Introductory Chapter: Antiphospholipid Antibodies - A Laboratory Criterion for the Antiphospholipid Syndrome, but Also Bystanders in Infections, Cancer, and Other Conditions

1. Introduction

Antiphospholipid syndrome (APS) is a systemic autoimmune thromboinflammatory disorder characterized by vascular thrombosis and pregnancy-related morbidity accompanied by persistent positive antiphospholipid antibodies (aPL) [1, 2]. APS is considered the most common acquired form of thrombophilia worldwide [3]. Obstetric APS is a complex entity that can affect both the mother and the fetus throughout pregnancy with high morbidity. The clinical complications of obstetric APS are diverse and include recurrent fetal loss, stillbirth, intrauterine growth failure, and preeclampsia [4]. In addition to thrombosis and pregnancy loss, other pathological manifestations regularly occur with APS including thrombocytopenia, destruction of heart valves, accelerated atherosclerosis, nephropathy, movement disorders, and cognitive decline [5]. Catastrophic APS (CAPS) is characterized by the rapid development of thrombosis in multiple organs and micro-thrombosis within a short period of time. Pediatric APS is a rare condition that is distinctly different from adult APS [6].

The classification of APS for clinical trials and studies is currently based on the international consensus statement established in Sapporo in 1999 and updated in Sydney in 2006, and includes a clinical criterion (vascular thrombosis or pregnancy morbidity) and a laboratory criterion (positive test result for aPL) [1] as shown in Figure 1. aPL are a heterogeneous family of IgG and/or IgM or, more rarely, IgA autoantibodies with an affinity for negatively charged phospholipids or protein-phospholipid complexes. Their persistent presence in sera has been associated with increased prothrombotic risk in various autoimmune diseases. The aPL that constitute the laboratory criteria for APS include lupus anticoagulant (LA), anticardiolipin antibodies (aCL), and anti-β2-glycoprotein I antibodies (anti-β2GPI) of immunoglobulin IgG and IgM classes. Extensive evidence has accumulated over the past decade that several other than those included in the APS classification criteria may be relevant to APS pathogenesis. Among them, antiprothrombin antibodies, especially antibodies against phosphatidylserine-prothrombin complex (aPS/PT), are supported by the most studies in the literature showing their strong correlation to LA activity and to clinical manifestations of APS [7, 8, 9]. An international multi-disciplinary initiative “APS action”, jointly supported by the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) is currently underway to establish a new diagnostic criterion for APS.

Figure 1.

Classification criteria for APS and definition of high and low risk profile. Created with BioRender.com.

APS can either be a disease in the absence of evidence of other autoimmune disease, or it can be secondary to another autoimmune disease such as systemic lupus erythematous (SLE) [10]. The profile of aPL, including type and titer, is an important factor determining the risk for thrombotic and obstetric events [11, 12]. The presence of LA, triple positivity or double positivity with positive LA, and the persistent presence of high titers of aCL and anti-β2GPI antibodies pose a high risk for the development of APS. In contrast, isolated positivity at low or medium titers of aCL or anti- β2GPI antibodies, particularly when transiently positive, poses a low risk.

A very rare, but life-threatening form of multiorgan thrombosis is known as catastrophic anti-phospholipid syndrome (CAPS) [13, 14, 15]. It is characterized by simultaneous thrombosis in multiple organs within a short period of time, that is, within a few days. Thrombosis often occurs at unusual sites, and small and medium-sized arteries are most frequently involved [16]. Less than 1% of patients with APS develop CAPS. CAPS is the first manifestation of APS in about half of diagnosed CAPS patients. The remaining patients have a history of APS. The mortality rate has decreased over time, mainly due to triple therapy (anticoagulation, corticotherapy and therapeutic plasma exchange—TPE—or intravenous immunoglobulin—IVIG), but it still exceeds 30% [17]. An international registry established in 2000 by the European Forum on Anti-Phospholipid Anti-bodies, and the last reported data (2016) includes 500 patients [17].

The other major clinical manifestations of APS are obstetric. These include unexplained death of one or more morphologically normal fetuses at or after 10-week gestation, premature delivery of one or more morphologically normal newborns before 34-week gestation due to either eclampsia, severe preeclampsia, or recognized features of placental insufficiency, and three or more unexplained, consecutive spontaneous abortions before 10-week gestation.

