Solid Phase Assays for Antiphospholipid Antibodies

Abstract

The diagnosis of antiphospholipid syndrome (APS) relies on the detection of circulating antiphospholipid antibodies (aPL). Currently, lupus anticoagulant (LA), anticardiolipin (aCL), and anti-β2-glycoprotein I antibodies (aβ2GPI) IgG or IgM are the laboratory criteria if persistently present over time. As aCL and aβ2GPI are two out of the three laboratory criteria, the detection of aPL by solid phase assays is an essential step in the diagnosis of APS. Advancement has been made to resolve some of the methodological challenges of aCL and aβ2GPI assays by providing guidelines how to measure aPL, as well as to gain a better understanding of their diagnostic role. However, solid phase assays for aCL and aβ2GPI still show substantive inter-assay differences, resulting in disagreement concerning positive/negative results, but also differences in titer of antibodies. This hampers the semiquantitative classification into low-medium-high positivity. The non-criteria aPL, such as antibodies against the domain one of β2GPI and anti-phosphatidylserine/prothrombin antibodies (aPS/PT) have roles in confirming the risk in APS, and can be useful, especially in patients with incomplete antibody profiles.

Keywords

antiphospholipid syndrome - anticardiolipin antibodies - anti-β2-glycoprotein I antibodies - prothrombin - β2-glycoprotein I - interference - classification

The presence of antiphospholipid antibodies (aPL) defines the antiphospholipid syndrome (APS).[1] The classification criteria act as surrogate markers for the diagnosis of APS, and are based on the combination of clinical symptoms and the presence of circulating aPL.[1] APS is an autoimmune disease with clinical symptoms characterized by thromboembolic complications and pregnancy morbidity, requiring long-term anticoagulant therapy.[1] [2] The primary form of the syndrome is defined in the absence of any underlying autoimmune disease, whereas the secondary form is associated with another autoimmune disorder, especially systemic lupus erythematosus (SLE).[2] Both forms of APS usually do not show diagnostic or management differences. Besides the main clinical presentation of thrombosis and pregnancy morbidity, the syndrome may be associated with a variety of other clinical symptoms: thrombocytopenia, cardiac valve disease, renal, neurological, pulmonary and dermatological manifestations.[2] The aPL are not specific for APS and can be present in different clinical settings as well as being sometimes detected in healthy individuals or in patients with other autoimmune disease.[2]

The autoantibodies associated with APS, the aPL, include anticardiolipin antibodies (aCL), anti-β2-glycoprotein I antibodies (aβ2GPI), and lupus anticoagulant (LA).[1] [3] The first two groups, aCL and aβ2GPI antibodies, are measured by solid phase assays.[4] aCL are directed against cardiolipin, which is a phospholipid contained in cell membranes. aβ2GPI are directed against β2-glycoprotein 1 (β2GPI), a cardiolipin-binding cofactor of aPL. LA comprises a mixture of various aPL, which is detected by the prolongation of phospholipid-dependent coagulation tests.[3] [5] Measurement of aCL and aβ2GPI results in an antibody titer, while LA testing is reported with a final conclusion as positive or negative, interpreted toward local cut-off values of the clotting times, depending on antibody characteristics and reagents used.[4] [5]

The Sydney criteria were intended to be classification criteria for clinical trials and studies to identify patients with high probability of APS. Meanwhile, the laboratory criteria are now used as diagnostic criteria as well.[1] These laboratory criteria were further defined over the past years, and are still subject of discussion and regular updates.[3] [6] The diagnosis of APS remains a challenge from the clinical side, since clinical criteria of APS, vascular thrombosis, and pregnancy morbidity, are common in the general population, and often not triggered by the presence of aPL. Thus, the requirement for reliable aPL assays for the diagnosis of APS is high. Additionally, detection of aPL is more than a diagnostic tool since the type of aPL and the level define the risk of vascular and obstetric features in APS patients.[7] [8] In particular, patients positive for the three groups of aPL (LA, aCL, aβ2GPI) or double positive (aCL and aβ2GPI), and those with medium/high titer of aPL, have the highest risk for adverse events.[9] All assays currently used for aPL detection show shortcomings, making the laboratory diagnosis of APS a real challenge.[10] [11] [12] In this state-of-the-art article, insights on the clinical relevance and the interpretation of aPL will be described, with focus on aCL and aβ2GPI detected by solid phase assays.

Type of Assays

aCL and aβ2GPI are measured by solid phase assays, a technique frequently used to detect autoantibodies. It is important to understand the limitations of the various techniques as well as their clinical relevance. This implies insight into the sensitivity and specificity of these assays but also the relevance in the clinical context and the relevance of the antibody level.

