Lupus Anticoagulant Detection under Magnifying Glass: Comparison
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Please note this is a comparison between Version 2 by Peter Tang and Version 1 by Tiziano Martini.

Diagnosis of antiphospholipid syndrome (APS) requires the presence of a clinical criterion (thrombosis and/or pregnancy morbidity), combined with persistently circulating antiphospholipid antibodies (aPL). Lupus anticoagulant (LA) is one of the three laboratory parameters (the others being antibodies to either cardiolipin or β2-glycoprotein I) that defines this rare but potentially devastating condition. For the search for aCL and aβ2-GP-I, traditionally measured with immunological solid-phase assays (ELISA), several different assays and detection techniques are available, thus making these tests relatively reliable and widespread. On the other hand, LA detection is based on functional coagulation procedures that are characterized by poor standardization, difficulties in interpreting the results, and interference by several drugs commonly used in the clinical settings in which LA search is appropriate. 

  • lupus anticoagulant
  • antiphospholipid antibody syndrome
  • thrombosis
  • anticoagulation
  • activated partial thromboplastin time

1. Introduction

The term “lupus anticoagulant” (LA) was coined by Feinstein and Rapaport in 1972, to designate an acquired inhibitor of blood coagulation found in the plasma of patients with systemic lupus erythematosus (SLE) [1]. LA indicates a heterogeneous family of autoantibodies directed against complexes formed by negatively charged phospholipids (PL) and proteins such as prothrombin, β2-glycoprotein-I (β2-GP-I), and others [2].
Given the absence of population-based studies, the true prevalence of antiphospholipid-antibody positivity (LA), anticardiolipin antibodies (aCL), or anti-β2 glycoprotein-I (β2GPI) antibodies in the general population is not known [3], but according to some authors the prevalence ranges between 1 and 5% [4].
Tests for aPL are positive in approximately 13% of patients with stroke, 11.5% with myocardial infarction, 9.5% of patients with deep vein thrombosis, and 6% of patients with pregnancy morbidity [4]. The absolute risk of a first thrombosis in antiphospholipid-antibody–positive patients who do not have other risk factors is reported as less than 1% per year. As arterial and venous thrombotic events in antiphospholipid-antibody-positive patients are often multicausal, the annual risk of a first thrombosis in patients with persistent antiphospholipid-antibody profiles and a systemic autoimmune disease or additional thrombotic risk factors may be as high as 5% [4].
LA is one of the three laboratory criteria (Sydney criteria) defining antiphospholipid syndrome (APS) [5], a systemic autoimmune disease defined by thrombotic or obstetrical events that occur in patients with persistent antiphospholipid antibodies [6]. The other two criteria defining APS are the presence of anti-cardiolipin antibodies (aCL) and anti-β2-glycoprotein-I antibodies (aβ2-GP-I). For the search for aCL and aβ2-GP-I, traditionally performed by solid-phase immunoassays (ELISA), several different assays and detection techniques are currently available, thus making these tests relatively reliable and widespread [7]. In contrast, LA detection is based on functional coagulation assays that are characterized by poor standardization, difficulty in interpreting the results, and interference by several drugs commonly used in the clinical setting in which LA search is appropriate [8]. 

2. Indications to the Tests: Patients’ Selection and Timing of Testing

Testing for LA is appropriate only in those patients in which there is a reasonable suspicion of APS [9]: for the intrinsic weaknesses of these functional tests, an accurate selection of patients is essential to avoid incidental findings, which are potentially able to disorient the clinician; regardless, it has been demonstrated that asymptomatic persistent carriers of LA and of the other antiphospholipid antibodies have a higher risk of future thromboembolic events [11,12][10][11]. The timing of testing is a crucial point to avoid situations that are potentially prone to misinterpretation of the results:
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Current guidelines recommend avoiding LA testing during an acute thrombotic event or an acute episode (e.g., an infection) [9], because of the interference of raised levels of coagulation factors (factor VIII) and of C-reactive protein on LA assays.
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During pregnancy, many coagulation factors are physiologically increased (especially factor VIII) [13][12], making LA testing results’ interpretation difficult; ISTH recommends that in this setting the results should be taken into consideration with caution [9].
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Ideally, LA testing should be performed in patients not taking any anticoagulant drug [9]; the search for LA in anticoagulated patients is currently a matter of great debate, because in these patients, anticoagulant therapy is often started very soon and the possibility of performing LA testing while on anticoagulation drugs assumes importance. A recent guidance of the ISTH Scientific and Standardization Committee for lupus anticoagulant/antiphospholipid antibodies faced this argument [14][13], concluding that LA detection during anticoagulation remains a challenge.

