The Antiphospholipid Syndrome: Diagnosis, Pathogenesis, Laboratory Testing, and Management

Welcome to the latest issue of Seminars in Thrombosis & Hemostasis, which is being devoted to the antiphospholipid (antibody) syndrome (APS), and covering its pathogenesis, clinical manifestations, management, and diagnosis (including laboratory testing). The history of APS as a clinically recognized entity goes back several decades.[1] [2] Now, some 30 years later, APS remains a challenge for both laboratory workers and clinicians in a wide range of specialities.[3] [4] In addition to the presence of appropriate clinical features, the diagnosis of APS also fundamentally requires the finding of positive antiphospholipid antibody (aPL) test result(s).[3] [4] [5] Testing for aPL comprises clot-based assays for the identification of lupus anticoagulant (LA)[4] [6] as well as various immunological assays (sometimes called “solid-phase” assays).[4] [5] [6]

There are a large number of immunological assays that have been developed for assessing aPL and thus APS,[4] but the most common, and those recommended by the classification criteria consensus guidelines[5] are assays for anticardiolipin antibodies (aCL) and anti-β₂-glycoprotein I antibodies (aβ₂GPI). In this context, positivity for immunoglobulin (Ig) G and/or IgM isotypes for aCL and/or aβ₂GPI, and/or positivity for LA, are noted within the latest 2006 consensus guidelines[5] as identifying “definite” APS (when matched to an applicable clinical feature[s]) for inclusion into research studies.

According to the literature reviews, positivity for IgG aCL, IgM aCL, IgG aβ₂GPI, and IgM aβ₂GPI, and LA, do not have equal “weighting” in terms of their association with adverse APS features such as recurrent thrombosis and pregnancy morbidity.[5] [7] [8] In addition, IgA immunoglobulin isotypes (aCL and aβ₂GPI), which are not included in the current laboratory criteria for APS, have also been proposed to have a potential role in the pathology of APS.[1] [4] [9] [10] [11] [12] Nevertheless, most laboratories cannot perform an encompassing immunological aPL test panel comprising IgG, IgM, and IgA for both aCL and aβ₂GPI, as well other potential solid-phase aPL. Therefore, most laboratories restrict their immunological-based testing to a core test panel comprising IgG aCL, IgM aCL, and IgG aβ₂GPI.[13] [14]

Although Seminars in Thrombosis & Hemostasis has published several papers related to APS in the past few years,[15] [16] [17] the last occasion in which APS was extensively covered was in 2008.[18] [19] [20] We begin this issue with a contribution from Willis et al on the topic of the pathogenesis of APS.[21] Notably, Harris and Pierangeli were involved in writing the previous article on pathogenesis of APS in the earlier APS issues,[22] and which has proved to be of great interest to our readership.[23] The article in this issue can therefore be considered an update of the earlier work. In summary, the presence of pathogenic aPL is the characterizing feature of APS, mediating the recurrent pregnancy loss and thrombosis typical of the disease, through their action on various antigenic targets. Despite the available knowledge regarding the mechanisms by which aPL induce a procoagulant phenotype in the vasculature and abnormal cellular proliferation, and differentiation in placental tissues to cause the typical clinical features, these processes still remain incompletely understood. It is known that inflammation serves as a necessary link between the observed procoagulant phenotype and actual thrombus development, and is an important mediator of the placental injury in APS patients. Even less well understood are the processes underlying the ontogeny of these pathogenic antibodies. This review seeks to highlight what is known about the mechanisms that contribute to the origin of pathogenic aPL, as well as the action of these antibodies on target antigens that produce the pathological features of APS. The authors also examine the feasibility of classifying patients in clinical phenotypes related to underlying pathophysiological mechanisms, and how this could impact the management of patients using novel “targeted” therapeutic strategies.