Already in 1985, the first ELISA (enzyme linked immunosorbent assay) for aCL was developed and resulted in the first description of the so-called anticardiolipin syndrome, later named APS with persistent positivity of LA and aCL as necessary conditions.[13] In the 1990s it was illustrated that aCL activity in most APS patients depends on a co-factor protein identified as β2GPI.[13] In the revised Sapporo classification criteria, the Sydney criteria, the aβ2GPI were included as separate laboratory criterion.[1]

The currently commercially available aCL assays coat the solid phase with cardiolipin and β2GPI, which increases the specificity of the assay. The β2GPI dependency of the aCL assays is mandatory to avoid detection of non-cofactor dependent aCL associated with infection and drugs.[14] Limited data on the clinical relevance of β2GPI-independent aCL are available and β2GPI binding antibodies are regarded as a main player in the pathophysiology of APS.[2]

By the introduction of the aβ2GPI assay into the criteria, it was thought that these antibodies were more specific and more reliable for standardization, and the residual role of the aCL was then debated. Soon, it became clear that aβ2GPI assays also suffer from the same problems of standardization.[13] β2GPI is regarded as the main cofactor for aPL, and its pathogenic role has been illustrated in in vitro experiments and animal models.[2] However, the clinical evidence of aβ2GPI is not that strong, as illustrated in a recent review where the presence of aβ2GPI had only a weak independent association with thrombosis and was, at best, inconsistently associated with obstetric complications.[15]

The methodologically correct aCL assays show a comparable sensitivity and specificity to aβ2GPI assays and aCL and aβ2GPI levels are highly correlated.[14] [16] [17] [18]

aβ2GPI antibodies bind to β2GPI complexed with cardiolipin in cardiolipin coated plates, or directly to β2GPI in β2GPI coated plates. β2GPI-dependent antibodies are responsible for positive results in the two solid-phase assays, aCL and aβ2GPI assays, both laboratory classification criteria for APS.[1] [3]

This can raise the question why we need both aCL and aβ2GPI assays. First, a meta-analysis confirmed the role of aCL as a marker related with an increased risk of thrombosis.[19] Second, in APS patients we expect no single positive result for one or the other assay. aCL measurement is of help in confirming the positivity of aβ2GPI of the same isotype, and vice versa. Positive aCL in the absence of aβ2GPI should be interpreted with caution. False positive and false negative results vary between assays and depend on solid phase assay conditions from different manufacturers.[18] Isolated aCL are usually not associated with APS-related clinical symptoms. Due to variability in immunological assays,[18] checking with another type of aβ2GPI assay can be useful to exclude or confirm the β2GPI dependence of the aCL. If the aβ2GPI are negative, the aCL antibodies are either co-factor independent or bind through cofactors different from β2GPI with unknown significance.[3] Positive aβ2GPI with a negative aCL may include aβ2GPI directed against domain four or five of the protein. These antibodies are usually positive in the aβ2GPI assays that are coated with the whole β2GPI molecule, and give negative result for aCL.[20] Antibodies against domain four or five of the β2GPI are regarded non-pathogenic, compared with the antibodies against the domain one of β2GPI (aDI) that are well correlated with thrombosis and obstetric complications.[21] [22] Regarding the assay limitations with possible discrepancies in aCL and aβ2GPI positivity, the current laboratory criteria with allowance for only one positive laboratory criterion sufficient for diagnosis of APS, may be questioned. To overcome over-diagnosis based on single positivity of aCL or aβ2GPI, both aCL and β2GPI have diagnostic value.

Traditionally, aCL and aβ2GPI are detected by ELISA[1]; recently, however, automated platforms with variations of the solid phase (e.g., magnetic microparticles, microspheres) and various detection systems (e.g., chemiluminescence, flow cytometry, multiplex systems) have been introduced into the market.[16] [17] [18] Automated systems have the advantage of the working conditions being more harmonized, providing a strict protocol on how to perform the assay, which may reduce the inter-laboratory variation for laboratories using the same system.[23] In general, ELISAs have shown large inter-laboratory variation and limited consensus in external quality control programs.[24] Head-to-head comparison of different platforms show comparable sensitivity and specificity for ELISA and automated systems[18] and correlation of titers between automated solid phase assays and ELISA is good.[17] [18] The automated platforms perform aCL and aβ2GPI IgG/IgM assays more rapidly and in a less labor-intensive fashion, providing up to four results at once instead of running multiple ELISAs. However, the availability and regulatory approval of automated systems requiring specific instrumentation may be a drawback for some geographical regions.

Interferences

Interfering factors of solid phase assays may include rheumatoid factor (RF), or substances (hemoglobin, bilirubin, triglycerides) interfering with the absorbance signal measured to determine the antibody level, which may relate to specific methodology. Manufacturers should indicate the level of IgM RF as well as the concentrations of hemoglobin/bilirubin/triglycerides and other interfering factors that may bias results.[4]

In contrast to LA detection that is prone to interference with the phospholipid-dependent coagulation tests, aCL and aβ2GPI solid phase assays do not suffer from confounding effects of anticoagulants or acute phase proteins that may otherwise interfere with results.[5] Although LA is an important laboratory criterion having a strong correlation with thrombosis and obstetric complications, the solid phase assays have the advantage not giving false positive results when measured during anticoagulation therapy.[25]

