Challenges of laboratory testing in patients suspected of antiphospholipid syndrome: practical implications for clinicians
Key words: anti-β2 glycoprotein I antibodies, anticardiolipin antibodies, antiphospholipid syndrome, interpretation, lupus anticoagulant
CC BY-NC-SA 4.0
Challenges of laboratory testing in patients suspected of antiphospholipid syndrome: practical implications for clinicians
CC BY-NC-SA 4.0
This paper focuses on laboratory tests necessary for a diagnosis of antiphospholipid syndrome (APS). The diagnosis starts with a selection of patients suspected of APS. Timing related to a clinical event is important to avoid false classification of APS patients. To correctly interpret the test results for antiphospholipid antibodies (aPL), it is necessary to understand all possible pitfalls and interferences. Lupus anticoagulant (LA) measurement remains a complex procedure with many challenges. The effect of anticoagulant therapy, the main confounder of LA measurement, can be overcome by removal agents for direct oral anticoagulants (DOACs) or by considering assays such as Taipan snake venom time / Ecarin clotting time not affected by antivitamin K therapy and anti‑Xa DOACs. However, both procedures have limitations. Solid‑phase assays for anticardiolipin antibodies (aCL) and anti-β2 glycoprotein I antibodies (aβ2GPI) show interassay differences. Diagnosis is based on the measurement of 3 groups of aPL: LA, aCL, and aβ2GPI, of immunoglobulin (Ig) G and IgM isotype. This allows the clinicians for developing antibody profiles that help identify patients at risk. Other aPL, such as antibodies against domain I of β2GPI and anti‑phosphatidylserine / prothrombin antibodies may be useful in risk stratification of APS patients, and in some specific situations of patients with an incomplete antibody profile, but are not needed for diagnosis. Laboratory diagnosis of APS remains challenging. To increase the diagnostic efficacy and reliability, an integrated interpretation of all results and an interpretative comment should be provided on the laboratory report. Therefore, a close interaction between clinical pathologists and clinicians is mandatory.
Introduction
Antiphospholipid syndrome (APS), also known as Hughes syndrome, is an autoimmune disorder in which the immune system mistakenly produces antibodies that increase the risk of blood clots. Nearly 35 years after APS was first described, our understanding of this complex disorder continues to evolve. Originally identified as an acquired autoimmune thrombophilia, APS is now known to involve mechanisms beyond coagulation‑mediated thrombosis. For example, complement activation might contribute to pregnancy complications.
The presence of specific autoantibodies is characteristic of APS. These antibodies, known as antiphospholipid antibodies (aPL), target blood proteins bound to phospholipids, essential components of the cell membranes. aPL include anticardiolipin antibodies (aCL), anti-β2 glycoprotein I antibodies (aβ2GPI), and lupus anticoagulant (LA). aCL target cardiolipin, a phospholipid found in the cell membranes, while aβ2GPI antibodies are directed against β2GPI, a protein that binds to cardiolipin. LA is a pool of autoantibodies detected by prolonging phospholipid‑dependent coagulation tests.1
APS can result in the formation of blood clots in both arteries and veins, causing serious health issues, such as deep vein thrombosis (DVT), pulmonary embolism (PE), stroke, or myocardial infarction (MI). In women, APS also increases the risk of pregnancy complications, including recurrent miscarriages, stillbirths, preterm delivery, and preeclampsia. Some people with APS have a low platelet count (thrombocytopenia), which can heighten the risk of bleeding. Other symptoms of APS may include heart valve disease or livedo reticularis. Different pathophysiological mechanisms are responsible for the obstetric and thrombotic complications in APS.1-4 While the thrombogenic properties of aPL are a main concern in thrombotic APS (TAPS), obstetric complications seem to depend more on a wide set of proinflammatory properties of aPL. Depending on the predominating clinical symptoms, 3 entities can be identified: TAPS, obstetric APS, and catastrophic APS (CAPS). CAPS is a rare presentation of APS and accounts for approximately 1% of APS patients. It is a lethal systemic disease characterized by multiple organ failure progressing within an extremely short period.5,6 APS is a complication in up to one‑third of patients with systemic lupus erythematosus (SLE).7 The stand‑alone presentation of APS, primary APS, is more common and occurs in the absence of other systemic autoimmune disorders.1 The exact cause of APS is unknown, but it is believed to involve a combination of genetic and environmental factors. Certain infections, medications, or other autoimmune conditions may trigger the production of aPL.1
The diagnosis of APS is based on a combination of clinical criteria, thrombotic disorders and pregnancy complications, and laboratory tests that detect aPL. The classification and diagnostic criteria include LA, aCL, and aβ2GPI immunoglobulin (Ig) G or IgM, if persistently present at least 12 weeks apart. A patient is classified as an APS patient, if at least 1 clinical criterion and 1 laboratory criterion are met.8-10
