPL assays. Interestingly, some authors have proposed antiPS/PT as a potential biomarker of seronegative APS. As a whole, testing for antiPS/PT has been suggested to be useful to— (1) better stratify the clinical risk; (2) identify patients negative for conventional aPL but with APS-like manifestations; and (3) characterize an isolated LA positivity. The detection of antiPS/PT has been suggested as a surrogate of LA when the functional coagulation assay cannot be performed because of an ongoing anticoagulation. However, it should be underlined that PS/PT dependent LA do not display the same diagnostic/predictive value as β2GPI-dependent LA, therefore the detection of anti-β2GPI is mandatory. In conclusion, even though aPS/PT assay deserves attention, it is difficult to demonstrate the diagnostic/predictive value of isolated aPS/PT and current evidence is not solid enough to recommend routine testing.