Antiphospholipid Antibodies

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Elizabeth M. Van Cott, MD
Charles Eby, MD

June 2016—CAP Press has released the second edition of its 2008 book, An Algorithmic Approach to Hemostasis Testing, edited by Kandice Kottke-Marchant, MD, PhD. She is chair of the Robert J. Tomsich Pathology and Laboratory Medicine Institute and section head of hemostasis and thrombosis, Cleveland Clinic Foundation. What follows is a sample chapter written by two of the 20 contributors, Elizabeth Van Cott, MD, of Massachusetts General Hospital, and Charles Eby, MD, of Washington University School of Medicine. Because of space limitations, we eliminated here their two sections on anticardiolipin antibody testing and other antiphospholipid antibody immunoassays and clinical features and diagnosis, as well as the suggested reading list.

Introduction. Antiphospholipid antibodies are acquired autoantibodies directed against phospholipid-protein complexes. These antibodies are associated with an increased risk for venous and arterial thrombosis as well as miscarriage. The two main types of antiphospholipid antibodies are lupus anticoagulants and antibodies against anticardiolipin–beta 2-glycoprotein complexes. The antiphospholipid antibody syndrome is diagnosed in patients with a history of thrombosis or certain obstetric complications plus a positive laboratory test for antiphospholipid antibodies that persists for more than 12 weeks.

Lupus anticoagulants are acquired autoantibodies that prolong a variety of phospholipid-dependent clotting tests. Although originally identified in patients with systemic lupus erythematosus, leading to the name lupus anticoagulant, this heterogeneous group of antibodies does not typically cause excessive bleeding, and although lupus anticoagulants are common in patients with systemic lupus erythematosus and other autoimmune disorders, most patients with a lupus anticoagulant do not have an autoimmune condition.

Anticardiolipin antibodies recognize a complex of cardiolipin (a phospholipid normally found in mitochondria) bound to a protein called beta 2-glycoprotein I (β2GPI). The in vivo function of β2GPI is not certain, but it is known to bind to anionic phospholipid membranes. Recent evidence shows that it inhibits von Willebrand factor adhesion to platelets. Lupus anticoagulants are more heterogeneous. Laboratory investigations have identified several target proteins (prothrombin, β2GPI, protein C, protein S, and annexin V) and have confirmed that the plasma from a patient with a lupus anticoagulant typically contains autoantibodies that recognize more than one epitope.

The pathologic mechanisms involved in antiphospholipid antibody-associated thrombotic complications remain obscure. Many different mechanisms have been proposed. One mechanism implicates antiphospholipid antibodies in the disruption of annexin V (annexin A5) binding to phospholipid membranes. Annexin V is a protein that prevents the formation of coagulation complexes on phospholipid surfaces; decreased binding caused by antiphospholipid antibodies could lead to increased coagulation complex formation and thrombosis. In addition, there is evidence that anti-β2GPI antibodies neutralize β2GPI inhibition of von Willebrand factor adhesion to platelets and also activate pathways in platelets, monocytes, and endothelial cells, which may have prothrombotic effects.

Laboratory Testing for Antiphospholipid Antibodies
Patients suspected of having antiphospholipid antibody syndrome should be tested for both lupus anticoagulants and anticardiolipin antibodies because patients with this syndrome can have lupus anticoagulants alone, anticardiolipin antibodies alone, or both. Some clinicians also order testing for anti-b2GPI antibodies. The case study on page 73 provides an example of the laboratory evaluation for antiphospholipid antibodies.

Lupus Anticoagulant Testing and Guidelines

In the hemostasis laboratory, the presence of a lupus anticoagulant (LA) is detected indirectly by:

• observing a prolongation of a phospholipid-dependent clotting test designed to be sensitive to LA;
• demonstrating an inhibitor effect and ruling out a coagulopathy by showing incomplete correction of the prolonged clotting time in a 50:50 mix of patient and normal pooled plasma;
• demonstrating phospholipid dependence, typically shown by shortening of the clotting time with the addition of more phospholipid; and
• evaluating for the possibility of a co-existing specific factor inhibitor, particularly against factor VIII, or an anticoagulant drug such as heparin, direct thrombin inhibitor, or direct factor Xa inhibitor.

Because of both LA heterogeneity and variation in methods, reagents, and instrumentation, no single clotting test provides adequate sensitivity for detection of lupus anticoagulants. Therefore, in 1995 the International Society on Thrombosis and Haemostasis (ISTH) Scientific Subcommittee on Lupus Anticoagulants/Phospholipid-Dependent Antibodies recommended that at least two sensitive screening tests for LA that assess different components of the coagulation pathway—intrinsic (activated partial thromboplastin time [aPTT], kaolin clotting time [KCT]), extrinsic (dilute prothrombin time [dPT]), and common pathway (dilute Russell viper venom time [dRVVT])—be employed and, if positive, mixing and confirmation steps be performed using the same test method. The ISTH updated their guidelines in 2009. Among the updates, two test methods were recommended: aPTT and dRVVT, and a mixing study was no longer considered required for integrated test systems that include screening and confirmation in a single procedure. In recent years, it has become more widely known that lupus anticoagulants often falsely correct to normal in mixing studies.