The primary aim of treatment for APS is to lower the risk of blood clots, and this is typically accomplished through the use of anticoagulants, even though there is currently no cure for the disease. In patients with milder forms of APS, or those at risk of pregnancy complications, a low dose of aspirin may be recommended. With appropriate treatment, APS symptoms can be managed in many patients, and the risk of complications is reduced.11,12 However, APS can be a serious condition with recurrent events, and it can be associated with other autoimmune diseases, specifically SLE. Regular monitoring and adherence to treatment are crucial for individuals with APS to maintain their health and prevent complications.12
The occurrence of thrombosis and pregnancy complications is frequently influenced by underlying factors not associated with aPL. Therefore, an accurate laboratory diagnosis, specifically detection of aPL, is essential for diagnosing APS. It is crucial to ensure an accurate diagnosis, as both over- and underdiagnosis can significantly impact therapy and the assessment of thrombotic risk and recurrence of thrombosis.11 Moreover, laboratory parameters are very important, since the type and level of aPL determine the risk in APS patients.8,9,13-18 To detect these antibodies, 2 types of laboratory tests are used: clotting assays for LA and immunoassays for aCL and aβ2GPI. We need assays with good diagnostic power for identifying aPL. However, the laboratory diagnosis is challenging. Multiple tests are necessary but assay standardization and harmonization of reporting results are lacking. Besides the analytical concerns, the interpretation of the laboratory results can be difficult.19
In clinical practice, the indication for testing is the choice of the clinicians based on clinical suspicion of APS. However, there is a significant heterogeneity in the clinical presentation of APS patients, and the occurrence of thrombosis and pregnancy complications can have many other causes.9 Consequently, the decision which patients to test is a challenge. Also, a clinician may not be fully aware of the limitations of the tests used for aPL measurement. On the other hand, a clinical pathologist in the laboratory may not know the patient’s clinical condition and potential interfering factors, such as the use of anticoagulants. It is essential to provide information on the patient anticoagulation status and clinical symptoms for accurate result interpretation. It is crucial to emphasize that preventing misdiagnosis of APS requires close collaboration between clinicians and laboratories. By integrating good laboratory practices with good clinical practices, we can significantly enhance the accuracy of APS diagnosis.
In this paper, we highlight the diagnostic challenges of APS by focusing on the laboratory tests necessary for the diagnosis. For the interpretation of the test results for aPL, understanding all pitfalls and interferences is necessary.
Who to test for antiphospholipid antibodies
Testing for aPL is advised in patients with clinical manifestations suggestive of APS and those with systemic autoimmune diseases in which aPL have a high prevalence, such as SLE.8,9,20 However, large‑scale testing for aPL is not advised in the populations with a low prevalence of APS, for example, elderly patients, to avoid incidental findings, false‑positive tests, or transient antibodies.7 Table 1 lists clinical situations demanding aPL testing.
a Testing for aPL to be considered in younger patients (<50 years) with unprovoked VTE, recurrent VTE, and VTE at unusual sites
b Testing for aPL to be considered in younger patients (<50 years), especially without a high‑risk profile according to cardiovascular risk factors
c Microvascular thromboses include documented livedoid vasculopathy, acute or chronic aPL nephropathy, pulmonary hemorrhage (pulmonary capillaritis and recurrent microvascular thrombosis leading to alveolar hemorrhage), myocardial disease (MINOCA diagnosed on histopathology or magnetic resonance imaging), adrenal hemorrhage (due to adrenal venous infarction diagnosed on imaging)
d Requires emergency testing for aPL in patients without previous APS diagnosis
e Positive aPL with a specific definition for SLE: positive LA or positive aPL based on ELISA for aCL (IgA, IgG, or IgM) at medium or high titer (>40 APL, GPL, or MPL, or >99th percentile) and positive aβ2GPI antibodies (IgA, IgG, or IgM, no threshold indicated)
Abbreviations: aβ2GPI, anti-β2 glycoprotein I antibodies; aCL, anticardiolipin antibodies; aPL, antiphospholipid antibodies; APL, immunoglobulin A phospholipid, APS, antiphospholipid syndrome; ELISA, enzyme‑linked immunosorbent assay; GPL, immunoglobulin G phospholipid; Ig, immunoglobulin; LA, lupus anticoagulant; MINOCA, myocardial infarction with no obstructive coronary arteries; MPL, immunoglobulin M phospholipid; SLE, systemic lupus erythematosus; VTE, venous thromboembolism |
Thrombosis |
Venous thromboembolism (identify risk factors to stratify provoked or unprovoked)a |
|
Arterial thrombosisb |
|
Both venous thromboembolism and arterial thrombosis in the same patient (simultaneous or within a short period of time <1 month), after exclusion of patent foramen ovale |
Microvascular thrombosisc |
Suspected catastrophic APSd |
Pregnancy morbidity |
Prefetal or fetal deaths: 3 or more consecutive prefetal (<10 weeks) and / or early (10–16 weeks) fetal deaths, or at least 1 fetal death (16–34 weeks) alone |
Preeclampsia with severe features OR placental insufficiency with severe features (<34 weeks) with or without fetal death or premature birth of a normal neonate before 34th week of gestation |
Preeclampsia with severe features AND placental insufficiency with severe features (<34 weeks) with or without fetal death or premature birth of a normal neonate before the 34th week of gestation |
Heart valve disease |
Cardiac valve thickening |
Vegetation |
