Antiphospholipid antibody testing

Abstract

Antiphospholipid antibodies (aPL) are a family of autoantibodies targeting phospholipid-binding proteins and are associated with several clinical settings, and most notably define the antiphospholipid syndrome (APS). These antibodies can be identified using a variety of laboratory tests, which include both solid-phase immunological assays and functional clotting assays that detect lupus anticoagulants (LA). aPLs are linked to a range of adverse medical conditions, such as thrombosis and complications affecting the placenta and fetus, potentially leading to morbidity and mortality. The specific aPL identified, along with the pattern of reactivity, correlates with the severity of these conditions. Therefore, laboratory testing for aPL is crucial for evaluating the risk of complications and for fulfilling certain classification criteria for APS, which are also applied as diagnostic markers in medical practice. This review provides an overview of the available laboratory tests currently for measuring aPL and discusses their clinical implications.

Resumen

Los anticuerpos antifosfolípidos (aPL) son una familia de autoanticuerpos que se dirigen contra las proteínas que se unen a los fosfolípidos y están asociados con varios contextos clínicos, y más notablemente definen el síndrome antifosfolípido (SAP). Estos anticuerpos pueden identificarse mediante una variedad de pruebas de laboratorio, que incluyen tanto análisis inmunológicos de fase sólida como análisis de coagulación funcionales que detectan anticoagulantes lúpicos (AL). Los aPL están vinculados a una serie de procesos médicos adversos, como la trombosis y las complicaciones que afectan a la placenta y al feto, lo que potencialmente conduce a morbilidad y mortalidad. El aPL específico identificado, junto con el patrón de reactividad, se correlaciona con la gravedad de estos procesos. Por lo tanto, las pruebas de laboratorio para aPL son cruciales para evaluar el riesgo de complicaciones y para cumplir con ciertos criterios de clasificación para el SAP, que también se aplican como marcadores diagnósticos en la práctica médica. Esta revisión proporciona una visión general de las pruebas de laboratorio disponibles actualmente para medir los aPL y discute sus implicaciones clínicas.

Introduction

In the early 1980s, the antiphospholipid syndrome (APS) was identified as a distinct syndrome first described in patients suffering from Systemic Lupus Erythematosus (SLE) with antiphospholipid antibodies (aPL) that were more likely to experience repeated pregnancy loss, neurological symptoms, arterial/venous thrombosis, and thrombocytopenia.¹ APS was later identified as a systemic disease in the years that followed. The main clinical manifestations of APS are described elsewhere in this special issue. In this review, we aimed to describe the current and upcoming evidence for laboratory testing in APS and their role in the management of this condition.

Antiphospholipid antibody assays

Testing for lupus anticoagulant (LA), anticardiolipin (aCL), and beta-2 glycoprotein I (B2GPI) antibodies is integral to the current classification criteria for APS.² For a patient suspected of having APS, identifying persistent circulating antiphospholipid (aPL) antibodies is crucial, given that the disease's clinical manifestations are common in the general population and often complicated by various underlying factors. The tests used to detect aPL must be both sensitive and specific to ensure an accurate diagnosis of APS. This precision is vital because overdiagnosis or misdiagnosis can significantly impact the treatment pathway for these patients. Therefore, the choice and efficacy of assays for aPL detection must be carefully considered, aligning with international guidelines.^3–5

Historically, the assays commonly employed for aPL detection have been described as suffering from methodological limitations and lack of standardization.^3–5 However, over the last decade significant improvement have been achieved in this field. To enhance laboratory testing for APS, the Scientific and Standardization Subcommittee on aPL of the International Society on Thrombosis and Haemostasis (ISTH) published guidelines in 2009 and subsequent updates for the detection of lupus anticoagulant,^4,7 which have successfully improved the standardization of this test internationally. Additionally, guidelines for the detection of aCL and B2GPI antibodies using different immunoassays have been issued to further elucidate the key concerns such as cut-off definition, inter and intra assay comparability, and clinical significance.^3–7