2. Clinical manifestations of APS are heterogeneous and nonspecific

The heterogeneity and non-specificity of potential clinical signs illustrates that APS is as a true systemic autoimmune disease and underscores the need for a better understanding of disease mechanisms that will enable a personalized approach to treatment. Despite some improvements in the diagnosis and prognosis of APS and the prevention of thrombosis reoccurrence, robust laboratory biomarkers are still lacking.

Because APS affects young patients in the most productive years of their lives, the consequences of organ or tissue damage lead to impaired health-related quality of life (HRQoL). There are several reasons why APS could adversely affect HRQoL. The clinical manifestations are diverse, and many of them damage vital tissues. In addition, APS may overlap with rheumatoid arthritis (RA) and SLE, both of which already significantly affect HRQoL. Another aspect that affects HRQoL in APS is treatment with high-dose anticoagulation indefinitely in patients with thrombosis and/or at high risk of thrombosis.

In the general population, the incidence of clinical manifestations present in APS is high and could often be triggered by other underlying factors. Consequently, the diagnosis of APS relies predominantly on laboratory measurements. However, current laboratory tests are hampered by technical limitations in the pre-analytical and analytical phases and by the fact that there is no standardization of these tests. Despite the many attempts to increase the specificity of laboratory criteria and the establishment of consensus criteria for serology, a high number of patients are still misdiagnosed. One of the most important reasons for this is the high heterogeneity of aPL in patients with APS. Thus, it remains to be clarified whether different manifestations are caused by subpopulations of autoantibodies against different epitope specificities that are currently detected by the same test(s). Unfortunately, most APS patients exhibit more than one type of aPL, making it difficult to assign pathogenic effects to one epitope specificity or another. In addition, the diagnosis of pediatric APS is even more challenging since it is such a rare condition. Diagnosis may be delayed or missed when adult APS criteria are used, because in pediatric APS, non-thrombotic clinical manifestations, such as thrombocytopenia, hemolytic anemia, and neurologic disorders such as migraine, epilepsy, and chorea, may precede thrombotic manifestations.

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 well-characterized 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, 24, 25, 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.

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.

4. Thrombotic and obstetric risk assessment

Risk stratification is a major challenge in the management of patients with APS, and a possible role of aPL as a risk or even prognostic factor for arterial/venous thrombosis and miscarriages has been intensively discussed [27, 29]. Single, double, and triple aPL positivity is not uncommon in patients with APS, and such multiple positivity is usually associated with a higher risk for the occurrence or recurrence of thrombotic or obstetric adverse event [30, 31]. Recently, two research groups proposed a quantitative index to quantify the likelihood of thrombosis in APS. One included the aPL profile, and 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. A recent study examined different scoring systems after 2 years of systematic review [9]. They showed that all non-criteria aPL, including IgA aCL, IgA anti-β2GPI, and IgA/IgG aPS/PT were significantly associated with both thrombosis and obstetric complications. They proposed a novel quantitative scoring 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.

5. Antiphospholipid antibodies in infections

It is known that aPL may be transiently elevated in sera during various infections, including skin infections (18%), human immunodeficiency virus infections (17%), pneumonia (14%), hepatitis C virus (13%), and urinary tract infections (10%) [32]. The presence of aPL in sera and also its clinical significance was first noted in patients with Treponema pallidum infection [33]. With the continued use of cardiolipin-based serologic tests for syphilis diagnosis, it became apparent that a small group of patients with autoimmune diseases, especially SLE, had “false-positive” tests. In 1983, researchers recognized that the presence of aPL in SLE patients was associated with thromboembolic events and recurrent miscarriage, and the term anticardiolipin syndrome and later antiphospholipid syndrome (APS) were coined [34, 35].

Since the global COVID-19 pandemic, a possible link between the presence of aPL and infection with the SARS-CoV-2 virus has been investigated. Several groups have reported the presence of aPL in patients with COVID-19 and have suggested the possibility of SARS-CoV-2 virus-induced APS [36, 37, 38]. Coagulopathy and thrombotic events, including deep vein thrombosis, pulmonary embolism, and stroke, are serious manifestations in critically ill patients with COVID-19.

Currently, the role of aPL in thrombotic complications in COVID-19 is still unclear. Similar to the severe coagulopathies associated with COVID-19, patients with CAPS may develop thrombosis in multiple organs within a very short period of time [39]. Because of the similarity between the course of COVID-19 and CAPS, it was hypothesized that SARS-CoV-2 infection could be a possible trigger for APS. Detailed analysis of 23 studies (with a total of 250 patients) of aPL at COVID-19 showed that the presence of LA, aCL, and anti-β2GPI was 64%, 9%, and 13%, respectively [40]. However, none of the included studies reported re-examination of aPL after 12 weeks, so it is not clear whether the aPL presence in COVID-19 patients was transient or persistent. The only study in which aPL testing was repeated after 1 month and in which aPS/PT was also measured included 31 patients with COVID-19 [41]. In this study, elevated aPL levels were confirmed in 74% of patients, but 9/10 of the LA-positive patients retested were negative the second time. This observation supports the frequent single LA positivity during the acute phase of COVID-19 infection.