Thrombocytopenia |
20–130 × 109/l |
Systemic lupus erythematosuse |
Suspected antiphospholipid syndrome
In patients with suspected APS, aPL testing should include LA, aCL, and aβ2GPI, to diagnose APS (laboratory domain of classification criteria) and to assess the risk profile for future aPL‑related events.9
The most frequent thrombotic manifestations of APS are venous thromboses (DVT and PE). Risk factors should be carefully analyzed to identify provoked venous thromboembolism (VTE), that is, with major risk factors, and unprovoked VTE.21 Testing for aPL is all the more indicated in younger patients (<50 years) with unprovoked VTE, recurrent VTE (unexplained by subtherapeutic anticoagulation, patient nonadherence, or malignancy), and VTE at unusual sites.20
Macrovascular arterial thrombotic events should be analyzed according to their site and associated risk factors.9 It is advised to test for aPL in younger patients (<50 years) with ischemic stroke, MI, or other macrovascular arterial thromboses (peripheral, splanchnic). It should be kept in mind that APS patients may experience MI with no obstructive coronary arteries (MINOCA), as described in the section on microvascular events. Occurrence of both VTE and arterial thrombosis in young patients (simultaneous or within a short time <1 month), after exclusion of patent foramen ovale, also warrants aPL testing. More generally, any combination of potential manifestations of APS (thrombotic, obstetric, and other) may necessitate aPL testing.
Microvascular thromboses include documented livedoid vasculopathy, acute or chronic aPL nephropathy, pulmonary hemorrhage (pulmonary capillaritis and recurrent microvascular thrombosis leading to alveolar hemorrhage), myocardial disease (MINOCA diagnosed on histopathology or magnetic resonance imaging), or adrenal hemorrhage (due to adrenal venous infarction diagnosed on imaging).
Suspected CAPS may require emergency testing for aPL. The features common for the patients with CAPS include: 1) clinical evidence of multiple organ involvement (3 or more organs, systems, and / or tissues) developing over a very short period (simultaneously or in less than a week), 2) histopathological evidence of multiple small‑vessel occlusions, and 3) laboratory confirmation of the presence of aPL, usually in high titer. More than one‑third of CAPS patients have not been diagnosed as APS before the occurrence of CAPS.6 This condition is characterized by a high risk of mortality (up to one‑third of the patients) or chronic organ damage. Therefore, an emergency diagnosis must be made to guide adequate management.
Clinical manifestations of pregnancy morbidity include prefetal or fetal deaths (3 or more consecutive prefetal [<10 weeks] and / or early fetal [10–16 weeks] deaths, or at least 1 fetal death [16–34 weeks] alone). New classification criteria also require considering preeclampsia and placental insufficiency.9 Two categories have been identified, that is, preeclampsia with severe features or placental insufficiency with severe features (<34 weeks) with or without fetal death, and preeclampsia with severe features and placental insufficiency with severe features (<34 weeks) with or without fetal death.
Among other clinical manifestations not included in the Sydney classification criteria, heart valve disease and thrombocytopenia are new domains in the American College of Rheumatology / European Alliance of Associations for Rheumatology (ACR/EULAR) classification criteria.8,9 Heart valve disease, defined as valve thickening or vegetations, may now lead to aPL testing for suspected APS.9 Among laboratory hematology parameters, mild, moderate, or sometimes severe thrombocytopenia has been defined as a new domain in APS. Therefore, thrombocytopenia of 20 to 130 × 109/l may lead to aPL testing.9
Systemic lupus erythematosus
SLE is a condition often associated with aPL and APS. Up to 40% of SLE patients may have aPL during the course of the disease. Moreover, up to 40% of APS patients also suffer from SLE.7 Positive aPL with a specific definition based on enzyme‑linked immunosorbent assay (ELISA) for aCL (IgA, IgG, or IgM) at medium or high titer (>40 IgA phospholipid [APL], IgG phospholipid [GPL], or IgM phospholipid [MPL], or >99th percentile) and positive aβ2GPI (IgA, IgG, or IgM, no threshold indicated), or a positive LA are included in the 2019 ACR/EULAR classification criteria for SLE. It should be noted that the antibody profile and thresholds are somewhat different from those included in the APS classification criteria9. This is partly due to the specificities of antibody profiles reported in SLE.22 The presence of aPL may impact clinical manifestations of SLE, in particular heart valve disease23 or autoimmune hemolytic anemia.24 The aPL profile will help stratify the risk for thrombosis and lead to primary or secondary antithrombotic prophylaxis.11,25
When to test for antiphospholipid antibodies
Timing of testing
Testing during the acute phase of the event is strongly discouraged because of high levels of factor VIII (FVIII) or C‑reactive protein20; the former may mask LA by shortening the activated partial thromboplastin time (aPTT), and the latter may give false‑positive results due to interferences with PL in the reagents of aPTT and dilute Russell viper venom time (dRVVT), both clotting assays used in LA measurement.26 During pregnancy coagulation factors increase and resolve after 6 weeks postpartum,27 returning LA results to baseline postdelivery.28 However, in specific situations, such as suspected CAPS, testing in the acute phase may be necessary despite these pitfalls.
