Laboratory Diagnosis of Lupus Anticoagulant and Antiphospholipid Antibodies

Introduction

Antiphospholipid (aPL) antibodies are a heterogeneous family of autoantibodies that are primarily directed against plasma proteins complexed with anionic phospholipids that are found on damaged/activated cellular membranes, including endothelial cells, trophoblasts, platelets, and monocytes. The formation of autoantibodies against many antigenic targets, interfering with many of the biochemical and cellular functions of phospholipids, has led to various pathological clinical manifestations in patients with high levels of these autoantibodies. The association of thrombosis and/or fetal loss with aPL antibodies is known as the antiphospholipid syndrome (APS). The current investigational criteria for APS ( Table 158.1 ) are defined by a constellation of clinical and laboratory manifestations. These criteria include histories of documented thrombosis and/or defined pregnancy complications together with positivity of specific laboratory tests (i.e., “APS criteria assays”), including positivity of coagulation tests identifying a lupus anticoagulant (LA), and medium to high titer positivity in immunoassays for anti-β2-glycoprotein I (anti-β2GPI) IgG or IgM and anticardiolipin (aCL) IgG or IgM. Rare patients with aPL antibodies may develop disseminated thrombosis in large and small vessels with resulting multiorgan failure known as catastrophic APS ( Table 158.2 ).

Table 158.1

Sydney Investigational Criteria for the Diagnosis of the Antiphospholipid Syndrome ^a

Modified from Miyakis, S., Lockshin, M.D., Atsumi, T., et al. (2006). International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). Thromb Haemost, 4 , 295–306.

Clinical

Vascular thrombosis (one or more episodes of arterial, venous, or small vessel thrombosis). For histopathologic diagnosis, there should be no evidence of inflammation in the vessel wall.
Pregnancy morbidities attributable to placental insufficiency, including (1) three or more otherwise unexplained recurrent spontaneous miscarriages, before 10 weeks of gestation, (2) one or more fetal losses after the 10th week of gestation, (3) stillbirth, and (4) episode of preeclampsia, preterm labor, placental abruption, intrauterine growth restriction, or oligohydramnios that are otherwise unexplained.

Laboratory

Medium or high titer aCL or anti-β2GPI IgG and/or IgM antibody present on two or more occasions, at least 12 weeks apart, measured by standard ELISAs.
Lupus anticoagulant in plasma, on two or more occasions, at least 12 weeks apart, detected according to the guidelines of the ISTH SSC Subcommittee on Lupus Anticoagulants and Phospholipid-Dependent Antibodies.

aCL , anticardiolipin; aPL , antiphospholipid; ELISA , enzyme-linked immunosorbent assay; Ig , immunoglobulin; β2GPI , β2-glycoprotein I.

a “Definite APS” is considered to be present if at least one of the clinical criteria and one of the laboratory criteria are met.

Table 158.2

Proposed Criteria for the Classification of Catastrophic Antiphospholipid Syndrome (APS)

Modified from Asherson, R.A., Cevera, R., de Groot, P.G., et al. (2003). Catastrophic antiphospholipid syndrome: International consensus statement on classification criteria and treatment guidelines. Lupus, 12 , 530–534.

1.
Evidence of involvement of three or more organs, systems, and/or tissues ^a

2.
Development of manifestations simultaneously or in less than a week

3.
Confirmation by histopathology of small vessel occlusion in at least one organ or tissue ^b

4.
Laboratory confirmation of the presence of antiphospholipid antibodies (lupus anticoagulant and/or anticardiolipin antibodies) ^c

Definite Catastrophic APS

All four criteria

Probable Catastrophic APS

All four criteria, except for only two organs, systems, and/or tissues involvement

All four criteria, except for the absence of laboratory confirmation at least 6 weeks apart due to the early death of a patient never previously tested for aPL before the catastrophic APS event

Criteria 1, 2, and 4

Criteria 1, 3, and 4 and the development of a third event in more than a week but less than a month, despite anticoagulation

a Usually, clinical evidence of vessel occlusions, confirmed by imaging techniques when appropriate. Renal involvement is defined by a 50% rise in serum creatinine, severe systemic hypertension (N180/100 mm Hg), and/or proteinuria (N500 mg/24 hours).

b For histopathological confirmation, significant evidence of thrombosis must be present, although, in contrast to Sydney criteria, vasculitis may coexist occasionally.

c If the patient had not been previously diagnosed as having an APS, the laboratory confirmation requires that the presence of antiphospholipid antibodies must be detected on two or more occasions at least 6 weeks apart (not necessarily at the time of the event), according to the proposed preliminary criteria for the classification of definite APS.

It is important to note that some patients may have typical clinical manifestations of APS but are entirely negative for APS criteria assays, (aCL or anti-β2GPI IgG/IgM and/or LA tests), a situation referred to as seronegative APS (SNAPS). Furthermore, there are other aPL antibody tests that are positive in patients with APS clinical manifestations, but are not yet accepted by consensus criteria. These tests are currently being referred to as “noncriteria laboratory assays.” In this chapter we will review the assays involved in identifying the presence of aPL antibodies.