Later in the pandemic, two independent reviews were published that examined the prevalence of aPL in COVID-19 patients and its clinical significance [42, 43]. The prevalence of LA ranged from 35 to 92% in ICU patients, aCL IgG in 52%, and IgM in 40% of patients, and anti-β2GPI IgG and IgM were found in up to 39% and up to 34% of patients, respectively. Between 1 and 12% of patients had a triple-positive aPL profile [43]. In the second review, the authors primarily examined studies of aCL and anti-β2GPI but also addressed non-criteria aPL [42]. They concluded that aPL positivity may be a feature of COVID-19, at least in some patients, but in general the identified “solid-phase” aPL are of low titer and cannot be well associated with the thrombotic aspects of COVID-19. Also, in the few studies in which persistence was examined, the results seemed to indicate transient positivity of aPL that occurred only during infection. Importantly, high-titer aPL or multiple APL positivity (including double and triple positivity) was in the minority for COVID-19. There is also one important study where antigen specificity of aPL in COVID-19 has been investigated. These researchers have found that, contrary to APS, which is characterized by high aPL titers with specificity against domain 1 on β2GPI, patients with COVID-19 exhibit low titers of anti-β2GPI, with specificity against domains 4 and 5 [44].

The risk of a recurrent thrombotic event in patients with APS is greatly increased in those who have multiple subtypes of aPL (LA, aCL, anti-β2-GPI, aPS/PT), that is, double-, triple-positive patients. In patients with COVID-19, double or triple aPL positivity appears to be rare and aPL positivity appears to be transient. A well-designed, age- and sex-controlled observational study compared the aPL profile of hospitalized COVID patients with that of a) patients with thrombotic APS and b) patients with culturally/serologically proven infections [45]. Their data showed that positive aPL values can be found in half of the patients with infections, as 53% of patients with COVID-19 and 49% of patients with other viral/bacterial infections had positive aPL values. Importantly, however, the aPL profile was different when comparing patients with overt APS and patients with aPL detected in the setting of infections. Therefore, author conclude, caution is required in interpreting and generalizing the role of aPLs in the management of patients with COVID-19.

6. Antiphospholipid antibodies in cancer

The relationship between thrombosis and cancer was first established by Trousseau in 1865. Since then, numerous studies have shown that thromboembolism is a common complication of cancer, occurring in 15% of all cancer patients [46, 47]. Despite extensive research and modern interventions, thromboembolic disorders are still a major cause of morbidity and mortality in these patients. The risk of thromboembolic events is four times higher in cancer patients than in the general population and this risk is further increased in patients undergoing chemotherapy [47, 48]. Much of this high risk is attributed to the cancer itself. However, patient-related factors such as age, performance status, body mass index, underlying comorbidities, and therapy are also the important factors. The biological origin of thromboembolic events is related to the pro-coagulant, hypoxic, and inflammatory state associated with tumors, especially in advanced stages [49]. Several mechanisms contribute to the hypercoagulable state observed in cancer, resulting in a complex interplay of various factors, including tissue factors, platelet and endothelial activation, coagulation abnormalities, procoagulants secreted by tumor cells, abnormal blood flow, and abnormal tumor angiogenesis [46, 50]. The question arises whether the presence of aPL further increases the thromboembolic risk in patients with malignancies.