The best timing for aPL testing and the variation of aPL over time in APS patients are not entirely clear.29 LA positivity may vary over time, regardless of the presence of SLE,28,30 and during pregnancy it may decrease or increase.31 aPL may increase with SLE activity and decrease with effective SLE treatment.30 Levels of aβ2GPI may vary in aPL‑positive patients treated with hydroxychloroquine and during thrombosis, with aβ2GPI titers decreasing around the time of thrombosis, due to deposition of these pathogenic aPL at the thrombotic site.32 Therefore, it is recommended to repeat the tests performed during pregnancy or soon after delivery, or during or shortly after a thrombotic event.
Nonetheless, testing during the acute phase may be needed in patients with thrombotic events, for example, in stroke patients suspected of APS, to guide therapy toward anticoagulation rather than antiplatelet drugs.20,33 The choice of anticoagulation therapy is defined by the antibody profile of aPL. In patients with venous thrombosis and triple positivity (LA, aCL, and aβ2GPI), DOACs are contraindicated but can be considered in venous thrombosis with single or double aPL positivity.12,34 Urgent testing is also needed to diagnose CAPS, which is characterized by multiorgan failure in patients meeting the laboratory criteria of APS.35
Repeat testing
Only persistent positive aPL have clinical relevance. The first positive aPL test should be repeated after 12 weeks to exclude transient positivity.10,20,36 Transient antibodies have been described in infectious diseases or on certain drugs and are not of clinical significance.10 aPL can arise transiently in patients with critical illness and various infections. These antibodies may rarely lead to thrombotic events that are difficult to differentiate from other causes of thrombosis in critically ill patients. Viral infection, most commonly with HIV and hepatitis C virus, can increase the risk of developing elevated aPL level and associated thromboembolic events. Patients with HIV were more likely to develop aCL and those with Epstein–Barr virus had elevated aβ2GPI levels.37,38
Retesting is crucial to prevent overdiagnosis and to confirm the antibody profile. In these cases, repeat testing should be suggested within a shorter time than 12 weeks. The retesting schedule is established at 12 weeks, as per the Sydney classification criteria.8 The 12‑week interval has been selected based on the recommendations of experts, and there is limited research on testing beyond this time period. In a retrospective study, the extended presence of aPL was observed in 96% of patients after 3 months. It was noted during a median follow‑up of 56 weeks, and was found to be independent of the antibody profile.39 This underlines the importance of annual retesting to validate persistence of positive results beyond this timeframe; however, more studies are needed to strengthen this recommendation.
Repeat testing can also be advised and indicated in the laboratory report when borderline or doubtful results are obtained for LA.20 In these cases, repeat testing should be suggested within a shorter time than 12 weeks. Repeat testing can be useful when discrepant results for aCL and aβ2GPI are obtained, and in patients with a high clinical suspicion of APS and negative results for aCL and aβ2GPI, testing with another test platform can be useful.40,41
Interference of anticoagulant therapy
One of the main confounders in LA testing is the interference of anticoagulant therapy, inherent to the test principle of clotting assays used for LA.20 Ideally, sampling for LA should be postponed until anticoagulation is discontinued to avoid false‑positive or false‑negative results.20,42 Immunoassays for aCL and aβ2GPI are not susceptible to the interference of anticoagulation.36 Requests for LA testing during anticoagulation, still the mainstay of therapy, are frequently made in daily practice. All anticoagulants affect clotting assays, although there are differences in their effects depending on the anticoagulant and the assay. If testing during anticoagulant therapy is unavoidable, the laboratory has several strategies to mitigate anticoagulant interferences. Methodologies to avoid interference of DOACs are available, but the effect of vitamin K antagonists (VKAs) still poses a problem, while heparins are generally not a problem.20,42
Heparins influence the clotting times of aPTT, but dRVVT is affected to a limited extent, since dRVVT reagents contain heparin neutralizers quenching the heparins up to 1 anti‑Xa IU/ml. Also, some LA‑specific aPTT and silica clotting time reagents contain heparin neutralizers. That makes LA testing reliable if the anti‑Xa level is within the therapeutic range.20 When feasible, it is advised to take samples at least 12 hours after the last dose of low‑molecular‑weight heparin (LMWH) was administered, and as near as possible to the next dose to guarantee low anti‑Xa activity in the sample.