When considering aPL testing, it is essential to emphasize that only patients with a high likelihood of APS should be tested for aPL. Today since a broader range of clinical disciplines order the aPL tests we have witnessed in the real-life scenario a decrease in the percentage of the pretest probability and of the posttest probability, as a consequence. This aspect has been properly addressed in the recently released EULAR/ACR Classification Criteria for APS.² Routine screening for aPL without any clinical indications of the disease is generally not recommended to prevent the incidental finding of aPL. Clinical scenarios that warrant a high suspicion of APS include, for instance, younger patients (below 50 years of age) with unprovoked thrombotic events, thrombosis at atypical sites, or those with thrombotic or pregnancy complications associated with a secondary autoimmune condition.⁷ The prevailing guidance is to perform all aPL tests simultaneously and to interpret the results collectively, considering that aPL represent a diverse array of autoantibodies with overlapping yet distinct features.^2–7

Lupus anticoagulant

The term ‘lupus anticoagulant’ (LA) is misleading, as it is neither a diagnostic test for lupus nor an anticoagulant. LA refers to an in vitro phenomenon where substances behave as anticoagulants; however, the positivity for LA actually confers a pro-thrombotic state in vivo. Due to its capacity to recognize various antiphospholipid antibodies (aPL), the LA assay is considered broadly reactive. To detect LA, a combination of at least two coagulation tests is employed. Commonly used tests include the diluted Russell viper venom time (dRVVT) and the activated partial thromboplastin time (aPTT). These functional coagulation assays measure the ability of aPL to prolong phospholipid-dependent clotting time. A positive result in at least one of these tests suggests the presence of LA. The traditional method for LA detection is a three-step process involving screening, mixing, and confirmation phases.^4,6 A LA-positive result is indicated by prolonged clotting time in the screening phase, which does not correct upon mixing the patient's plasma with normal plasma, and is then corrected by adding excess phospholipids during the confirmation phase. This confirms the specificity of the anticoagulants for phospholipids—that is, aPL. Due to the nature of functional assays, no coagulation test is 100% sensitive, necessitating the use of two tests with differing approaches to enhance sensitivity. The dRVVT and aPTT are recommended to increase standardization in LA testing, requiring reliable, repeatable, sensitive, commercially available, and quality-controlled assays. The mixing step is critical to avoid false positives. However, this step has been debated due to the time and reagents it consumes, and because it may correct coagulation in cases of low antibody titers.^4,6 One significant limitation of LA coagulation tests is their vulnerability to interference by anticoagulant therapy (Refs. 70–78). Elevated levels of C-reactive protein or factor VIII can lead to false-positive or false-negative results, respectively.⁸ In addition to the diluted Russell viper venom time (dRVVT) and activated partial thromboplastin time (aPTT), other phospholipid-dependent coagulation assays are generally not recommended due to variations in reagent compositions, poor reproducibility, or limited availability.^9–11 The British Society for Haematology (BSH) includes the dilute prothrombin time (dPT) as an alternative to aPTT in its guidelines,^9–11 and the Clinical and Laboratory Standards Institute (CLSI) cites it for second-line testing.^9–11 The dPT is sensitive in detecting lupus anticoagulant (LAC), but its variability in reagents limits its use.^9,12,13 The kaolin clotting time (KCT), which differs from aPTT by lacking an exogenous phospholipid source and uses kaolin as a contact activator, has fallen out of favor due to standardization challenges, the absence of confirmatory tests, and incompatibility with certain optical clot detection analyzers.^10–14

The Taipan snake venom time (TSVT) coupled with ecarin time (ET) is an emerging assay for LAC assessment.^5,15 It has demonstrated promising sensitivity for LAC detection with reduced interference from oral anticoagulation treatments in multicenter studies. The TSVT assay employs oscutarin C from Coastal Taipan viper venom to convert prothrombin to thrombin in a manner dependent on phospholipids and calcium, but independent of Factor V.^15,16 Meanwhile, ET, using venom from the Indian Saw-Scaled viper, activates prothrombin without the need for phospholipid cofactors.¹⁵ The TSVT/ET combination can function as both a screening and confirmation assay, with ET being phospholipid independent.¹⁵ These tests are less impacted by vitamin K antagonists (VKAs) and direct oral anticoagulants (DOACs) that inhibit Factor Xa, potentially offering a reliable solution for LA testing in patients on anticoagulation therapy. Recently automated algorithms with launch of the mixing step and confirmation step based on predefined cutoff values and calculations improved the standardization process of the test interpretation reducing the intralaboratory and interlaboratory variation.¹⁶ However, more standardized collaborative research is necessary before these assays can be widely recommended.⁴