3. Preanalytical Phase

3.1. Sample Characteristics

The International Society on Thrombosis and Haemostasis (ISTH) and the Clinical and Laboratory Standard Institute (CLSI) produced guidelines that provide detailed information on the preanalytical phase of LA testing [9,15][9][14]. Venous blood must be collected into 0.109 mmol/L sodium citrate with blood/anticoagulant ratio 9/1 and rendered platelet-poor (final platelet count < 10 × 109/L) by double centrifugation (2000× g for 15 min or similar) at room temperature. Filtration by cellulose acetate filters is not recommended, although it is very high performing in removing all the platelets, because it causes the loss of von Willebrand factor and other coagulation factors [16][15]. A correct centrifugation (apparently a banal procedure) is essential for the success of the tests, because the activated platelet surface expresses negative-charged PL, which is able to bind the antiphospholipid antibodies, providing a false-negative result, especially with low-titres antibodies [17][16]. This problem can be exacerbated using frozen plasma (in situations in which an immediate analysis of the sample is not possible) because repeated freeze/thaw cycles may disrupt the platelet membrane, releasing an excess of PL [18][17]. For this reason, the aforementioned guidelines suggested only one cycle of freezing and thawing, freezing the plasma within 4 h of the collection, rapidly thawing it at 37 °C for 5 min in water bath by total immersion, and analyzing it within 4 h; according to CLSI guidelines [15][14] the frozen plasma can be stored for 14 days at −20 °C and is stable for 6 months at −70 °C. A recent study, performed with respect to the CLSI guidelines, reported the persistence of dilute Russell’s viper venom time (dRVVT) and silica clotting time (SCT) positivity before and after one freezing/thawing cycle (thus demonstrating the stability of frozen plasma) [18][17].

3.2. Interference

During an acute thrombotic event, the raising of factor VIII levels can shorten activated partial thromboplastin time (aPTT), producing false-negative LA screening by aPTT tests [19][18], while dRVVT is not influenced by factor VIII levels, as factor X is directly activated by the diluted Russell’s viper venom [7]. Increased levels of factor VIII can also be found in pregnancy, cancer, surgery, and acute episodes such as inflammation and infection. In these situations, the results of LA testing should be taken into consideration with caution. The association of LA positivity with a phlogistic condition, not always accompanied by a clinical APS phenotype, was recently confirmed in patients with COVID-19 [20,21,22][19][20][21]. C-reactive protein (an acute-phase reactant), through its affinity with the PL present in the reagents, can prolong PL-dependent clotting tests and lead to false-positive LA tests [23][22]. Furthermore, several drugs (antibiotics, antiarrhythmics, chlorpromazine) and vaccines (hepatitis B) can occasionally be associated with a LA positivity [24][23]. Anticoagulants of any species are able to prolong the clotting times of PL-dependent tests, leading to difficulty in interpreting LA testing results. LA search while on anticoagulation drugs is discussed later.

4. Lupus Anticoagulant Detection Procedure

4.1. General Principles: Three Steps Approach

LA detection is based on the in vitro functional behavior of these autoantibodies, which are able to affect some coagulation assays, producing a prolongation of clotting times. The heterogeneity of these antibodies and the variability of the effect they provoke in several coagulation assays explain why there is not a single test that is sensitive to all the LA, but it is necessary to use a combination of several assays for a correct diagnosis [25][24]. The so-called “three-step approach” consists of performing a sequence of three coagulation assays:
  • A screening assay, namely a PL-dependent test: aPTT, dRVVT (as recommended by ISTH guidelines [9], see above); if present in the patient plasma, LA is able to bind and inhibit PL, prolonging the clotting time beyond the upper limit of the reference range;
  • A mixing assay, in which the coagulation test is repeated on a mixture of normal plasma and the patient’s plasma; if LA is present in the patient plasma, the increase of coagulation factors provided from the normal plasma will not be able to correct the prolongation of the clotting time;
  • A confirmatory assay, in which the coagulation test is repeated while increasing the PL concentration; if LA is present in the patient’s plasma, the excess of PL is able to quench these antibodies, causing a shortening of the clotting time.
This sequential approach allows one to exclude the situations in which coagulation abnormalities other than LA are present, narrowing down the diagnostic possibilities until the presence of LA is confirmed or excluded. Therefore, this stepwise procedure can reduce costs, avoiding unnecessary mixing and confirmatory tests if the screening test is normal; however, in daily practice, screening and confirmatory tests are often performed at the same time, followed (if necessary) by the confirmatory test. The (possible) weak points of the three-step method and its possible modifications are discussed above.