Since the use of DOACs has become increasingly popular over the last years,43 and DOACs strongly interfere with LA testing, many studies have been performed to evaluate the best strategy for LA testing during DOAC therapy.44 DOACs give false‑positive results in aPTT and dRVVT test systems, even if taken at low concentrations, making sampling at trough levels no solution to avoid interference.45 Currently, DOAC neutralizers are well‑studied and widely used.44 These are handy tools but should be used with caveats. These adsorbent materials (commercially available Tablets or activated charcoal) are not added to the reagents but require an extra step in the sample preparation. Pretreatment of the sample with adsorbents may affect clotting times, resulting in false‑positive or negative LA results, and can lead to incomplete removal of DOACs.46-49 Consequently, pretreatment of plasma with adsorbents is only advised in DOAC‑treated patients. Testing the DOAC level before and after the neutralizer may provide some evidence of DOAC‑free LA testing.20 Comparable results were obtained for a DOAC filter, which efficiently removed DOACs from plasma in most of the cases, with minor alterations in clotting times.50,51 This confrms that DOAC‑removing agents or devices should not be used in samples not containing DOAC.20 This sample pretreatment makes the LA detection procedure even more complex and costly. The use of these products also leads to loss of plasma sample volume, which is often already limited given the sample requirements for a full investigation of APS. The best strategy remains to avoid testing for LA in patients on DOACs, or, if unavoidable, to undertake testing at trough levels. On a pragmatic empirical basis, LA testing may be undertaken at least 48 hours after the last dose, and longer in patients with renal impairment, although DOAC levels should also be checked.20 Alternative assays, such as Taipan snake venom time (TSVT) and Ecarin clotting tests (ET) are less affected by anti‑Xa DOACs. However, recommendations for their general use await more evidence.44 Moreover, using DOAC removal agents has the advantage of eliminating the influence of anti‑Xa and anti‑IIa DOACs in patients treated with these agents.
The effect of VKA cannot be neutralized, as in vivo the vitamin K–dependent coagulation factors are reduced affecting all clotting assays exploring the coagulation pathways that include FII, FVII, FIX, and FX. This factor deficiency can be compensated in vitro by adding normal plasma. However, this strategy is no longer recommended because it may lead to false‑negative or false‑positive results.20,42 TSVT and ET are less affected by VKAs. LA test results with the TSVT/ET test panel significantly overlap with the recommended dRVVT and aPTT test panels, where a positive result is diagnostic. Still, a negative result of TSVT/ET does not exclude LA.52-55 This means that TSVT/ET could be an additional assay performed after dRVVT and aPTT, specifically in patients on VKAs. However, these tests are not yet recommended in the guidelines for LA measurement, and their general use awaits more evidence and availability of reagents.20 Altogether, this means that testing during VKA therapy remains a challenge, without ideal strategies to circumvent the interference with the aPTT and dRVVT. LA results in patients on VKAs should always be interpreted by experienced staff.9,20 Similarly as for DOACs, a temporary switch from VKA to another anticoagulant (mostly LMWH) with lower assay interference might be an option. However, bridging strategies are not preferred by clinicians and should be evaluated case by case.
How to order testing for antiphospholipid antibodies
The type and combination of aPL define the risk for clinical events, for both thrombosis and pregnancy morbidity.9,56 Therefore, it is important to test for the 3 groups of aPL (LA, aCL, and aβ2GPI) in patients with thrombosis or pregnancy complications that may be associated with APS.