The phospholipid-dependent tests used to detect LA (the activated partial thromboplastin time, dilute Russel viper venom test, or silica clotting time) are prolonged by most anticoagulants as well as by LA, thus making the interpretation of screening, mixing, and confirmation steps very difficult.[5] APS patients are candidates for long-term anticoagulation and require laboratory evaluation to assess the antibody profile. Despite that some strategies to overcome interference of anticoagulants exist (neutralization of anticoagulants, alternative tests), LA detection remains a challenge during anticoagulation therapy.[25] Although strongly discouraged to test in the acute phase or during anticoagulant therapy, many patients are referred for laboratory testing at the start of, and during, anticoagulation therapy.[5] [25] One of the reasons here being that the antibody profile, and presence versus absence of aPL, may influence the decision to discontinue or to extend anticoagulation duration, and may define the choice of anticoagulant since vitamin K antagonists are preferred over direct oral anticoagulants in triple positive patients.[26]

Agreement between Solid Phase Assays

Variability between aPL detecting assays is hypothesized to result from preanalytical, analytical, and postanalytical conditions, calibration and assay-specific issues.[12] Technical aspects on how to measure aPL with solid phase assays and how to interpret results are addressed in guidance documents[4] but cannot prevent existing differences amongst the large variety of commercial and in-house assays.

Best illustrated by external quality control programs,[24] [27] this inter-assay variation remains a major concern. In a real-world setting on a large cohort of APS and non-APS patient samples, we have recently shown that with commercially available solid phase assays, even with eliminating inter-laboratory and inter-operator variation, there is still variable detection of aCL and aβ2GPI IgG/M between platforms.[18] In this study, a discrepancy of 36, 60, 53, and 36% for aCL IgG, aCL IgM, aβ2GPI IgG, and aβ2GPI IgM positivity was observed, respectively. Detection of aCL IgG and aβ2GPI IgM resulted in the best agreement. Remarkably, discrepant samples displayed lower median aPL titers.[18] Nevertheless, these may impact on the identification of samples as negative or positive, where results are around the cut-off values. Despite the discrepancies in positive/negative results, this was not reflected in the clinical performance of the platforms by odd ratios for thrombosis and/or pregnancy morbidity considering the results of all four aPL together, being globally concordant among the tested platforms.[18] Therefore, if both IgG and IgM are needed, it is recommended to measure aCL and aβ2GPI IgG and IgM within the same platform.[18]

Nevertheless, detection of patients positive for aCL or aβ2GPI is assay dependent and this poor agreement between available platforms hampers a uniform classification of positive aCL/aβ2GPI antibodies. When clinical suspicion is high for APS, and results of aPL are not in line with what is expected, consideration of retesting with another type of solid phase platform can be useful. A sample assigned positive in one assay does not automatically test positive in the same type of assay from a different manufacturer or in another laboratory. Clinicians are not always aware of the sensitivity and specificity of laboratory tests. Test results should always be related to clinical symptoms and an interaction of the laboratory and clinician is mandatory.[11]

Besides differences in agreement (positivity vs. negativity), also large variation in titers has been described.[28] The differences in titer can be explained by the difference in calibration of the assays. Results of aCL and aβ2GPI are expressed in arbitrary units and derived from the assay-specific calibration curve converting the test signal into antibody units.[4] Manufacturers use a variety of calibrators, often using secondary standards and working calibrators.[10] [18] Ideally, these calibrators should be traceable to a primary standard. If aCL assays are calibrated against the Harris standards, results are expressed in GPL or MPL, 1 GPL or MPL referring to 1 μg of IgG or IgM antibody. No reference material is available for aβ2GPI, therefore, results are expressed in arbitrary units.[4]

Harris/Louisville standards and Koike monoclonal antibody standards were developed to introduce an international standard for the aCL assays. Patient-derived material such as Harris/Louisville standards is finite and may have batch-to-batch variation. In contrast, production of monoclonal antibodies is indefinite and reproducible over time, but have the disadvantage that these may not be representative for the reactivity of patient's polyclonal aPL, and they are not always positive in all aPL assays. The Koike standards are not commercially available anymore. Human monoclonal antibodies derived from APS patients can offer an alternative.[10] Recently, a patient derived reference material for aβ2GPI has been developed but is not available yet.[29] The lack of a “gold standard” reference material makes comparison of titers across different platforms very difficult.

Criteria for Positive and Negative Results

In the current Sydney classification criteria, only medium and high levels, i.e., greater than the 99th percentile or 40 units IgG or IgM for aCL and greater than the 99^th percentile for aβ2GPI, are regarded as relevant.[1] Patients with definite APS usually have values of aCL far exceeding 40 GPL.[30] Lower levels of antibodies are observed in certain clinical settings, especially pregnancy morbidity.[31] Awaiting more clinical studies and international consensus, the same threshold (99^th percentile) for aCL and aβ2GPI is recommended for patients with thrombosis and patients with obstetrical complications.