The second article is by Pengo and colleagues,[24] who provide a comprehensive overview of the currently reported clinical and cohort studies of aPL and APS. In patients with APS, the presence of multiple (triple) aPL test positivity carries a much higher risk of thrombosis recurrence and/or pregnancy-related morbidity than the presence of single or dual aPL test positivity. The same holds true for aPL “carriers,” namely subjects with laboratory but no clinical criteria for APS. Thus, very different risk categories exist among patients with APS as well as in carriers of aPL. Triple aPL positivity apparently identifies the important pathogenic autoantibody (directed against epitopes in domain I-II of β₂GPI). Pengo and coworkers believe that it is in this category of patients that trials on new therapeutic strategies should focus.

Gillis and Wong then focus our attention on the rarer clinical associations of aPL.[25] Thrombosis in certain vascular beds, such as the cerebral circulation, the veins of the lower legs and cutaneous vessels, and/or fetal loss, are common manifestations of APS. However, aPL have been found in association with a large range of other clinical conditions, and these constitute a rather heterogeneous group which are the focus of this review. Thus, aPL may rarely be found in association with thromboses of vascular beds other than those commonly associated with APS. In this case, the combination of thrombosis and aPL still satisfies the criteria for APS, and management of this group of patients is the same as that of APS associated with the more common manifestations of the disease. Alternatively, aPL may be detected in a range of conditions in which thrombosis cannot be clearly demonstrated, such as duodenal ulcer and transverse myelopathy. The approach to management of patients who have aPL in association with these conditions is less clear, although in some cases interventions to remove the associated antibody have been associated with amelioration of the conditions. Lastly, in several studies, aPL have been detected in a proportion of patients with conditions occurring commonly in the normal population—these findings have to be treated with caution in view of inconsistent findings between the reported results and methodological limitation of studies purporting to show positive results.

Cervera and Espinosa then update our readership on catastrophic antiphospholipid syndrome (CAPS).[26] These authors were involved in the similar report for this journal for our 2008 issue.[27] Although less than 1% of patients with APS develop the catastrophic (CAPS) variant, its potentially lethal outcome emphasizes its importance in clinical medicine. However, the rarity of this variant makes it extraordinarily difficult to study in any systematic way. An international registry of patients with catastrophic APS (“CAPS Registry”) was therefore created in 2000 by the University of Barcelona group headed by Cervera (www.med.ub.es/MIMMUN/FORUM/CAPS.HTM) to collate all the published case reports as well as newly diagnosed cases of CAPS from all over the world. Currently, this database documents the entire clinical, laboratory, and therapeutic data of more than 350 fully registered patients, and data from this registry is the focus of the current review of CAPS.

Les et al then take us through the controversial area of intensity and duration of anticoagulation therapy in APS.[28] Antithrombotic drugs are the therapeutic cornerstone for these patients, but choosing the specific agent (vitamin K antagonists or antiplatelet drugs), the intensity of anticoagulation (e.g., international normalized ratio [INR] range 2.0 to 3.0 or 3.0 to 4.0), and the duration of treatment, is often debated. A recent consensus document recommends warfarin to an INR range of 2.0 to 3.0 for patients with a first venous thromboembolic event. Higher anticoagulation intensity is recommended for patients presenting with arterial events. Combined therapy with warfarin and aspirin is another possibility in these patients, but some authors recommend standard intensity warfarin or aspirin, either as monotherapy. In general, a more intense regimen is warranted for high-risk patients. On the basis of an increased risk of recurrence during the first 6 months following warfarin withdrawal, long-term anticoagulation is considered the standard treatment. Nevertheless, Les and colleagues suggest that anticoagulation regimes of shorter duration could be given in selected cases of venous thromboembolism who have transient risk factors and a low-risk profile.

Galli, who also contributed to the 2008 APS issues,[29] then takes us through the interpretation and recommended testing for aPL.[30] During the last three decades, efforts have been made to improve the harmonization and reproducibility of laboratory detection of aPL and several guidelines have been published. The prognostic significance of aPL is also being clarified through the fine elucidation of their antigenic targets and pathogenic mechanisms. Several clinical studies have consistently reported that LA is a stronger risk factor for both arterial and venous thrombosis, compared with aCL and aβ₂GPI. In particular, LA activity dependent on the first domain of β₂GPI and triple aPL positivity are strong risk factors for future thrombotic episodes and obstetric morbidity. Hopefully, this increasing knowledge will help improve diagnostic and treatment strategies for APS.