LA analysis is performed in venous blood collected into citrate tubes. The laboratory processes the citrated plasma by double centrifugation and then freezes the plasma within 4 hours of collection if LA analysis is postponed, which is the case in most laboratories performing LA analyses in batches. To guarantee good sample quality, the samples must be timely delivered to the laboratory after venipuncture.20 Both serum and citrated plasma can be used for aCL and aβ2GPI. It is preferable to conduct all 3 aPL (aCL, aβ2GPI, LA) tests in the same sample. However, it is common to use coagulation assays for LA testing and solid‑phase assays for aCL/aβ2GPI testing, with assays to be carried out in different laboratory departments: the hemostasis / hematology laboratory and the immunology / biochemistry laboratory. Therefore, most laboratories use serum to perform solid‑phase assays.17
Routine coagulation tests including the prothrombin time, aPTT, thrombin time, and fibrinogen help identify the presence of anticoagulant therapy, clotting factor deficiencies, or specific coagulation factor inhibitors, and acute‑phase reactants. All these conditions can influence the clotting assays used for LA detection.20 The immunoassays (solid‑phase assays) used for aCL and aβ2GPI are not prone to these interferences. One of the main confounders in LA testing is the presence of anticoagulant therapy, and the results should always be interpreted with reference to the ongoing therapy. Therefore, it is mandatory that clinicians provide this information in their request for aPL testing.20 In the laboratory, medical records can be consulted to check for the anticoagulation status of the patients, but for samples sent from outside the hospital, or for laboratories not associated with a hospital, this information is lacking when not provided by the clinician. Examining test patterns in patients with unknown anticoagulant therapy status can be a valuable tool for identifying the specific anticoagulant in use. For instance, the activity in anti‑Xa assays will only be evident for heparins and anti‑Xa agents (including apixaban, rivaroxaban, and edoxaban), while the thrombin time is only affected by anti‑IIa agents (such as dabigatran and unfractionated heparin). Each anticoagulant has a unique impact on coagulation assays, and no 2 anticoagulants exhibit the same profile of effects.44 This again emphasizes how essential it is for clinicians to closely collaborate with their laboratory to avoid incorrect classification of patients as suffering from APS. Anticoagulation does not influence immunoassays for aCL and aβ2GPI, but the assays are susceptible to interference from rheumatoid factor (RF), or compounds such as hemoglobin, bilirubin, or triglycerides that interfere with the absorbance signal measured to determine the antibody level. Although not required while ordering aPL testing, these parameters can be useful for the laboratory.36 Similarly, hemolysis, icterus, and lipemia mainly affect the aPTT by shortening it and producing potential false‑negative results for LA.20
Result reporting
Lupus anticoagulant
LA is reported with a final conclusion as positive / present or negative / absent. The conclusion is the result of a multistep procedure of a screening, mixing, and confirmation step in 2 phospholipid‑dependent coagulation systems, the aPTT and dRVVT. LA positivity is confirmed if all 3 steps in at least 1 test system (aPTT or dRVVT) are positive, that is, beyond the locally established cutoff value. The screening and mixing step results are expressed as normalized ratios (dividing the clotting time of the patient sample by the clotting time of a pooled normal plasma analyzed in the same run) to reduce the batch‑to‑batch variation. The confirmatory step can be expressed by a ratio (screen clotting time / confirm clotting time) or by percent correction [(screen‑confirm)screen] × 100. Some of the integrated tests are designed to measure a difference in clotting times in a mixture of patient and normal pooled plasma. The mixing and confirmation steps are performed in every sample with a prolonged screening test.20
Laboratory testing for LA remains complicated with many pitfalls in the test procedure as well as in the interpretation. Warnings for interference and comments on retesting should be provided along with the conclusion. Detailed quantitative results for each step can be reported, allowing clinicians to interpret them according to the local cutoff value.20 In APS patients with persistent positive LA, we noticed that LA positivity was lower in only LA‑positive patients than those positive for all 3 groups of aPL (LA, aCL, and aβ2GPI), the so‑called triple‑positive patients.57 Similarly, LA was stronger in thrombotic triple‑positive patients than triple‑positive carriers,58 suggesting that stronger LA better corresponds with a high‑risk profile. Further attempts to quantify LA by the degree of prolongation of aPTT/dRVVT showed no correlation with the clinical relevance, and previous studies indicated that quantification of LA is only partially informative regarding the prediction or exclusion of thrombosis in patients with APS.13,53,59 Therefore, further studies are needed to resolve this problem. The presence of LA is currently confirmed without considering the strength of LA in relation to the levels of aPL or its avidity. Also, if laboratories provide detailed analytical results on the 3 steps, a final conclusion should be given as positive or negative for LA without grading the positive result.20
Anticardiolipin and anti-β2 glycoprotein I antibodies
aCL and aβ2GPI IgG and IgM are reported quantitatively with their titer and units, along with the local cutoff value of the method used in the laboratory.36 A large variety of assays, ELISA and non‑ELISA, are available and show different characteristics.41 Automated systems have an advantage over manually performed ELISA, as they are easier to operate and less prone to pipetting errors, and consequently show lower interlaboratory variation. They offer all 4 tests (aCL and aβ2GPI IgG and IgM) in 1 batch, rather than multiple ELISA tests, which enhances diagnostic accuracy.34 The identification of patients testing positive for aCL or aβ2GPI depends on the assay used, and this complicates a consistent classification of positive aCL and aβ2GPI antibodies. All professionals in the field must be aware of the variation in sensitivity and specificity of these immunoassays. It is important to note that if a sample tests positive in one test, it may not necessarily test positive in the same type of test made by a different manufacturer or conducted in a different laboratory. It can be helpful to retest using a different type of solid‑phase platform if unexpected negative results are obtained. It is essential for effective capture that results are connected to clinical symptoms, making collaboration between the laboratory and clinician a necessity.