Although titers between systems correlate, the numerical values of the antibody titers may differ.[17] [18] [28] Titers of aCL and aβ2GPI measured with chemiluminescence techniques are much higher compared with ELISA.[28] The nonparametric 99^th percentile cutoff appears to be more specific than the >40 GPL/MPL value. Because of the variation in titers in different commercial kits, it is advised that each laboratory uses the 99^th percentile of a local normal population to determine the cutoff value, also for aCL.[4] However, as this requires in general a recommended minimum of 120 normal samples, some laboratories are challenged to do so. Nevertheless, it seems nearly impossible to recommend one numerical value (being >40 GPL/MPL[1]) as general criterion for positivity. So far, each test result above the cutoff should be regarded as positive and reported quantitatively along with the local cutoff value.[4] Calculation of an in-house cutoff value by the 99^th percentile should be done, and to obtain a reliable cutoff value at least 120 normal donors should be used.[4] [32] Since the high number of normal donors is not feasible for every laboratory, an alternative is the transference of the manufacturer's cutoff values, which is often applied for the solid phase assays.[4] Before transference of the manufacturer's cutoff values a verification should be performed on a cohort of 20 normal donors. Applying the CLSI C28-A3 guideline, manufacturer's cutoffs may be accepted if no more than two out of 20 tested normal values fall outside those of the manufacturer's reference range, if so, a second cohort of 20 normals should be tested.[33] If more than 10% of the normal values are outside the manufacturer's reference range, cutoff values should be calculated in-house. In addition, transference of manufacturer's cutoff values assumes that these cutoffs are established by appropriate statistical models using a sufficiently large donor population. Of note, most manufacturers suggest in the package insert that in-house cutoff values should be calculated.

Medium and high aPL titers are considered to be more strongly correlated with clinical outcomes of APS than low titers. Increasing likelihood ratios (LR) with increasing autoantibody titers has already been reported for other autoimmune diseases.[34] Increasing LR for thrombotic and obstetric APS based for aCL IgG was observed with increasing titers. The same trend was observed in the thrombotic test population for aβ2GPI IgG, but less pronounced in the obstetric test population. For aCL IgM and aβ2GPI IgM no prominent difference in LR was observed for diagnosis of thrombotic or obstetric APS.[35]

This raises the issue of classification into low-medium-high positive. Qualitative reporting of results categorizing aCL and aβ2GPI IgG/M titers in low, medium, and high titers could increase harmonization of interpretation and therefore, could be very useful for the clinician.[8] [11] Even more if qualitative gradation of results is interchangeable between different systems. An international multi-disciplinary initiative has been started to develop new classification criteria to identify patients with high likelihood of APS for research purposes, and recognize the difference in semiquantitative reporting between solid phase platforms.[6] So far, no standardized method to define these ranges is available. Reports using gradation of positivity by defining intervals are rare,[36] and did not sufficiently investigate whether this can be applied to different solid phase systems. A recent study investigated whether traditionally established aCL and aβ2GPI thresholds of 40 and 80 MPL/GPL are appropriate to categorize results as moderate and high titers for ELISA and automated solid phase platforms.[35] Agreement for the 40/80 threshold and otherwise defined thresholds for low, medium, and high categorization were studied. Threshold levels of 40/80 units showed poor agreement between ELISA and automated platforms for classification into low-medium-high positivity, especially for aCL and aβ2GPI IgG. Use of 40/80 units as medium-high thresholds was acceptable for aCL and aβ2GPI IgG ELISA, confirming the Sydney criteria that were based on standardized ELISA techniques.[1] Semiquantitative reporting of aCL and aβ2GPI IgM had less impact on increasing probability for APS, for both ELISA and automated systems. The use of 40 and 80 units as medium and high thresholds is acceptable for aCL IgG and aβ2GPI IgG ELISA but cannot be applied to analytical solid phase platforms with chemiluminescence and multiplex flow immunoassay methodology. Agreement for semiquantitative interpretation of aPL IgG between ELISA and chemiluminescence and multiplex flow immunoassay techniques improved by defining thresholds based on receiver operator curves. However, this is difficult and not feasible for many laboratories. A more accessible method to define platform-specific thresholds, is the calculation of thresholds following comparison of parallel measurement of monoclonal antibodies by an automated solid phase platform and ELISA.[35]

Isotype of aCL and aβ2GPI

For both aCL and aβ2GPI, IgG and IgM antibodies are included in the current criteria.[1] [3] IgG aCL and aβ2GPI show a stronger association with clinical events and are often associated with IgM positivity, as illustrated in a multicenter study and two recent reviews.[37] [38] [39] This multicenter study comparing four solid phase platforms demonstrated that this was independent of the platform used.[37] Isolated aCL or aβ2GPI IgM is rare in thrombotic APS and more frequent in obstetric APS. Although, in contrast to thrombotic complications, in pregnancy morbidity aCL and aβ2GPI IgM are independent risk factors. Data of this multicenter study support testing for IgG and IgM, especially in women suspected of obstetric APS, aCL and aβ2GPI IgM have an added value for diagnosis. In thrombotic patients suspected of APS, first line testing for IgM has no added value for diagnosis, but can be used in risk stratification since positivity of IgM, on top of IgG and LA, increases the risk.[37] A stepped approach starting with aCL and aβ2GPI IgG, and if positive also performing IgM, can be a good option to estimate the risk profile. For non-criteria clinical manifestations, the association is mainly shown for LA and aCL IgG, except for thrombocytopenia that is also significantly associated with IgM.[40] [41] [42] Several studies confirmed that the presence of aCL and aβ2GPI of the same isotype reinforces the clinical probability of APS.[30] [43] [44]