Additional functional assays

While current coagulation assays for LA measurement are subject to interference, they are also labor-intensive and complex to interpret. It is important to pursue further development of functional assays or to identify solid-phase immunoassays as alternative biomarkers to conventional LA tests. There is increasing interest in thrombin generation assays (TGAs), which offer a more comprehensive view of the coagulation process than clotting time-based assays. TGAs measure thrombin production in plasma following the addition of tissue factor and phospholipids, capturing both procoagulant and anticoagulant activities. This generates a thrombogram that provides several derived parameters (reference 108). TGAs are particularly sensitive in identifying LA-positive patients and may even measure LA intensity within a single test.^17–20 However, TGAs are still labor-intensive, lack standardization, and are not yet robust enough for routine clinical use.^17–20 Recent guidelines on executing TGAs aim to standardize methodologies, potentially aiding their integration into clinical diagnostics and management, including for APS.^17–20 The introduction of automated systems could revolutionize routine practice, offering time efficiency and reduced inter-laboratory variability. These automated TGAs could consolidate phospholipid-dependent testing into one assay, simplifying LA detection, which currently requires multiple tests.¹⁷

Antibodies against β2GPI and anticardiolipin antibodies

Solid-phase immunoassays are utilized for the detection of aCL and anti-β2GPI antibodies. The identification of these antibodies, particularly the IgG or IgM isotypes at medium to high titers, is considered diagnostic.^2,21 LA assays have the capability to screen for all aPL antibodies, irrespective of the cofactor protein involved. The immunoassays measure antibodies against cardiolipin (via the aCL antibody assay) or β2GPI (via the anti-β2GPI antibodies assay), with β2GPI being the principal cofactor protein for aPL.^12,21 aCL antibody assays that are methodologically robust and include anti-β2GPI in the reagents achieve similar sensitivities and specificities to those of anti-β2GPI assays alone.^12,21

Which assay?

aCL and aβ2GPI antibodies are traditionally detected using solid-phase immunoassays, such as ELISA, which are standardized by the APS classification criteria.⁵ Over the years, alternative methods like chemiluminescent, fluorescence enzyme, and multiplex flow immunoassays have emerged. More recently, semi-automated analyzers for aCL and aβ2GPI testing have been introduced, offering increased consistency and reduced variability in diagnostics, although they’re not universally accessible.^5,12,21 Fundamentally, these assays adhere to the immunoassay principle where an antigen is immobilized on a solid phase and exposed to patient antibodies. The binding is then detected by conjugated anti-human IgG or IgM antibodies, eliciting a measurable response.^5,12,21 Despite using similar principles, assays vary in materials and methodologies, leading to significant inter-assay variability.^5,12,21 Thus, it's crucial to maintain assay consistency for patient follow-up and consider retesting with different platforms in cases of high clinical suspicion but negative results.

Which isotype?

It is noteworthy that the detection of identical isotypes of both aCL and anti-β2GPI antibodies increases the probability of the APS diagnosis.^22–24 Although IgG-type antibodies are more closely associated with thrombosis than the IgM isotype, recent literature reviews have not conclusively determined how many APS cases would be overlooked if IgM testing were omitted.²² The relevance of IgA antibodies for anti-β2GPI and aCL remains controversial, and thus, routine measurement of this isotype is not currently recommended.^2,5 IgA testing may not be widely useful for initial diagnostic screening but could be considered in confirming APS or in patients with a high clinical suspicion of APS who are negative for the criterion aPL.^22–24 Variability in calibration and assay properties leads to inter-assay differences in the results of aCL and anti-β2GPI antibody tests.^22–24 This variation is partly due to different solid-phase coatings in the assays, which can influence the amount of antigen available for antibody binding.^5,21–26 However, advancements in technology, such as the adoption of automated systems that standardize operating procedures, have also reduced interlaboratory discrepancies over the years.^25,26