Both aCL and aβ2GPI have diagnostic value and show significant association with APS‑related clinical events.10 Discrepant results can be observed for aCL and aβ2GPI. aCL are supposed to be β2GPI‑dependent, and if aβGPI antibodies are negative, aCL antibodies recognize proteins different from β2GPI of unknown significance.60 Positive aβ2GPI with negative aCL may include aβ2GPI directed against domain 4/5 of the 5 domains of β2GPI that are regarded nonpathogenic, contrary to the antibodies directed against domain I of β2GPI that strongly correlate with thrombosis and obstetric complications.61,62 However, single moderate or high titer of aCL IgG and single aβ2GPI IgG are sufficient to classify an individual as an APS patient.9
Both IgG and IgM isotypes of aCL and aβ2GPI are associated with APS, but IgG aCL and aβ2GPI show a stronger association with clinical events, and are often associated with IgM positivity. In the ACR/EULAR classification criteria, isolated aCL or aβ2GPI IgM are given little weight.53-55 Many studies have shown that the coexistence of aCL and aβ2GPI of the same isotype strengthens the clinical likelihood of APS.18,63 aCL and aβ2GPI IgA isotype is not included in the APS classification criteria and diagnostic workup for APS,8-10 and there is not enough evidence to include IgA for diagnostic testing in APS.64
Titers are reported derived from the calibration curve that differs between the systems.36,65 This results in different titers obtained in different systems, and in newer automated systems antibody titers are much higher as compared with ELISA.40,66 Therefore, it is recommended to report aCL and aβ2GPI results with their numerical value along with the local cutoff value.10 Medium and high aPL titers strongly correlate with clinical outcomes of APS. Historically, 40/80 MPL/GPL thresholds corresponding to medium / high levels were established for ELISA, and are still used in the classification criteria.8,9,67 However, these thresholds are method‑dependent and largely differ between ELISA and non‑ELISA, as well as between different non‑ELISA methods.68 The semiquantitative reporting in ranges of low / medium / high that is used in classification criteria could also benefit APS diagnosis and risk estimation in daily practice. Categorizing titers of aCL and aβ2GPI IgG/M titers could be very useful for a clinician to estimate the risk for clinical events. So far, no standardized methods are available to define ranges of low / medium / high titers for non‑ELISA methods. Reports using a gradation of positivity by defining intervals are rare.68-70 A recent study showed that 40/80 value is reliable for ELISA but that each method needs its specific thresholds to be defined.68,70 The clinical relevance of increasing titers was proven by increasing likelihood ratios indicating that the higher the titer of aCL or aβ2GPI IgG, the higher the risk for thrombosis and pregnancy morbidity.68 Categorization for IgM seems to have lower impact on thrombotic risk estimation.68 Awaiting more studies, results should be reported with their titer, although the laboratory can give an estimation of titers corresponding to low / medium / high levels based on literature for their specific method.68,70
Laboratory diagnosis of antiphospholipid syndrome
Diagnosis of APS starts with the presence of clinical criteria that can be observed in different domains, comprising thrombosis, obstetric complications, cardiac valve lesions, and thrombocytopenia.9 To consolidate the diagnosis, a positive aPL test is required, containing LA, aCL, or aβ2GPI IgG or IgM. Results of LA, aCL, and aβ2GPI should be interpreted together to assess the clinical significance.10 The combination of the 3 groups of aPL may determine the probability of developing a clinical event. Triple positivity is considered as the highest risk for thrombosis and pregnancy morbidity, followed by double positivity.9,18,71 Single positivity of one aPL is associated with lower risk, especially for isolated aCL or aβ2GPI.9,57 Although in the classification criteria, it is sufficient to have 1 positive aPL out of the 3, combining the aPL may benefit risk assessment.9 Triple‑positivity is defined as LA, aCL, and aβ2GPI positive, double‑positivity indicates aCL and aβ2GPI positivity, and single positivity means only 1 aPL positive.18 Although aCL and aβ2GPI might be of different isotypes (IgG or IgM) in triple- and double‑positive patients, the clinical relevance is higher with aPL of identical isotypes.18,63 In the final report, besides the individual results of LA, aCL IgG/IgM, and aβ2GPI IgG/IgM, the antibody profile should be discussed.20 Importantly, the persistent positivity of aPL is a requirement that should be commented on in the laboratory report.20 The laboratory physician has access to aPL results requested on samples received from outside the hospital that were performed before referring the patient. Although triple‑positive patients usually have a persistent antibody profile on follow‑up testing after 12 weeks,39,72 retesting for confirmation after 12 weeks is still recommended.10 Reproducing the same test results as for the initial positive test renders the test result more reliable and confirms the antibody profile particularly important in single‑positive patients and in the context of interferences that affect the test result.10 The results should always be related to the clinical context and interpreted with reference to the anticoagulation status of the patient. A close interaction between the laboratory and the clinician is required.