In 2014, the Clinical and Laboratory Standards Institute (CLSI) published its first guideline for LA testing. When compared to the 2009 ISTH guidelines, key differences include:

• Screening and confirmatory assays have priority over mixing studies
• No restriction on the test method used such as dPT or KCT (although both the aPTT-based and dRVVT-based methods are the preferred first-line assays)
• Using normalized ratios for all applicable LA tests
• Reporting results as “indeterminate” is acceptable if testing as a whole does not clearly distinguish between the presence and absence of LA in the patient

Multiple methods and reagents have been proposed for LA testing, and laboratories use a variety of combinations of in-house and commercial assays. As a result, proficiency testing results for LA have been poor for borderline- and weak-positive samples, whereas better agreement has been reported for negative or strongly positive LA plasmas. Presently, a lupus anticoagulant standard does not exist, although b2GPI monoclonal antibodies have been used successfully for LA proficiency testing. The strength or potency of a positive LA plasma has not been shown to correlate with the risk of thrombotic complications, and therefore LA test results are usually reported as positive or negative.

In order to fulfill the consensus laboratory criteria for antiphospholipid antibody syndrome, persistence of a lupus anticoagulant must be confirmed by repeated testing at an interval of ≥12 weeks. LA test results that are initially weakly positive are often transient, reinforcing the importance of confirmatory testing.

Preanalytic Variables. There are numerous preanalytic variables that can affect LA test results. Residual platelets in the test plasma (and control plasma used for mixing steps), especially if samples are frozen and thawed at a later time for testing, can produce platelet microparticles that absorb and neutralize LA antibodies, thus producing a false-negative screening or mixing test. To avoid this interference, quality control measures should be undertaken to ensure that residual platelet counts in plasma are less than 10,000/µL. Filtration of test plasma through a 0.22-µm filter before freezing or after thawing has been used to remove residual platelets and microparticles, but these filters can prolong clotting times by removing certain coagulation factors and also remove large von Willebrand factor multimers. Therefore, use of filters is not currently recommended. The normal pooled plasma used in mixing studies should not be lyophilized.

Patients with inherited or acquired coagulopathies, or who are taking an oral vitamin K antagonist (warfarin), may have false-positive LA screen results due to decreased plasma levels of coagulation factors. Ideally, repeating the screening test on a 50:50 mix of test and control plasma will produce a substantial correction with factor deficiencies and warfarin therapy, but false-positive mixing studies can occur with warfarin. In general, although lupus anticoagulants are associated with a positive 50:50 mixing study, some weak lupus anticoagulants will have a negative mixing study result.

Plasma that contains heparin may produce false-positive LA screening tests and complicate the interpretation of mix-and-confirm steps. Performing a thrombin time and, if prolonged, performing a thrombin time after treating the specimen with protamine or heparinase to neutralize heparin, or a reptilase time, which uses a snake venom insensitive to heparin, will identify heparin-contaminated samples. An anti-Xa assay, which detects all heparin types, can also be used. If heparin is present in a test plasma, options include neutralizing or degrading heparin in the test plasma before performing LA testing, using commercial LA reagents that contain a heparin-neutralizing material, or obtaining a new sample that is free of heparin. Even if the LA reagent contains a heparin neutralizer, caution should be used because heparin concentrations often exceed the heparin-neutralizing capability of the reagent (typical neutralization up to 1 U/mL anti-Xa activity).

Direct thrombin inhibitors (DTIs), such as dabigatran, argatroban, bivalirudin, and hirudin, may cause false-positive LA results by blocking the active site on thrombin molecules. A thrombin time is a sensitive screen for DTIs as well, but it will remain prolonged after a heparin neutralization step if a DTI is present. Because DTIs cannot be neutralized or absorbed, LA testing should not be performed when the presence of a DTI is suspected, and a new specimen should be obtained after the DTI therapy has been discontinued. See Figure 24-1 for a suggested testing algorithm. Similarly, the new factor Xa inhibitors, such as rivaroxaban, apixaban, and edoxaban, can also cause false-positive LA results. A positive anti-Xa assay with a normal thrombin time can be used to detect the presence of a factor Xa inhibitor, as shown in Figure 24-1. Currently, Food and Drug Administration-approved calibrators are not available for rivaroxaban or apixaban. Nevertheless, the presence of a factor Xa inhibitor is detected by an anti-Xa assay even if heparin, low-molecular weight heparin, or fondaparinux is used as the calibrator, as the assay cannot distinguish which anticoagulant is inhibiting factor Xa. However, without rivaroxaban or apixaban calibration, the result would not provide concentration of these oral factor Xa inhibitors; it would just reveal whether or not factor Xa inhibition is detected. Because these new factor Xa inhibitors cannot be neutralized, LA testing should not be performed when a patient is taking one of these drugs.