IgA aCL and aβ2GPI are not included in the current criteria for APS.[1] [3] Many studies have illustrated the association between APS-related clinical symptoms and the presence of aCL/aβ2GPI IgA, especially in SLE.[45] However, there is no strong evidence of the added value of IgA aPL[46] [47] since isolated IgA is very rare, and IgA aPL are usually found in association with IgG and/or IgM. The clinical significance is unknown as IgA aPL are in most cases linked to non-criteria clinical manifestations of APS.[8] [46] [47] [48] So far, there is not enough evidence to recommend testing for IgA aCL and/or IgA aβ2GPI to increase the diagnostic accuracy of APS. aPL are included in the classification criteria for SLE, including IgG, IgM and IgA aCL and aβ2GPI. Overall, aPL have a low relative weight in the classification criteria for SLE, but no difference is made between the isotype.[49]

Other Antiphospholipid Antibodies

Amongst the “non criteria” aPL, anti-phosphatidylserine/prothrombin antibodies (aPS/PT) and antibodies toward the domain one of β2GPI (aDI) have been most frequently studied.

aDI antibodies are a subgroup of aβ2GPI antibodies, directed against the domain one of the protein β2GPI that consists of five domains. A high association with thrombosis has been shown for antibodies recognizing a specific epitope (G40-R43) within the domain one of β2GPI.[50] However, the different methods to detect aDI IgG vary in specificity for this subgroup of antibodies.[51] A commercial chemiluminescence-based assay (only available for IgG) has been evaluated in many studies with various results regarding association with thrombosis and pregnancy morbidity.[51] The original in-house ELISA test measured a more specific population of aDI directed against the G40-R43 epitope, compared with the commercial aDI probably measuring all aDI antibodies against any epitope on domain one of β2GPI.[22] No higher risk association for thrombosis was found when aDI was added to the current aPL panel or when aβ2GPI was replaced by aDI.[22] [52] The absence of an added value may result because of reduced exposure of the epitope G40-R43 in the chemiluminescence assay.[51] One recent study indicated aDI as an independent risk factor.[53]

Clinical studies have illustrated that aDI are frequently present in triple positive patients (positivity for LA, aCL, and aβ2GPI) known as high-risk patients. Titers of aDI in these patient groups are higher compared with double positive patients (positive for aCL and aβ2GPI).[22] [54] [55] [56] The high correlation between aDI and triple positivity in obstetric and thrombotic patients, confirm the higher risk for clinical events in APS.[22] [54] However, there is not 100% overlap with aβ2GPI and up to 20% of the patients positive for antibodies against the whole β2GPI molecule can test negative for specific aDI, depending on the assay used for aβ2GPI.[22] [57] Instead of being a replacement of aβ2GPI, aDI can be used as a confirmatory test and is useful for proving the specificity of the aβ2GPI antibodies. Especially in patients with an incomplete antibody profile defined as double positive patients (aCL and aβ2GPI positive) or single LA or single aβ2GPI positive patients, aDI may confirm the risk. Testing for aDI in these patients could confirm or exclude the association of pathogenic aβ2GPI autoantibodies.[58]

aPS/PT antibodies seem to represent a strong risk factor for thrombosis, both arterial and/or venous.[59] Persistently positivity for aPS/PT has been shown in patients and asymptomatic carriers with triple positivity, a marker of clinical severity, making aPS/PT effective as part of risk scoring.[60] [61] Equal as for aDI, in triple positive patients, titers of aPS/PT are higher than in double or single positive patients.[60] [62] [63] aPS/PT IgG are frequently present in APS patients.[61] [64] aPS/PT of isotype IgM are frequently present in isolated LA (non-APS) patients, as well as in asymptomatic carriers.[61] [62] [65] aPS/PT IgG and IgM are more frequently found in triple positive patients compared with single LA positive patients.[63]

aPS/PT antibodies might be a surrogate for LA because of their strong correlation with LA,[61] [65] [66] particular in conditions where LA assays show methodological shortcomings as in anticoagulated patients, this might be helpful.[58] [67] However, not all single LA positive patients are aPS/PT positive.[63] [65] To replace LA with a suitable and similarly predictive marker that can be measured with an immunological method needs further investigation.