Lakos then provides a nice overview of interference in aPL assay testing.[31] Both immunoassays and coagulation assays are prone to interferences, and clinicians need to be aware of the limitations of these assays. Interference is a clinically significant bias in the measured analyte concentration due to the effect of another component or property of the sample. Besides immune-mediated interferences (such as heterophile or human anti-animal antibodies, rheumatoid factor, high immunoglobulin levels, or factor inhibitors), aPL assays are uniquely affected by anticoagulants and the presence of residual platelets in the test plasma. Interferences are usually analyte- and assay-specific and may go unrecognized in routine laboratory practice. Despite advances in our knowledge on the mechanisms of interferences in aPL assays, Lakos believes that it is unlikely that total elimination will be possible.

Willis et al then discuss current international initiatives in aPL testing.[32] These tests have been available from as early as the 1980s, and since then several novel assays have been developed that have varied usefulness in the diagnosis and prognosis of APS patients. For almost three decades there has been an ongoing effort to standardize the aCL, aβ₂GPI, and LA assays, but there are still reports of significant intra- and interlaboratory variation in the results of all three assays. There have also been numerous issues with the implementation of novel (noncriteria) aPL tests in standard testing panels, due to either lack of standardized testing methods or limited evidence of their clinical utility in APS patients. At the recent 13th International Congress on Antiphospholipid Antibodies (APLA 2010, 13–16 April 2010, Galveston, TX), two task forces were set up to address these problems. This review gives a general description of current problems hindering the standardization of aPL tests and the implementation of novel assays as standard components of aPL testing panels. It also highlights the approach used by APLA 2010 Task Forces to address these problems and presents their recommendations.

Kershaw and colleagues then take us through the process of the laboratory identification of LA from the perspective of a “real-world” tertiary hospital-based laboratory.[33] The main laboratory characteristic of LA is their ability to prolong phospholipid-dependent clotting times in vitro. The laboratory demonstration of LA requires a systematic approach combined with an awareness of the many variables that can affect test results. The ideal testing procedures are those that are sensitive enough to detect weak LA, and specific enough so as not to produce incorrect conclusions. International guidelines have been published to assist laboratories in applying correct testing processes. The most recently published guidelines[6] from the International Society on Thrombosis and Haemostasis (ISTH) update the criteria for detecting the presence of LA that were presented in the 1995 guidelines.[34] Some of the specific recommendations relate to the key areas of determining cut-off levels for screening, mixing, and confirmatory procedures. Kershaw and coworkers believe that the more challenging aspects of testing for LA include maintaining sensitivity and specificity of the assays, especially in the presence of anticoagulant therapy.

Tripodi then continues the theme of LA testing, and focuses on the often debated question of “to mix or not to mix in LA testing?.”[35] Mixing patient and normal plasma has been used for many years to assist with making decisions on which direction to proceed for further investigation of abnormally prolonged coagulation tests: namely, either individual coagulation factor measurement, or to search for circulating anticoagulants. Mixing tests, however, gained wide acceptance only after LA was described and, therefore, have been included in several ISTH guidelines for LA detection.[6] [34] Important though they may be considered, the dispute between those who advocate the use of mixing tests and those who deny their superiority (or their need) for LA detection is difficult to resolve. This article aims to provide a balanced view on this dispute. Based on the limited information provided by the literature one may conclude that the disadvantages of performing mixing studies are the fact that (1) they are time-consuming; (2) they require a suitable source of normal pool plasma; and (3) weak LA may be lost at the time of the study due to the dilution of the index plasma into normal pooled plasma. On the contrary, performing mixing tests makes the occurrence of false-negative LA in patients who present with the so-called “lupus cofactor phenomenon” relatively unlikely. This would tend to justify their performance even though the frequency of the occurrence of this phenomenon in LA-positive patients is still unknown.