A high prevalence of aCL, anti-β2GPI, LA, anti-phosphatidylcholine, anti-phosphatidylserine, anti-phosphatidylinositol, anti-phosphatidylethanolamine, and anti-prothrombin antibodies has been observed in patients with various types of hematologic malignancies and solid tumors [47]. Therefore, the already increased risk of thrombosis in cancer patients is even higher for carriers of aPL. The reported prevalence of elevated aPL levels in cancer patients varies from less than 5%, which is similar to the prevalence observed in healthy individuals, to as high as 70% [47]. This dramatic range is due in part to different methods being performed, differences in study design, and inconsistent definitions of aPL positivity in the medical literature. In general, aPL tests are highly heterogeneous and poorly standardized. In addition, most studies examined the prevalence of aPL only once and did not repeat the test after 3 months, so the frequency may be overestimated. A recent systematic review of observational studies found an increased risk of developing aPL in patients with gastrointestinal, genitourinary, and lung cancer, leading to thromboembolic events and death [51]. In addition, a 17-year observational study of 1592 non-thrombotic women with three consecutive spontaneous abortions before the 10-week gestation or fetal death at or after 10-week gestation showed that the risk of cancer was significantly higher in women with a history of obstetric APS than in the general population [52]. Recently, one research group investigated the presence of criteria and non-criteria aPL in patients with uterine malignancies [53]. The authors found that non-criteria aPL (against phosphatidic acid, phosphatidylserine, annexin V, and prothrombin) are more common in patients with uterine malignancies (UM) than in patients with non-cancerous gynecological diseases (NCGD). In contrast, the criteria aPL did not differ significantly between UM and the NCGD group. It is interesting to note that several studies associate non-criteria aPL, especially antiprothrombin antibodies, with obstetric complications, while they could not confirm the association with either anti-β2GPI or LA [11, 12].

In conclusion, aPL levels appear to be elevated in patients with various malignancies, increasing their risk for thromboembolic events. In the future, it would be important to conduct well-designed large-scale population studies as well as longitudinal studies on patients with various cancers to determine the true risk and confirm whether the increased prevalence of aPL positivity is transient. Although aPL positivity may help assess the risk of blood clots, there are currently no strong data to recommend aPL screening in cancer patients.

7. Antiphospholipid antibodies in healthy individuals

Low aCL levels are found in up to 10% of healthy individuals, and the prevalence of a positive aPL test increases with age [10]. High aPL levels and persistent positivity are rare in healthy individuals (less than 1%). There are no recent studies investigating the level of criterion-related or non-criterion-related aPL in the general population. The clinical significance of aPL in healthy individuals remains unclear. It is important to emphasize that not every positive test for aPL is of clinical significance, and patients with aPL are at different risk for adverse events related to aPL. A rare prospective study in which healthy blood donors were tested for aPL twice 1 year apart showed 10% positivity for aCL and 1% positivity LA at the first measurement. Of note, less than 1% of subjects were still positive after 1 year [54]. Therefore, in parallel with other cardiovascular risk factors such as hypertension, elevated cholesterol, diabetes, smoking or obesity, patients with aPL have a higher risk of adverse events. It is known that aPL can occur transiently during infections or other occasions. This is an important reason why aPL should be tested twice within 12 weeks, which is also embodied in the international classification criteria for APS.

Recently, an administrative database study of aPL in the general population was published that characterized patterns of aPL testing in a sample from the United States using laboratory data from 2010 to 2015. They identified 33,456 individuals with at least one aPL test. Of these, only 6391 (19%) had all three tests (LA, aCL, aGP1) performed. Confirmatory aPL tests were performed at least 12 weeks later in 77, 45, and 41% of initially positive LA, aCL, and aGP1, respectively. Of those retested, only 255 (10.6%) had a confirmatory positive aPL test. The most important finding is the low rate of a confirmatory positive aPL test ≥12 weeks after the first test, indicating that aPL testing is often be incomplete. Further investigation in the form of large-scale population studies as well as longitudinal studies is needed to better understand the clinical relevance of aPL in healthy individuals from different backgrounds.

8. Conclusion

The heterogeneity and non-specificity of the possible clinical symptoms highlight that APS is a true systemic autoimmune disease and emphasizes the need for a better understanding of the disease mechanisms that will allow a personalized treatment approach. In the general population, the incidence of clinical manifestations in APS is high and could often be triggered by other underlying factors. Therefore, the diagnosis of APS relies predominantly on laboratory measurements. Despite the many attempts to increase the specificity of laboratory criteria and to establish consensus criteria for serology, a high number of patients are still misdiagnosed. Treatment of APS requires an interprofessional team approach involving multiple specialties. Family physicians play an important role in identifying patients with APLS. Hematologists and rheumatologists play a critical role in diagnosis, treatment, and follow-up. Involvement of other specialties such as neurology, nephrology, cardiology, and dermatology may also be necessary if a particular organ system is affected. In addition, anticoagulation clinics can play an important role in monitoring therapeutic warfarin levels and INR levels with close follow-up. Last but not the least, pharmacists can help in the management of these patients, especially in identifying drug–drug interactions. Close communication between the interprofessional team and close monitoring of the patient is essential in the management of APS.