Similarly, as for aCL and aβ2GPI antibodies, a standardized ELISA for aPS/PT is not available and reference sera are lacking. Automated systems are not available yet, and a limited number of commercial ELISAs for aPS/PT is available. A comparison of a homemade and a commercial assay showed reproducible and accurate results.[68] Recently reported, a newly developed ELISA looks promising to detect pathogenic antiprothrombin antibodies by coating the ELISA plate with a novel prothrombin variant.[69] Although searching for aPS/PT in daily practice is not recommended yet, in some conditions aPS/PT might help, especially in confirming the risk. Adding aPS/PT to the triple positive profile, further consolidates the diagnosis of APS. When the aPL profile shows double positivity, positive aPS/PT may suggest a false negative LA, and when aDI and aPS/PT are negative, this indicates a lower risk for thromboembolic events.[58]

[Table 1] summarizes some of the potential uses of the non-criteria aPL tests.

Table 1 Use of non-criteria aPL tests

1. Scope of non-criteria aPL

aPL other than aCL and aβ2GPI are not included in the laboratory criteria for APS  • aPS/PT antibodies correlate with clinical symptoms of APS, are strongly associated with LA and frequently present in APS patients.  • aDI β2GPI IgG antibodies correlate with clinical symptoms of APS and are associated with triple positive high-risk population.  • aPS/PT and aDI β2GPI IgG antibodies confirm patients at risk.

2. Role of aPS/PT and aDI

• In triple positive patients (LA, aCL and aβ2GPI), positive aPS/PT consolidates the diagnosis of APS.  • In double positive patients (aCL and aβ2GPI), positive aPS/PT may indicate a false negative LA.  • Presence of aDI may indicate the presence of pathogenic aβ2GPI antibodies. When aDI and aPS/PT are negative, this indicates a lower risk for thromboembolic events.  • If the only positive test is aβGPI antibodies aDI and anti-domain four or five (4/5) antibodies (if available) should be analyzed; anti-domain 4/5 antibodies are not associated with thromboembolic events.  • aPS/PT may be used in isolated LA patients to confirm the single LA positivity.

Abbreviations: aPL, antiphospholipid antibodies; APS, antiphospholipid syndrome; aCL, anticardiolipin antibodies; aβ2GPI, anti-β2-glycoprotein 1 antibodies; aPS/PT, anti-phosphatidylserine/prothrombin antibodies; aDI, antibodies toward the domain one of β2GPI (aDI).

Combination of aPL

Although it is sufficient to have one aPL positive to define or diagnose APS, guidelines strongly advise to classify APS patients into categories according to type and number of tests positive.[1] [3] To assess the risk profile, results of all three groups of aPL, LA, aCL, and aβ2GPI should always be interpreted together.[3] [4] [5] Therefore, all three tests should be performed preferable on the same sample. LA testing with coagulation assays and aCL/aβ2GPI testing with solid phase assays tend to be performed in different laboratory departments: the hemostasis/hematology laboratory and the immunology/ biochemistry laboratory. Consequently, most of the laboratories use serum to perform solid phase assays.[10] Both serum and citrated plasma can be used for aCL and aβ2GPI, on the condition that assay specifications (including cutoff values) are validated for the corresponding sample type.[4]

Combined positivity for LA, aCL, and aβ2GPI antibodies (i.e., triple positivity) has been shown to be associated with a high risk of both a first event and recurrence, and for a first event in asymptomatic carriers.[9] [43] [70] [71] [72] Double or triple positivity for aPL is a risk factor for future thrombotic events, especially in individuals with an underlying autoimmune disease, whereas single positivity does not seem to carry an elevated risk of thrombosis.[71] Compared with triple positives, the risk in double positives (aCL and aβ2GPI) is slightly lower.[71] Triple positive patients usually have a persistent antibody profile on follow-up testing after 12 weeks.[73] [74] But also, double and single positive patients may be persistent positive, as illustrated in a retrospective study illustrating no significant lower persistence in the single positive patients (93.3%) compared with the double and triple positive patients (96.8 and 97.3%, respectively).[74]

Isolated aCL and aβ2GPI are regarded of less clinical relevance, either because of the non-cofactor characteristics of aCL or aβ2GPI directed against epitopes not determining LA activity.[14] [20] [75]

If inconsistencies are observed between aCL and aβ2GPI results, retesting with another type of solid phase assay may be helpful when clinical suspicion is high for APS, as well as testing for aPS/PT and aDI (see above).