Antiphospholipid Syndrome Criteria Assays

The current APS criteria assays developed from two laboratory anomalies. First, the biologic false-positive serologic test for syphilis, which was known to be associated with autoimmunity, and the test’s antigenic target cardiolipin led to the quantitative immunoassays to measure antibodies against cardiolipin and its cofactor β ₂ -glycoprotein I. Subsequent clinical trials associated elevated aCL and anti-β2GPI with thrombosis, spontaneous abortion, and neurologic disease. Meanwhile, patients with systemic lupus erythematosus (SLE) were found to have a lupus anticoagulant, an antibody-mediated inhibition of phospholipid-dependent coagulation, which caused prolonged partial thromboplastin time tests. The identification of a lupus anticoagulant (LA) in coagulation-based testing was subsequently associated with thrombosis and pregnancy complications. Both immunoassay and coagulation-based tests became crucial to identifying the aPL antibodies of APS.

However, these current APS criteria assay are inherently limited because they were not designed to measure known disease mechanisms. The aCL IgG and IgM assays are the most sensitive, but least specific assays, anti-β2GPI IgG and IgM assays are more specific but less sensitive, and LA assays, of which the dilute Russell viper venom time (dRVVT) is the most common, are the least sensitive but the most specific. Nevertheless, these assays have proven to be useful surrogates for thrombotic risk. Strong positivity for more than one of the APS criteria assays is indicative of an increased risk for clinical events.

Prevalence of Antiphospholipid Antibodies and Appropriateness of Testing

The prevalence of aPL antibodies in the asymptomatic “normal” population has generally been estimated at 3%–10% with a prevalence of 1%–5% for LA, 1%–5% for aCL antibodies, and 3% for anti-β2GPI antibodies. In one group of healthy young women, who served as controls in a study, 18% had elevated aCL antibodies and 13% tested LA positive. The development of aPL antibodies can occur after viral infections, syphilis, Lyme disease, hepatitis C, alcoholic liver disease, HIV infection, multiple sclerosis, and in patients taking medications such as chlorpromazine or procainamide. These elevations in aPL antibodies are generally not associated with thrombosis.

A significant proportion of SLE patients are positive for aPL antibodies; estimates range between 12% and 30% for aCL antibodies and 15% and 34% for LA antibodies. aPL antibodies have been associated with virtually all other autoimmune conditions. In some patients, the presence of aPL antibodies may herald the development of SLE.

Because of the prevalence of aPL antibodies in normal and reactive conditions, clinicians should determine the appropriateness of testing in their patients to avoid misdiagnosis. Patients in the high appropriateness group include patients with unprovoked and unexplained venous thromboembolism, arterial thrombosis in young patients (<50 years of age), thrombosis at unusual sites, late pregnancy loss, and any thrombosis or pregnancy morbidity in patients with autoimmune diseases (SLE, rheumatoid arthritis, autoimmune thrombocytopenia, autoimmune hemolytic anemia). The moderate appropriateness group includes asymptomatic patients who are incidentally found prolonged aPTT—often during routine testing—and young patients with recurrent spontaneous early pregnancy loss and provoked VTE. Patients in the low appropriateness group include elderly patients with venous or arterial thromboembolism.

Lupus Anticoagulant Tests

The various LA tests use different coagulation systems to report the inhibition of phospholipid-dependent blood coagulation reactions. These include modifications of the activated partial thromboplastin time (aPTT) with LA-sensitive and LA-insensitive reagents, the dRVVT, the kaolin clotting time, the tissue thromboplastin inhibition time, the hexagonal phase array test, and the platelet neutralization procedure. The results of LA tests can be variable among laboratories; although most of laboratories agree on identification of plasmas containing strong positive LA activity. The Subcommittee on Antiphospholipid Antibodies of the International Society of Thrombosis and Hemostasis has specific criteria for standardizing the diagnosis of LAs. They recommend performing two different coagulation tests with different assay principles (aPTT and dRVVT) as screening assays, then using mixing studies on abnormal results and repeat testing using phospholipids to confirm the presence of a phospholipid-dependent antibody.

Activated Partial Thromboplastin Time Tests

Different commercial aPTT reagents vary widely with respect to their sensitivities to LA, so it is important to know the characteristics of the particular reagent(s) that is being utilized. In general, LA-sensitive aPTT reagents are used in routine coagulation testing, so the identification of a prolonged aPTT in the absence of anticoagulation, bleeding, or known factor deficiency is a good screening test for an LA. When the aPTT is prolonged and not “correctable” by mixing studies with normal plasma, an LA should be suspected. The effects of incubation with normal plasma may be helpful in differentiating LAs from coagulation factor inhibitors. A factor VIII inhibitor usually requires incubation with normal plasma for 1–2 hours at 37°C to show prolongation on aPTT, whereas LA-containing plasmas usually prolong the aPTT immediately after mixing and show no further prolongation with incubation.

LA is confirmed by reassaying the aPTT with higher amount of phospholipids (e.g., hexagonal phospholipids) or frozen washed platelets as the source of phospholipid (i.e., platelet neutralization procedure ). It is important to note that both types of anticoagulants—i.e., LA and specific coagulation factor inhibitors—may sometimes coexist and yield a confusing laboratory picture. LAs may cause artifactual decreases in certain coagulation-based factor levels that are based on aPTT reagents; these patients are sometimes erroneously misdiagnosed as having multiple coagulation factor deficiencies. This problem can be handled by repeating the coagulation by using an LA-insensitive aPTT reagent or chromogenic assays for coagulation factor assays.

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