Several other aPL apart from LA, aCL, and aβ2GPI have been identified. Some of these are well studied, such as the antiphosphatidylserine / prothrombin (aPS/PT) antibodies, and the antibodies against the domain I of β2‑glycoprotein I (anti‑domain I), both measured with immunoassays. These aPL have been shown to be related to thrombotic events and pregnancy complications,73,74 but have no place in the first‑line diagnosis.75 Measurements of aPS/PT and anti‑domain I antibodies are useful in the laboratory workup of APS in specific situations.64 Their major added value is their role in risk assessment in patients also positive for criteria aPL (LA and / or aCL and / or aβ2GPI),75 and not in patients negative for criteria aPL with APS‑associated clinical symptoms,64 as single positivity for aPS/PT and anti‑domain I antibodies is rare.64 Positive anti‑domain I results can be useful in confirming specificity of aβ2GPI antibodies toward domain I of β2GPI, particularly in patients with double positivity for aCL and aβ2GPI, as well as patients with single positivity for LA or aβ2GPI. Testing for anti‑domain I antibodies in these patients can help confirm or exclude the engagement of pathogenic aβ2GPI autoantibodies.71 aPS/PT can be helpful when LA testing is unreliable or doubtful. In contrast to LA measurement that is prone to interferences of acute‑phase proteins and anticoagulant therapy, aPS/PT that are measured by solid‑phase assays were suggested to be a good surrogate.76 In double‑positive patients (aCL and aβ2GPI‑positive) positive aPS/PT may suggest false‑negative LA. Considering the large overlap of LA and aPS/PT in APS patients, and the knowledge that aPS/PT is responsible for LA activity, we can assume that these patients have a similar risk profile as triple‑positive individuals.71,77 When aPS/PT are negative in double‑positive patients, it suggests a lower risk for thromboembolic events. While there is significant overlap, both a meta‑analysis and supplementary papers have revealed that there is not a complete overlap with LA. Consequently, aPS/PT cannot fully substitute for LA.74,78,79
Conclusions
A diagnosis of APS is complex and relies on a combination of clinical and laboratory criteria. One of the key components is the detection of antibodies to phospholipid‑binding proteins, including LA, aCL, and aβ2GPI IgG and IgM. APS diagnosis starts with a consistent request for testing aPL in patients who are suspicious of having APS at a distance from a clinical event. Each of these tests has its own methodological concerns that must be taken into account for accurate interpretation. For LA, various clotting assays are used, and their results can be influenced by a range of factors, including anticoagulant therapy. Close collaboration between the clinician and the laboratory is crucial to ensure that the clinical context and any ongoing anticoagulation therapy are considered when interpreting LA results. This helps to avoid both false‑positive and false‑negative conclusions. Similarly, solid‑phase assays for aCL and aβ2GPI exhibit interassay variations in sensitivity and specificity. Interpretation of these results should benefit from semiquantitative grading, but there is no standardized classification into low, medium, or high titers across different methods, because titers differ largely between methods. Therefore, it is important to await further studies, and results above the local and method‑dependent cutoff values should generally be regarded as positive. It is also essential to consider that not all tests have equal diagnostic importance, and antibody profiles based on the combination of LA, aCL, and aβ2GPI help in identifying patients at a risk for APS‑related clinical symptoms. Other aPL, such as those against the domain I of β2GPI and aPS/PT antibodies, are not included in the diagnostic criteria for APS, as they are not necessary for the first‑line diagnosis.
Furthermore, it is important for clinicians to communicate the anticoagulation status and therapy of the patient, such as with hydroxychloroquine, to the clinical pathologists. The clinical pathologist should, in turn, provide a report with interpretative comments, including a warning about potential interference and a suggestion to repeat testing to confirm positivity. In summary, the accurate interpretation of APS‑related antibody tests requires a close and ongoing collaboration between the clinician and the laboratory, taking into account the clinical context, therapy, and the intricacies of different testing methods.
Table 2 summarizes the key messages for clinicians on the interpretation of aPL testing.
Abbreviations: DOAC, direct oral anticoagulant; FX, factor X; others, see Table 1 |
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Repeat testing is:
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- Schreiber K, Sciascia S, de Groot PG, et al. Antiphospholipid syndrome. Nat Rev Dis Primers. 2018; 4: 18005. | Crossref
- Gris JC, Guillotin F, Chea M, et al. Antiphospholipid antibodies in pregnancy: maternal and neonatal implications. Semin Thromb Hemost. 2023; 49: 337‑347. | Crossref
- Alijotas‑Reig J, Esteve‑Valverde E, Anunciacion‑Llunell A, et al. Pathogenesis, diagnosis and management of obstetric antiphospholipid syndrome: a comprehensive review. J Clin Med. 2022; 11: 675. | Crossref
- Kaneko K, Ozawa N, Murashima A. Obstetric anti‑phospholipid syndrome: from pathogenesis to treatment. Immunol Med. 2022; 45: 79‑93. | Crossref
- Li X, Liu Z, Zhang S. Catastrophic antiphospholipid syndrome presenting initially as severe abdominal pain. Pol Arch Intern Med. 2023; 133: 16373. | Crossref
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