Repeat Testing and Reporting of Results

If a test is positive on a first occasion, testing should be repeated after 12 weeks to confirm the persistence, as aPL are persistent over time according to the classification criteria.[1] [3] Transient positive results are usually found in association with infections and drugs[76] [77] and are thought not to be of clinical significance, therefore re-testing was originally meant to avoid over-diagnosis of APS patients who were not persistently positive.[3] Viruses can induce autoimmune diseases, in addition to genetic predisposition and environmental factors, including formation of transient aPL. Particularly, coronaviruses are mentioned among the viruses implicated in autoimmunity. Since the first description of a COVID-19 infected patient who was positive for aPL, many case reports, case series, and cross-sectional studies were published describing the association of aPL and COVID-19 infection. Interestingly, a high prevalence of aCL and aβ2GPI IgA has been described. However, few studies investigated the persistence over time of the aPL, and the role in thrombotic events was not consistent in published literature.[78] The type of aPL as well as the prevalence of different aPL over time vary, possibly linked to the inflammatory phase of the disease. Routine screening for aPL in COVID-19 patients is not recommended, as their pathogenic role in thrombosis in these patients is still unclear, although possible. Therefore, testing for aPL in COVID-19 patients with thrombosis or underlying autoimmune conditions can be performed, as recommended in the general guidelines for testing for aPL.[5] The presence of type and persistency of aPL would change the anticoagulant treatment decisions, as it is in other thrombotic patients with aPL.[8] Also in these patients, it is mandatory to confirm the identified aPL after 3 months whenever possible.

Limited information is available on antibody titer fluctuation over time. Decrease in levels of aCL over time has been described in association with belimumab (anti B-cell therapy), and decrease in aCL and aβ2GPI levels in patients treated with hydroxychloroquine (antimalarial).[79] [80] There is little information on the value or therapeutic consequences of retesting persistently positive patients annually. This could be potentially useful as part of risk assessment related to fluctuation of titers or change of antibody profile over long-term period.[5]

Results of aCL and aβ2GPI around the cutoff value should be interpreted with care and preferably repeated. Imprecision of the solid phase method should be considered before classifying a sample as positive or negative, as an imprecision of 10% is acceptable, this implies that a 10% difference around a value near the cutoff value may influence the classification as positive or negative.[4]

Repeat testing of aCL and aβ2GPI, as well as LA, is necessary to confirm positivity over time, in the context of patients with clinical criteria of APS, to manage these patients adequately as APS patients regarding anticoagulation therapy.[3] [8] As described above, aCL and aβ2GPI are also part of the classification criteria of SLE, patients often presenting without the clinical features of APS.[49] Classification criteria for SLE do not specify that aPL should be persistent positive. Although, to identify SLE patients at risk for thrombosis, it was demonstrated that repeat testing with persistent positivity is associated with a higher risk, as demonstrated for LA, an observation that can be probably extrapolated to aCL and aβ2GPI.

The report on aPL testing should include the recommendation of repeat testing after 12 weeks. Overall, a report with an explanation and an integrated interpretation on all aPL results should be provided. Therefore, a close interaction between the clinical pathologist (laboratory expert) and the clinician is necessary.[11] If insufficient clinical information is available, this should be mentioned on the report possibly hampering a correct interpretation of results. In daily practice, based on the clinical suspicion of APS, clinicians request testing for aPL. However, they may not be aware of the limitations of the tests used for measurement of aPL and the difficulties in interpretation. On the other hand, the clinical pathologist in the laboratory may not be aware of the clinical symptoms or the use of anticoagulants, the latter extremely important for the interpretation of LA. Information on the anticoagulation status and the clinical symptoms of the patient is mandatory for good interpretation of results and should be provided. In addition to the repeat testing after 12 weeks, retesting should be suggested on the report, depending on the clinical symptoms (criteria vs. non-criteria, place and type of thrombosis, underlying disease, aPL test results not in line with the clinical picture) and the demographic characteristics of the patient (age, other thrombotic risk factors, possible interference of anticoagulant therapy), the antibody profile (all three groups of aPL should be considered, in incomplete profiles aCL/aβ2GPI could be retested with another assay), discrepancies in aCL/aβ2GPI (disagreement in positivity of aCL/aβ2GPI or isotype), borderline positive or negative titer of aCL/aβ2GPI, and possible analytical interferences.

Besides discussion of the individual results of LA, aCL, and aβ2GPI in view of the local cutoff values stated in the report, finally, an overall conclusion should be made considering LA, aCL, and aβ2GPI results.

Conclusion

The detection of aPL by solid phase assays is an essential step in the diagnosis of APS. The laboratory diagnosis of APS remains challenging. Progress has been made to address some of the methodological challenges of aCL and aβ2GPI assays and in better understanding their diagnostic role. Assays for all three groups of aPL, LA, β2GPI-dependent aCL, aβ2GPI IgG and IgM should be performed to increase diagnostic utility, with an integrated interpretation of all results. Positivity should be confirmed on a second occasion after 12 weeks. In the interpretation of aPL results, antibody profiles help identify patients at risk. All assays must be performed according to the guidelines and only high titers (>99^th percentile) should be considered. Differences in agreement between solid phase assays remain, and differences in titer hamper the semiquantitative classification into low-medium-high positivity.