The next two articles in this issue are focused on internal quality control (IQC) and external quality assurance (EQA) in testing for aPL.[36] [37] IQC and EQA describe processes that are critical for ensuring the quality of laboratory test results, and thence, for aPL tests, the appropriate clinical diagnosis and management of APS. In brief, IQC is a process that helps control the quality of laboratory test results on a test-by-test basis; IQC should include samples that provide values around the assay critical cut-off values, and there is added value in the inclusion of non-kit/assay controls. EQA is a process that helps laboratories assess their performance against those of their peers. In the first of these articles, Favaloro et al discuss aCL and aβ₂GPI testing,[36] and provide some updated findings from the Royal College of Pathologists of Australasia (RCPA) Immunology Quality Assurance Program (QAP), by covering test results for the past 3 years (2009 to 2011 inclusive). Findings show similar trends to past years, indicating limited improvement in cross-laboratory test results and interpretations. In summary: (1) EQA participants reported greatly varying numerical test data for both aCL and aβ₂GPI, with interlaboratory coefficients of variation (CVs) >50% with most test challenges; (2) there was considerable overlap in the semiquantitative interpretation (negative, positive, low positive, moderate positive, strong positive) that different participants ascribed to identical numerical test results; (3) there was limited consensus among participants as to whether test results for individual EQA specimens were either positive or negative for aCL and/or aβ₂GPI. In the second article, Favaloro et al[37] focus on LA testing and provide some updated findings from the RCPA Haematology QAP over the same recent 3-year time period (2009 to 2011 inclusive). In brief: (1) essentially all laboratories currently perform LA testing using activated partial thromboplastin time (APTT) and dilute Russell viper venom time (dRVVT) methods, and about a third also employ the kaolin clotting time (KCT); (2) KCT usage has dropped slightly, from around 50% of laboratories in 2009, to around 35% in 2011, presumably reflecting take up of the latest consensus recommendations; (3) other methodologies such as silica clotting time (SCT) and the platelet neutralization procedure (PNP) are only used by <5% of laboratories; (iv) interlaboratory CVs are in general moderate, and substantially better than those reported for solid-phase assays such as aCL and aβ₂GPI, with median (range) values being 11.6% (9.2 to 25.5%) for APTT ratios, 16.7% (10.1 to 19.2%) for KCT ratios, and 11.7% (5.7 to 17.4%) for dRVVT ratios; (5) CVs increase slightly with increasing LA positivity; (6) most laboratories correctly interpreted test findings for LA, reporting normal samples as normal, and LA positive samples as positive (albeit with varying gradings of positivity); (7) however, some laboratories found interpretation to be challenging for some samples, namely a weak LA sample (which was reported as normal by around 50% of laboratories) and a very strong LA sample (which was reported as normal by around 10% of laboratories; primarily comprising those that did not perform mixing studies).

The final article in this issue of Seminars in Thrombosis & Hemostasis is by de Groot and Urbanus,[38] and provides a pensive appraisal of current and future testing for APS. The presence of aPL is essential to diagnose the APS, but the three assays currently available (LA, aCL, and aβ₂GPI) all suffer from several important drawbacks. First, the assays lack standardization, because the results of the assay partially depend on the laboratory that performs the assay. Second, the assays may or may not detect the autoantibody population that is responsible for the clinical manifestations that characterize the syndrome. Finally, the assays do not predict the risk of recurrence. de Groot and Urbanus believe that there is an absolute need for novel assays that generate prognostic information that can be used for a more tailored treatment of patients with APS. Important information has only recently become available on the protein against which the autoantibodies are directed, β₂GPI. Based on the progress made in terms of our understanding of the physiology of β₂GPI, it should be feasible to design assays that better predict the consequences of the presence of aPL in the circulation, and this should be a matter of priority for the near future.

We would like, as always, to thank all the authors of this issue of Seminars in Thrombosis & Hemostasis for their original and comprehensive articles, and hope that our readers find the content of considerable interest.

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