Lupus anticoagulant: screening methods

This screening comes under the scope of the screening for antiphospholipid syndrome (APS), an autoimmune disease characterised by the presence of antiphospholipid Abs (APAs) in the plasma of patients at risk or who have already suffered vascular thrombosis and/or recurring obstetric complications.

Screening for LA must be pertinent, i.e., it must only be performed on patients who have or are likely to have an APS.

The identification of APAs, which is an essential step in diagnosing an APS, is based on three different tests:

• a phospholipid-dependent clotting test, which allows for the identification of lupus anticoagulants LAs);
• an ELISA test, identifying anticardiolipin (aCL) Abs;
• an ELISA test revealing anti-β2GPI antibodies.

These tests are different but not independent and they do not have the same clinical significance. aCLs essentially recognise β2GPI and LAs bind to anti-β2GPIs and anti-prothrombins. At a clinical level, there are numerous arguments in favour of the pathological nature of the antiβ2GPIs and multiple studies have shown that the clotting test identifying LAs is the test of choice when seeking to identify clinically-significant APAs, in particular dRVVT. Finally, triple APA positivity yields a far higher thrombotic risk than simple positivity; the estimated risk is 5.3 % versus 1.36 %.

Identification of APAs is essential in diagnosing APS, but it must be clinically pertinent in regard to patient handling. Indeed, in the event of positive APAs, there is a high risk of thrombotic or obstetric recurrence with, consequently, long-term AVK treatment and an according exposure to risk of haemorrhage. It is therefore important to avoid over-diagnosis.

This then goes hand-in-hand with the biological difficulties in identifying LAs, due to:

• the heterogeneity of the epitope specificities of the tests;
• the diversity of phospholipid content in reagents (variable EEQ laboratory performance, in particular for weak LA);
• failure to comply with current recommendations.

For aCLs and anti-β2GPI Abs, IgG isotypes are always of greater interest than IgMs and care must be taken to avoid false positives, notably in IgMs, where there is rheumatoid factor, cryoglobulin or hypergammaglobulinemia.

Finally, other non-conventional APAs may be screened for, targeting the beta2-GP1 protein, but to date, their place in diagnostics and risk stratification remains uncertain: antiphosphatidylserine/prothrombin Abs, very much related to triple APA positivity, as well as anti-domain 2 (IgG) or anti-domain 4/5 Abs of the beta2-GP1 protein. Only very few specialised laboratories search for these Abs.

The recommended way to search for an LA type CAC is to follow four steps:

1. screening: highlighting of the prolongation of clotting tests for the intervention of phospholipids;
2. highlighting of an inhibiting action due to the absence of correction of prolongation after mixing the plasma to be tested with normal plasma;
3. confirmation of the inhibitor’s dependence on phospholipids (PLs): correction of clotting time through the addition of PL;
4. exclusion of other associated coagulopathies.

Screening for and diagnosing LA in practice

Screening is carried out some time after the acute thrombotic episode. The diagnosis of LA is based on:

1. the prolongation of clotting time of the two PL-dependent screening tests;
2. non-correction after mixing with specimen plasma;
3. confirmation of the PL dependency of the inhibitor by the “normalising” effect of a strong concentration of PL;
4. exclusion of any other coagulopathy.

The same sample should also be used to screen for the presence of aCL and anti-β2GP1 antibodies.

Sensitivity to anticoagulant treatments

• Screening for LA under unfractionated heparin: some commercial reagents for dRVVT or aPTT contain a heparin inhibitor (polybrene or heparinase) in quantities of up to 0.6 or 0.8 U/ml (check thrombin time normality).
• On low molecular weight heparin (LMWH): the search for LA is recommended at least 12 hours after the last administration (interference depends on the ratio of anti-IIa/anti-Xz of the LMWH).
• On AVK, the search should ideally be performed 1 to 2 weeks after discontinuing AVK. It is only recommended if the INR is < 1.5. For an INR ranging between 1.5 and 3, screening for LA is accepted, as long as a mixing study is performed.
• On DOAs: the search is not recommended due to the number of false positives; today, there are neutralisation kits available based on active carbon, which can therefore amend the presence of DOAs in these patients.

A positive result must be confirmed at least 12 weeks after the first test and interpreted in respect of the complete APA profile of the patient (patients at high risk of APS if LA is associated with another APA). Caution must be applied with current anticoagulant treatments and blood tests run during an acute phase of a thrombotic episode (the influence of the CRP risks false positive results).