From calibration to cut-off choice

Calibration curves are mandatory in each ELISA run or for new reagent lots in automated systems to accurately determine aPL titers. Recalibration may be necessitated by internal quality control outcomes. Evaluations of each calibration must adhere to the manufacturer's specifications, rejecting any that do not align or exhibit a correlation coefficient below 0.90.²⁷ Reference materials for assay calibration lack uniformity, with manufacturers supplying various non-standardized calibrators.^5,28,29 In the 1980s, Harris et al. developed polyclonal patient-derived calibrators for aCL, expressed in IgG and IgM phospholipid units (GPL/MPL), which are the precursors to the now-known “Harris standards”.^30–32 The “Koike standards” or “Sapporo standards,” which are monoclonal antibody (MoAb) standards, offer batch consistency and virtually limitless production, albeit not reflecting the polyclonal nature of APS antibodies.^30–32 While aCL results can be reported in GPL/MPL if validated against Harris standards, aβ2GPI testing lacks a universal unit, leading to a wide range of reporting units.³⁰ Harmonization efforts are underway to establish WHO standards in IU/mL for both aCL and aβ2GPI.^33,34 The current APS classification criteria regard aCL IgG or IgM detection as significant at moderate to high titers (>40 GPL/MPL or >99th percentile), with similar parameters for significant aβ2GPI levels.^34–36 The choice of 40 GPL/MPL as a cut-off for aCL was based on its correlation with APS-related symptoms,^35,36 but variability often exists between this and the 99th percentile.^37,38 Due to high inter-assay variability, using a single numeric threshold is not advised, and the International Society on Thrombosis and Haemostasis-Scientific Standardization Subcommittee (ISTH-SSC) recommend defining laboratory-specific cut-offs based on the non-parametric 99th percentile of reference individuals.²⁷ For clinical interpretation, higher titers of IgG aCL are more closely linked to APS events.^39,40 While a semiquantitative interpretation has been proposed, numerical discrepancies across assays prevent a standardized approach.^5,40 However, a semiquantitative interpretation for IgG aCL and aβ2GPI may be clinically valuable, provided that assay-specific thresholds are clearly defined.⁴¹ Harmonizing these ranges could be facilitated by paired analysis using standard materials or patient samples across different assays, including those traceable to the original Harris standards.⁴¹

Antibody profile

When evaluating the antibody profiles in patients, the presence of LA is considered a primary risk factor for thrombotic events associated with APS.⁴² However, since patients may test positive for only one type of aPL antibody, it is essential to conduct tests for all three antibodies—LA, aCL, and anti-β2GPI)—to establish a complete antibody profile. The initial guidelines for aPL profiling were established in the 2006 Sydney APS classification criteria, which included patient classification based on single or multiple aPL positivity. These criteria support the concept that the aPL profile influences the probability of APS-related clinical events.^2,42 A subsequent revision has suggested taking into account the diversity and quantity of positive test results.²

Research indicates that patients with multiple positive aPL tests, especially those who are ‘triple positive’ for LA, aCL (IgG or IgM), and anti-β2GPI antibodies (IgG or IgM), have the strongest association with thrombotic expressions of APS.^43–45 Furthermore, ‘triple positivity’ is associated with recurrent thrombosis, and when detected in asymptomatic individuals, it is linked to a heightened risk of a primary thrombotic event.^43–45 Patients with triple-positive APS typically retain this profile over time and demonstrate consistent outcomes three months following initial testing.^43–45 Nevertheless, it is common practice to recommend retesting for aPL after three months to avoid misdiagnosis, such as mistaking transient antibody elevations—possibly due to infections—as chronic APS.^46,47 It is also critical to validate the reliability of positive results through confirmation testing, particularly in light of suboptimal standardization and potential interferences that may affect the accuracy of test outcomes.^46,47