4.2. Choice of Assays

The most recent ISTH guidelines [9] recommend using at least two tests as screening, based on different principles:
  • A LA-sensitive test derived from aPTT (such as SCT, silica clotting time);
  • The dRVVT (dilute Russell viper venom time).
The aPTT is based on activation of the intrinsic pathway. Its sensitivity to LA depends on the combination of two aspects: the type of activator and the concentration of PL. It is recommended to select an aPTT that uses silica as activator [9]. Silica clotting time (SCT) is a phospholipid-dependent test that contains colloidal silica as activator and a very low concentration of PL [37][25], thus making it very sensitive to LA. Ellagic acid has shown acceptable sensitivity in some aPTT reagents [26]. Further information about aPTT is available in [38][27].
Dilute Russell viper venom time utilizes a potent activator of factor X, the Russell’s viper venom. Russell’s viper, also known as Daboia russelii, is a venomous snake species, a member of the Viperidae family, found in South Asia, including India, Pakistan, Sri Lanka, Bangladesh, and other neighboring countries. Its potent venom interferes with the blood clotting process as it contains enzymes that directly activate some coagulation proteins (factor X, factor V, prothrombin, fibrinogen, and plasminogen [39][28]), making this test sensitive to deficiencies (congenital or acquired) of these factors, while it is independent of a deficiency of intrinsic pathway factors (XII, XI, IX, VIII) [37][25]. However, up to 20% of patients with factor VIII inhibitors can show a positive dRVVT test [40][29]. dRVVT is recommended for its specificity and robustness [9].
Both aPTT-based assay and dRVVT should be performed in highly standardized conditions, and their results should be expressed as the ratio between a patient’s clotting time and a normal pooled plasma (NPP) clotting time [8]. As aPTT and dRVVT are both positive in only a small fraction of patients, the recommendations indicate considering the LA screening positive whenever one of the two tests is positive.
Beyond aPTT and dRVVT, several phospholipid-dependent assays exist but are not recommended because of their limited commercial availability, poor reproducibility, and variability in reagents’ composition.
Moreover, there is one relatively novel test that needs to be mentioned in the LA research that is based on the analysis of the clot formation curve: clot waveform analysis (CWA). CWA is performed using a coagulation analyzer that measures the changes in light transmission as a blood clot forms. The clot formation curve is then analyzed for specific features that are characteristic of LA. CWA has been shown to be a sensitive and specific test for LA detection. It is also a relatively simple and inexpensive test to perform, making it a potential alternative to traditional LA testing methods, such as the APTT and the dRVVT. CWA can be used to detect LA in patients with a prolonged aPTT or dRVVT and has the potential to revolutionize the diagnosis of LA. However, it is little used in laboratories and its clinical utility is still being evaluated [41][30]. Therefore, it is not included in this revisewarch.