The variety of available solid phase assays on the market may complicate the decision of what solid phase assays to implement in the laboratory. The selection of the assays should rely on some important minimal performance characteristics. Imprecision can be assessed by internal quality control material or patient samples, and should be <10%.[4] As no golden standard assay exists, the new assay should be validated from an analytical and a clinical point of view. Agreement between assays should be explored as well as the clinical performance assessing the association of an assay with thrombotic or pregnancy complications.[10] Since an extensive clinical validation is not feasible for all laboratories, published reports in peer reviewed journals may help, critically considering the study design. Consulting external quality assessment exercise reports before decision making, may demonstrate what the imprecision of the assays is, and how assays perform. A laboratory with less experienced laboratory technologists may benefit from an automated system instead of ELISA, having more harmonized protocols and being easier to perform. An alternative can be sending the samples to a centralized fully equipped laboratory with experience in aPL testing. Equally, if laboratories are not able to measure all aPL, this issue can be resolved by sending samples to a reference laboratory performing all aPL on the same sample, as well as offering the analysis of the non-criteria aPL, of value in some special circumstances. Once implemented, laboratories should include internal quality control material (commercial or patient based) for their assays and should participate at external quality assessment exercises. By participating in such schemes, laboratories can assess their performance against those of the peers.

The non-criteria aPL, such as antibodies against the domain one of β2GPI and aPS/PT antibodies have their role in confirming the risk in APS, and can be useful, especially in patients with incomplete antibody profiles.

[Table 2] summarizes the key messages on testing for aPL with solid phase assays.

Table 2 Key messages on measurement of aCL and aβ2GPI

1. Scope of aCL and aβ2GPI

• aCL and aβ2GPI IgG and IgM are two out of the three classification criteria for APS, both measured with solid phase assays.   ○ Identification of all three groups of aPL (LA, aCL, aβ2GPI) enables risk stratification.   ○ Triple positive patients (LA, aCL and aβ2GPI positive) and double positive patients (aCL and aβ2GPI positive) are at high risk to develop clinical symptoms associated with APS.   ○ Medium and high aPL IgG titers are more strongly correlated with clinical outcomes of APS than low titers. IgM aPL titer correlates less with clinical outcome.   ○ IgM aCL and/or aβ2GPI are significantly correlated with thrombosis and pregnancy morbidity, but higher odd ratios are obtained for IgG compared with IgM positivity.   ○ Presence of aCL and aβ2GPI of the same isotype reinforces the clinical probability of APS.   ○ Isolated aCL/aβ2GPI IgM is rare in thrombotic APS and is not an independent risk factor. IgM positivity on top of LA and IgG increases the risk.   ○ Isolated aCL/aβ2GPI IgM is an independent risk factor for obstetric APS.   ○ All three parameters (LA, aCL, aβ2GPI) should be performed at once on the same sample.

2. Analytical aspects for aCL and aβ2GPI

• ELISA or automated systems with difference in solid phase or detection method can be used.  • A β2-dependent aCL assay is mandatory to avoid detection of non-cofactor dependent aCL.  • aCL and aβ2GPI correlate and show comparable sensitivity and specificity for diagnosis of APS.  • If aßGPI are negative and aCL positive, then aCL are antibodies recognizing proteins different from β2GPI, whose significance is not known.  • Detection of aCL and aβ2GPI IgG/M varies between test platforms.  • Numerical values vary between test platforms, hampering a uniform semiquantitative classification into low-medium-high titers. Local cutoff values (> 99th percentile) are recommended.  • It is recommended to measure aCL and aβ2GPI IgG and IgM within the same platform.  • When clinical suspicion is high for APS, and results of aPL are not in line with what is expected, consideration of retesting with another type of solid phase platform can be useful.  • In single aCL, single aβ2GPI or single LA positivity retesting of aCL and aβ2GPI is advised, with consideration of another type of solid phase platform.  • Imprecision of the method of solid phase assays should be considered, especially for results around the cut-off.  • Solid phase assays for aCL and aβ2GPI are not prone to interference of anticoagulant therapy, but some interfering substances have been described, such as rheumatic factor, bilirubin, hemoglobin or triglycerides.

3. Interpretation and report of results

• aCL and aβ2GPI assays should be interpreted together with coagulation LA tests to assess the clinical significance.  • Individual results of aCL IgG/IgM and aβ2GPI IgG/IgM and the antibody profile should be discussed.  • Results are interpreted according to the local cutoff values stated in the report. aCL and aβ2GPI are reported with their titer. Each result above the local cut-off should be considered as positive.  • Report with an explanation of the results should be given and interpretive comments should be included (e.g., positive results of aCL or aβ2GPI need to be confirmed on a second occasion after 12 wk to confirm persistent positivity).  • To prevent misdiagnosis the diagnostic workup for APS requires collaboration between the clinician and the laboratory.

Abbreviations: aCL, anticardiolipin antibodies; aDI, antibodies toward the domain one of β2GPI (aDI); aPL, antiphospholipid antibodies; aPS/PT, anti-phosphatidylserine/prothrombin antibodies; APS, antiphospholipid syndrome; aβ2GPI: anti-β2-glycoprotein 1 antibodies; LA, lupus anticoagulant.

Conflict of Interest

None declared.

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Publication History

Article published online:
08 June 2022