Non criteria antiphospholipid antibodies

In clinical practice, when screening for aPL, standard test panels typically include only those antibodies specified in the current classification criteria. Non-criteria tests refer to a group of assays that are largely non-standardized, and there is no international agreement on their utility for patients with APS.^48–51 Nevertheless, specifically, the SSC-ISTH recommendations did not encompass the testing for anti-domain 1 (anti-D1) antibodies, a subset of IgG anti-β2GPI antibodies, due to the lack of appropriate clinical trials and the absence of a standardized commercial assay at that time.^48–51 However, research using specialized assays⁵³ has shown a noteworthy association between anti-D1 antibodies and thrombosis. With advancements in laboratory technology, a commercial chemiluminescence immunoassay has been developed to detect anti-D1 antibodies. This assay has been employed in various studies, confirming a significant odds ratio for thrombosis and underscoring the relevance of anti-D1 antibodies in risk stratification for APS patients. These antibodies, particularly the IgG isotype of anti-D1, are predominantly detected in patients with triple-positive aPL profiles and tend to appear at high titers.^50,51

Despite these findings, anti-D1 antibodies are not considered an independent risk factor for APS clinical manifestations, as evidenced by a limited number of studies.^48–51 Thus, they are viewed more as a confirmation of increased thrombotic risk rather than as an alternative to anti-β2GPI antibodies.

However, excluding triple/tetra-positive APS in which high-risk patients are clearly identified, aβ2GPI-D1 antibodies might be useful in the incomplete aPL profiles when deciding the type and length of anti-thrombotic treatment. Similarly, the ratio of anti-D1 to anti-D4/5 antibodies as a tool to identify those subjects carrying the “less pathogenic” anti-b2GPI antibodies should be explored.⁵¹

Antibodies to prothrombin

Antibodies to prothrombin can be detected by ELISA, utilizing either prothrombin coated onto irradiated plates (aPT) or the phosphatidylserine/prothrombin complex as antigen (PS/PT). Both tests have been associated with the clinical manifestations of APS. Current evidence indicates they originate from distinct populations of autoantibodies, yet they can be simultaneously detected in a single patient.

The link between APS and antibodies to prothrombin, identified as aPT or aPS/PT, has been evaluated with varying outcomes.^52,53 Nevertheless, recent evidence supports the clinical relevance of aPS/PT in diagnosing APS.^52,53 Atsumi and colleagues made early strides in elucidating the clinical significance of aPS/PT, finding that its presence increased the risk for APS 3.6-fold in a cohort of 265 Japanese patients with systemic autoimmune diseases.⁵⁴ Subsequent studies have corroborated the association of aPS/PT with APS clinical manifestations.^56,57 Among the retrieved studies mentioned in the available systematic review,^55,56 Zigon et al. reported that aPS/PT is a strong independent risk factor for aPL-related obstetric complications in a cohort of 156 patients with systemic autoimmune diseases.^55,56 Furthermore, Sanfelippo et al. demonstrated the utility of testing for aPS/PT in a large cohort of 728 patients suspected of APS, even in the absence of aCL or anti-β2GPI antibodies. They found that 41 individuals with elevated levels of aPS/PT experienced thrombotic events in 50% of the cases where medical histories were available.^55,56 This suggests that aPS/PT testing can help identify APS in patients who might otherwise remain undetected using current testing protocols. Bertolaccini team's assessed various aPL specificities to identify the profile with the highest diagnostic accuracy for APS. This investigation included 230 patients with SLE, testing for six aPLs in 23 possible combinations. The combination of LA, anti-β2GPI, and aPS/PT showed the highest diagnostic accuracy for APS overall, and specifically for thrombosis and pregnancy loss, with the greatest specificity compared to other combinations, including those defined by current classification criteria.⁵² In a systematic review analyzing data from over 7000 patients and controls across 38 studies on aPT and 10 studies on aPS/PT, we found that aPS/PT was associated with both arterial and/or venous thrombosis, with a stronger correlation than aPT⁵⁵ and was confirmed in a subsequent analysis.⁵⁶ Ongoing research aims to further delineate aPS/PT and their mechanisms. Nonetheless, further laboratory and clinical studies are imperative to definitively determine the significance and prognostic value of these antibodies in routine clinical practice. The potential inclusion of aPS/PT as an additional serological diagnostic tool for APS is actively debated, particularly regarding the identification of APS patients who test negative for classical aPL.