4.3. Screening Tests

The aim of the screening test is to evidence a prolongation of clotting time that could be related to the presence of LA. Because of the in vitro competition between LA and coagulation factors for phospholipid-binding sites, the phospholipidic component of the test is importantly diluted to accentuate the inhibitory effect of any LA antibody present [25][24]. The characteristics of the aPTT and the dRVVT are crucial for LA detection. They exist under many different commercial brands, which differ from each other in their composition of phospholipids and activators; these differing compositions determine differences in the sensibility and specificity of the procedure: a general principle is that reagents containing a low concentration of phospholipids are more sensitive to LA [8]. In addition, the representativeness of the different phospholipids classes can influence the assay performance [42,43,44][31][32][33]: the higher the relative content of phosphatydilserine, the less the sensitivity of the assay to LA. The specificity of LA screening test is hard to define, because of the difficulty of finding a test able to certainly individuate the “real” true positives (the “true LA”). There is some evidence that the association between LA detection and clinical events is stronger for dRVVT than aPTT [45][34]. Currently, there are no strict recommendations about the best composition of the phospholipids of the screening assay.
The CLSI guideline states that the dRVVT is the preferred test for LA detection, due to its high sensitivity and specificity. The aPTT and SCT are less sensitive than the dRVVT for LA detection [15][14].

4.4. Mixing Tests

A mixing test with screening reagent must be performed if the screening test clotting time is prolonged. A mix should be obtained utilizing a 1:1 proportion of patient plasma and pooled normal plasma (PNP), without incubation, within 30 min [9]. In theory, the coagulation factors contained in PNP will correct the prolongation of the screening test, restoring a clotting time that falls in the range of normality, if affected by a deficiency of one (or more) factors; if an inhibitor is present, the clotting time will remain prolonged [46,47][35][36]. PNP is very important for the standardization of the mixing test; it should be platelet-free, its content of each coagulation factor should be close to 100%, and it should be obtained from at least 20–40 healthy donors (males and females) [9,48][9][37]. PNP can be prepared “in house”, by mixing normal plasmas in a plastic receptacle, splitting the mix into small aliquots, and putting them into plastic tubes. The aliquots will then be rapidly frozen for storing at −70 °C (a temperature that ensures the stability of each coagulation factor for about 6 months). The aliquots of PNP can be thawed just before performing the test by rapidly exposing them at 37 °C in a thermostatic bath, which is gently tilted, and used within 2 h. There are several commercial lyophilized plasmas that show the same characteristics of home-made PNP and can be employed for performing the mixing test [49][38].

4.5. Confirmatory Tests

The confirmatory test consists of the execution of a screening test that has been made insensitive to LA: this is obtained by employing concentrated phospholipids in the reagent, to provide an antigen excess that is able to quench LA, abolishing the competition between LA and coagulation factors for phospholipids binding sites. This will result in a shortened clotting time of the screening test, which will fall into the reference range [25][24]. If an inhibitor other than LA is present, it will not be affected by the elevated phospholipids concentration, resulting in a confirmatory test that is unvaried compared to the screening test.
An alternative to adding an excess of phospholipids can be the use of reagents whose phospholipids component is innately LA-insensitive [50,51][39][40]: hexagonal phase phosphatidylethanolamine, purified inosithin, or phosphatydilserine [43,44,52,53][32][33][41][42].
ISTH guidelines recommend that confirmatory test(s) must be performed by increasing the concentration of phospholipids used in the screening test(s) [9].

4.6. Interpretation of Results

Clotting times of screening, mixing, and confirmatory procedures are prone to inter- (between same-principle tests offered by different manufacturers) and intra-assay (between batches of the same reagent) variability [25][24]; for this reason, it is recommended to normalize a screening, mixing, and confirmatory clotting time, generating a ratio between test clotting time and NPP clotting time [9,36,48,54,55][9][37][43][44][45].

5. LA Detection in Anticoagulated Patients

Anticoagulants are able to prolong the clotting time of screening, mixing, and confirmatory tests, thus making the interpretation of these results difficult for the clinician. Until a few years ago, LA detection during the treatment of an acute thrombotic episode was not an essential matter, as it was possible to delay it until the discontinuation of a regular course of VKA (vitamin K antagonists) therapy. The therapeutic option represented by DOACs (direct oral anticoagulants), currently the standard of care of anticoagulation for the vast majority of patients, brought to light the importance of LA detection before starting anticoagulant treatment. Recent clinical trials of patients with APS randomized to receive VKA or rivaroxaban (a direct factor Xa inhibitor) showed a significant excess of thrombosis recurrence in rivaroxaban patients [65,66][46][47]. The premature interruption of the TRAPS trial [65][46] led the EMA (European Medicines Agency) to warn against the use of DOACs in APS patients, recommending the exclusion of APS in patients with acute thrombosis after unspecified causes for whom treatment with a DOAC is indicated, shifting anticoagulation to VKA if there is an anti-phospholipids-antibodies positivity [67][48]. A recent guidance from the Scientific and Standardization Committee (SSC) for lupus anticoagulant/antiphospholipid antibodies of the ISTH [14][13] stated that “LA testing in patients on anticoagulation should be undertaken with the cognizance that anticoagulants may prolong the clotting time of the tests used for LA detection and that this effect may give rise to false-positive or false-negative LA”.

