Laboratory methods to detect antiphospholipid antibodies Steven A. Krilis 1,3 and Bill Giannakopoulos 1,2,3 1 Department of Infectious Diseases, Immunology, and Sexual Health and 2 Department of Rheumatology, St George Hospital, Kogarah, New South Wales, Australia; and 3 Department of Medicine, St George Clinical School, University of New South Wales, Kogarah, New South Wales, Australia This chapter reviews several important themes pertaining to the antiphospholipid syndrome (APS), including a description of the clinical features, a discussion of the main autoantigen, beta 2-glycoprotein I ( 2 GPI), and insights into the characteristics of the pathogenic anti- 2 GPI autoantibodies. Evidence-based considerations for when to test for APS are explored, along with the clinical significance of patients testing positive on multiple APS assays, so-called triple positivity. A detailed review of recently published laboratory guidelines for the detection of lupus anticoagulant and the solid-phase anticardiolipin and anti- 2 GPI ELISAs is undertaken. Finally, a brief review of nonclassification criteria laboratory assays with potential future diagnostic utility is presented. Learning Objectives ● To understand that the term antiphospholipid is a misnomer: the major autoantigen in APS is  2 GPI ● To understand that aCL, anti- 2 GPI, and LAC triple positivity does not predict thrombotic risk Clinical features of APS The core clinical manifestations of the antiphospholipid syndrome (APS) can be divided into thrombotic and obstetrical. 1 The former includes both venous and arterial thrombosis and can affect any part of the vascular bed. The most common site of venous thrombosis is in the lower limbs; the most common site of arterial thrombosis is the cerebral circulation. 2 The obstetrical manifestations include recurrent first-trimester miscarriages and/or a single second- trimester fetal death and/or early-onset severe preeclampsia. 1 Thrombocytopenia, autoimmune hemolytic anemia, heart valve thickening and dysfunction, and livedo reticularis, which are not part of the formal clinical classification criteria for APS, have been associated with patients diagnosed with APS. 1,2 In view of the lack of specificity of the clinical manifestations of APS, the laboratory investigations constitute a critical necessity to establish a diagnosis of APS and to allow for classification in clinical APS studies. The laboratory investigations are designed to detect elevated levels of pathologically relevant autoantibodies, which are generically described as antiphospholipid (aPL) autoanti- bodies (aAbs). 1 The accurate laboratory testing for APS carries significant implica- tions for individuals who have suffered from one of the core clinical manifestations. In the setting of venous thrombosis, the implication of being diagnosed with APS is consideration of indefinite anticoagu- lation, with the associated cumulative risk of bleeding complica- tions. This strategy is distinct to the management plan of non-APS venous thrombosis, although the evidence for this is not strong. 3,4 Likewise, in the setting of an individual having an arterial thrombo- sis such as a stroke, a diagnosis of APS entails consideration for treatment with either an antiplatelet agent such as aspirin or an anticoagulant such as a vitamin K antagonist (VKA; i.e., warfarin). 5 The term aPL aAbs is a misnomer. The aAbs that characterize APS do not directly bind to phospholipids. The main APS autoantigen is beta 2-glycoprotein I ( 2 GPI), an abundant plasma protein that binds to anionic phospholipids. 6 The detection of anti- 2 GPI aAbs with the anti- 2 GPI ELISA has recently been included as part of the laboratory classification criteria for APS (Figure 1). 1 There is an extensive body of evidence to suggest that these aAbs may be directly pathogenic. 7 There may also be other less common autoantigens to which APS aAbs bind, such as prothrombin (PT), annexin A5, and phosphati- dylethanolamine. 8,9 It is recommended that testing for these nonclas- sification criteria APS aAbs should only be undertaken in the research setting. 1 Laboratory tests for APS There are 2 broad categories of assays that are used to diagnose APS: lupus anticoagulant (LAC) assays and ELISAs. LAC assays There are several in vitro coagulation-based assays that have as their common point the measurement of the time to clot in vitro of the patient’s plasma relative to the time taken by healthy control samples. This is known as a screening test. If prolongation of the patient’s clotting time is demonstrated, then 2 further tests are undertaken, first to assess whether the prolonged clotting time is reversed with mixing (1:1 ratio) the patient’s plasma with pooled plasma from healthy controls and second to assess whether the prolongation in clotting time seen in the screening test is reversed with the addition of excess anionic phospholipid. The latter test is known as a confirmatory test. These in vitro coagulation-based assays are collectively termed LAC assays. A plasma sample is defined as testing positive for LAC if it has a prolonged in vitro coagulation time that is not reversed with mixing studies, but is reversed when extra phospholipid is added. 10 There are several different types of LAC assays available. The dilute Russell viper venom test (dRVVT) assay uses a component of the venom derived from the Russell viper in conjunction with dilute phospholipids to activate factor X to factor Xa. In contrast, the activated partial thromboplastin time (aPTT), the kaolin clotting time (KCT), and the silica clotting time (SCT) assays activate the contact activation and NONTRADITIONAL LABORATORY ASSAYS OF HEMOSTASIS:WHAT THE CONSULTING HEMATOLOGIST SHOULD KNOW Hematology 2014 321