Risk assessment in antiphospholipid syndrome

The clinical symptoms associated with aPL may range considerably among individuals. While some individuals carrying aPL may remain asymptomatic, others may experience pregnancy complications, thrombotic events, or both. Research is ongoing to ascertain the significance of different aPL profiles in predicting the risk of thrombosis or obstetric complications. Notably, triple positivity, persistently isolated medium-to-high titers of aCL, and LA presence have been identified as significant risk factors for thrombosis.^43–47 This is corroborated by data suggesting that adverse pregnancy outcomes are also associated with the presence of LA and triple positivity.^43–47

The Global Antiphospholipid Syndrome Score (GAPSS), established in 2013, incorporates various risk factors alongside the presence of aPL, acknowledged as a risk factor for APS.⁵⁷ GAPSS amalgamates independent predictors of pregnancy morbidity and thrombosis, including autoimmune antibody profiles such as anti-nuclear antibodies (ANA), extractable nuclear antigen antibodies (ENA), and anti-double stranded DNA antibodies (dsDNA), among others. Independent risk factors within the GAPSS include both criteria and non-criteria aPL, such as aCL, a-β2GPI), aPS/PT, LA, and the IgG/IgM isotypes of these antibodies, all linked to an elevated risk of thrombosis and/or pregnancy morbidity PM. In GAPSS, each risk factor is assigned specific points: IgG/IgM aβ2GPI (4 points), IgG/IgM aPS/PT (3 points), LA (4 points), hyperlipidemia (3 points), and arterial hypertension (1 point) (Table 1). Higher GAPSS scores have been associated with an increased incidence of thrombosis and/or pregnancy loss, particularly in patients with SLE, where the GAPSS model was initially developed.⁵⁷ Subsequently, a study involving 62 patients with primary APS applied the GAPSS scoring system.⁵⁸ It was observed that APS patients with a history of thrombosis alone exhibited higher GAPSS scores compared to those with only prior pregnancy losses. Additionally, patients with a GAPSS score of 11 or higher were found to have an elevated risk of future thrombotic events. Another investigation highlighted a relationship between GAPSS and a history of APS manifestations, especially thrombosis, suggesting its potential as a quantitative measure for APS.⁵⁸ More recent research involving patients with APS and SLE identified that a GAPSS score above 16 predicted an increased likelihood of thrombotic events.⁵⁸ The GAPSS usefulness in clinical settings is described in Table 2.

Table 1.

The Global AntiPhospholipid Syndrome Score (GAPSS) is a risk assessment tool designed to evaluate the risk of thrombotic/obstetric events in patients with antiphospholipid syndrome (APS).

Risk factor	Points
Anticardiolipin antibodies (IgG/IgM)	5
Lupus anticoagulant	4
Anti-β2 glycoprotein I (IgG/IgM)	4
Anti-phosphatidylserine/prothrombin (IgG/IgM)	3
Hyperlipidemia	3
Arterial hypertension	1

Table 2.

Clinical Application of Global Antiphospholipid Syndrome Score (GAPSS).

The GAPSS is particularly useful in clinical settings to:

Determine the necessity for more aggressive management and/or more strict follow-up.

Monitor patients with aPL for changes in their risk profile.

Educational tool about individual risk factors and the importance of their managing.

aPL: antiphospholipid antibodies.

Future directions

The future directions of aPL antibody testing are focused on overcoming the current challenges with the tests used to diagnose APS. Three main issues have been identified: the lack of standardization in assays, uncertainty whether the assays detect the autoantibodies responsible for clinical manifestations, and the inability of current assays to predict recurrence risk. The goal is to develop novel assays that can provide prognostic information for a more tailored treatment of patients with APS. Advances in our understanding of the target protein, β2GPI, suggest that it may be possible to design assays that better predict the clinical outcomes associated with aPL.

Ethical considerations

N/A.

Funding

None.

Conflict of interest

None.