6. LA and Other Coagulation Factors Assays

Other coagulation assays that use aPTT-based methods, as one stage measurement (OSA) [90][49] for factors VIII, IX, XI, and XII, may show interference due to LA presence, resulting in falsely low factor activity [91,92,93,94,95,96,97,98][50][51][52][53][54][55][56][57]. In these cases, some clotting factor activity is usually measurable, determining a non-parallelism between patient plasma and standard plasma during OSA performance [97][56]. Nevertheless, a very low or undetectable factor activity, without non-parallelism, has been reported in several cases [95,97,98,99][54][56][57][58]: these effects can be seen in all OSA factor assays and pose the problem of differential diagnosis with both a multiple factor deficiency and a factor-specific inhibitor. A fundamental aid in these circumstances comes from performing chromogenic factor assays (CSA) [90][49], which do not suffer from these interferences, showing normal clotting factor activities [92,96,97][51][55][56].

7. LA and Other Inhibitors

LA can be hard to distinguish from other antibody-mediated coagulation alterations, such as factor VIII autoantibodies characterizing acquired haemophilia A (AHA). Even if these inhibitors require incubation to express their activity (2 h at 37 °C), if they are present at very high titre, they immediately neutralize factor VIII of NPP in the aPTT mixing procedure, thus making them indistinguishable from LA [8]. Furthermore, some factor VIII inhibitors show a behavior similar to LA in confirmatory tests [100][59]. Importantly, LA can cause false positives in Bethesda assays [40][29]. dRVVT is very useful for excluding the presence of LA, as this test is less sensitive to factor VIII deficiency; however, it has been demonstrated that up to 20% of patients with factor VIII inhibitors show a positive dRVVT [40][29]. Italian, British, and international guidelines on the diagnosis of AHA indicate the need to exclude the presence of LA and investigate an isolated prolonged aPTT that does not correct with a mixing test [101,102,103][60][61][62].

8. Diagnostic Algorithm(s)

The “traditional” diagnostic algorithm, structured by the sequence of screening, mixing, and confirmatory procedures, recently became a matter of debate because of the pitfalls of mixing tests, which are challenging the central role of this assay in LA diagnostic work-up. The principal limitation regarding mixing is the unavoidable dilution effect on the antibody titer, which in some cases can make the LA undetectable, leading to false-negative results [104][63]. False-negatives mixing tests can also arise for reasons other than dilution, such as reagents’ sensitivity and specificity to LA: some LAs, undoubtedly positive in screening tests with both aPTT and dRVVT, show positivity in the mixing for one test but not for the other [105,106][64][65]. Finally, NPP characteristics can influence the results of mixing tests, as some NPPs have shorter clotting times, requiring a stronger LA for a mixing test be positive [107][66]. Moving from these premises, British Society of Hematology (BSH) guidelines [36][43] state that mixing tests improve specificity and, in the absence of other causes of prolongation of clotting times, samples that give positive results in screening and confirmatory tests on undiluted plasma should be considered positive. CLSI guidelines [15][14] move forward, giving a new priority order to the three steps: screening, confirmatory, and then mixing only if it will enhance diagnostic decision making. ISTH and BSH guidelines recommend that mixing studies should be considered an essential component of the LA diagnostic pathway, to identify the inhibitory nature of LA [9,36][9][43]. Besides the “classical” algorithm, an “iterative” algorithm can be found, in which an elevated screening test is followed by the confirmatory test. If it is normal and the % correction is above the cut-off, LA detection is positive; if the confirmatory test is prolonged and the % correction is above the cut-off, a mixing test is performed; if mixing is prolonged, LA detection is positive [25][24]. Integrated tests, as already said, concomitantly perform screening and confirmatory procedures, showing LA positivity if the ratio between these assays is elevated.

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