**3. Current laboratory criteria are unable to identify all patients with APS**

While aPL circulate in relatively stable concentrations in the blood, thrombosis occurs only occasionally. The persistent presence of aPL is thought to shift the hemostatic balance toward a prothrombotic state, but then a "second hit" is required to trigger the thrombotic event itself. Although this two-hit model is generally accepted, much remains to be learned about how exactly aPL predispose to thrombosis *in vivo* and how this predisposition interacts with the second hit. The pathogenic mechanisms responsible for thrombosis and obstetric complications in APS are based on a combination of factors, including inhibition of natural anticoagulant pathways, disruption of the annexin A5 anticoagulant shield on the phospholipid surface, activation of cellular elements, hemostatic reactions, and inflammation, particularly complement activation. Binding of aPL to the surface of vascular cells (endothelial cells, platelets, monocytes, trophoblasts) triggers activation of these cells, resulting in an increase in surface expression, production, and activity of procoagulant molecules and triggering the release of extracellular vesicles (EVs). EVs are submicron particles that are constitutively released by almost all cell type. In response to stimuli, such as cell activation by inflammation and/or apoptosis, their release into the cell surroundings is triggered to an even greater extend. EVs carry a diverse cargo (bioactive lipids, proteins, and nucleic acids) and might reflect the cell of origin and even its activation status. An increase in circulating EVs, particularly endothelial EVs, is considered a hallmark of vascular dysfunction and cardiovascular disease. Moreover, increased levels of EVs in the absence of an acute thrombotic event suggest a chronic state of vascular activation in APS. EVs could therefore be a useful biomarker to identify patients with aPL at the highest risk for complications. The lack of standardized approaches to isolate and/or characterize EVs has been a major limitation in determining their role in various diseases, including APS. Few studies have investigated EVs in APS patients. These studies have been limited to characterization of medium- to large-sized EVs, with significantly higher concentrations of endothelial and platelet EVs detected in the plasma of APS patients compared with healthy controls [9]. Despite wellcharacterized in vitro models of APS pathology, the field of EVs remains largely unexplored and may therefore provide insight into the APS mechanism. To our knowledge, no study has investigated whether EVs isolated from the vicinity of aPL-stimulated cells have the potential to activate distant endothelial cells in a similar manner.

In the general population, the incidence of clinical manifestations which can be attributable to APS is high and could often be triggered by other underlying factors. Therefore, the diagnosis of APS relies primarily on the laboratory measurements of aPL. Methods for their determination differ and have not yet been standardized. The common weaknesses of aPL determination are high inter-assay and inter-laboratory variations, problems in interpretation and clinical evaluation of test results, and their low diagnostic specificity. Elevated aPL levels can be associated with many other conditions such as infections, malignancies, and also the use of certain medications. The lack of reliable, robust diagnostic markers for APS thus limits patient identification and treatment and challenges researchers to find better diagnostic markers. A systematic review of observational studies that excluded patients with autoimmune diseases found a pooled prevalence rate of aPL in up to 23.3% of patients with stroke, 23% with myocardial infarction, 15.8% with deep vein thrombosis, and 13% of women with pregnancy adverse events [18].

Many investigators are exploring the usefulness of testing for non-criteria aPL specificities to identify APS in patients with thrombosis and/or pregnancy morbidity, particularly in those who are repeatedly negative on currently used tests. Among them, IgA aPL and antiprothrombin antibodies are most commonly proposed to assess the risk of thrombosis and pregnancy morbidity in patients with suspected APS [19]. A number of studies have shown that antiprothrombin antibodies represent distinct antibody subsets with overall diagnostic relevance for APS [7, 20]. Similar to anti-β2GPI, antiprothrombin antibodies, particularly aPS/PT, have a considerable value as a biomarker for both diagnostic evaluation and prediction of the clinical manifestations of APS. In 2017, a large international multicenter study found that IgG aPS/PT to be more prevalent in patients with APS than in patients without the syndrome. A positive test for these antibodies conferred a 10-fold higher risk of APS [21]. There is debate about the feasibility of including aPS/PT in risk assessment for APS to increase the accuracy of diagnosis in seronegative APS patients [7, 20, 22]. Our research group has extensively studied the clinical significance of antiprothrombin antibodies, showing that aPS/ PT have the highest percentage of LA activity compared with aCL or anti-β2GPI [8, 9, 23–26] and that they are strongly associated with thrombosis and adverse pregnancy outcomes independently of other aPL [8, 26]. In fact, aPS/PT were the only antibodies associated with pregnancy complications (recurrent pregnancy loss) occurring before 10-week gestation and with some late complications (preeclampsia and eclampsia), indicating their important role in the pathogenesis of obstetric APS.

*Introductory Chapter: Antiphospholipid Antibodies - A Laboratory Criterion… DOI: http://dx.doi.org/10.5772/intechopen.103846*

Recently, two research groups proposed a quantitative index to quantify the likelihood of thrombosis in APS. One included the aPL profile, the aPL score (aPL-S) [27], whereas the other included both aPL and conventional prothrombotic risk factors, the global APS score (GAPSS) [28]. Both groups included LA and IgG and IgM isotypes of aCL, anti-β2GPI, and aPS/PT. In contrast to risk stratification for thrombotic events, which has been well studied in aPL-positive patients, studies assessing the risk for obstetric complications are scarce. Our recent study investigated different scoring systems after 2 years of routine and systematic measurement of criteria and non-criteria aPL [9]. We showed that all non-criteria aPL, including IgA aCL, IgA anti-β2GPI, and IgA/IgG aPS/PT were as well significantly associated with thrombosis and obstetric complications. We proposed a new quantitative scoring [9] to evaluate the risk of adverse pregnancy events in aPL-positive patients, namely the obstetric risk score—ORS. The ORS showed much higher diagnostic accuracy for obstetric complications compared with any single aPL measure.
