Antiphospholipid antibody levels in early systemic lupus erythematosus: are they associated with subsequent mortality and vascular events?

Abstract Objectives aPL are present in between 20 and 30% of patients with SLE. They can cause vascular events (VE) or pregnancy morbidity. aCL and anti-beta-2-glycoprotein I (anti-β2GPI) are measured in clinical practice. Domain I (DI) of β2GPI is the main site for aPL binding. We investigated the prevalence of IgG anti-DI, aCL and anti-β2GPI antibodies in early SLE and their association with mortality and development of VE. Methods Samples from 501 patients with SLE that had been obtained and stored early during their disease were tested for IgG anti-DI, aCL and anti-β2GPI antibodies by ELISA. LA status and history of VE were obtained by reviewing medical records. Kaplan–Meier analysis was used to investigate mortality and occurrence of VE, comparing groups with and without aPL in early disease. Results Of 501 patients, 190 (38%) had at least one of these aPL, of whom 112 had anti-DI alone. Of 276 patients with complete vascular history, 83 had experienced VE. The 39 patients who were double or triple-ELISA-positive for any combination of the three aPL were more likely to have or develop lupus anticoagulant (P<0.0001) than those who were single-ELISA-positive or negative. In Kaplan–Meier analysis, they showed a trend towards developing more VE (P = 0.06). Conclusion IgG anti-DI antibodies were present in early serum samples from 29% of patients and were more common than IgG aCL or anti-β2GPI. There was some evidence suggesting that double or triple-ELISA-positivity for these antibodies identified a group with worse outcomes.

Introduction aPL were first described in the context of SLE and multiple reports over the last 30 years have shown that they are present in the blood of 2030% of patients with SLE [1]. Only a minority of these patients, however, develop vascular thrombosis and/or pregnancy morbidity, which are the cardinal features of the APS [2]. There are three tests for aPL used commonly in clinical practice; the aCL ELISA, the anti-beta-2-glycoprotein I (antib 2GPI) ELISA and the LA test [2]. In many lupus clinics, these tests are requested routinely for screening in all newly diagnosed patients with SLE. It is not clear, however, what to do in the case of patients who have positive aPL results at diagnosis, whereas they have not yet suffered thrombosis or pregnancy loss. It is recognized that features such as high-titre positivity for IgG aCL and/or anti-b2GPI, and triple positivity for LA, aCL and anti-b2GPI [3,4] are all factors that increase risk of thrombosis [5], but not to the extent that routine anticoagulation for those with these serological profiles is recommended as primary prophylaxis to prevent thrombosis.
b2GPI consists of five domains of which the N-terminal domain (Domain I, DI) is believed to contain the major immunological epitope [6,7]. Thus, assays for anti-DI antibodies have been proposed as an alternative test that could have greater predictive power for occurrence of thrombosis and pregnancy morbidity [710]. However, there is little evidence concerning the prevalence of anti-DI antibodies early in the disease course of patients with SLE and whether presence of these antibodies with or without aCL and anti-b2GPI positivity delineates patient groups with higher risk of mortality or vascular events (VE) over many years of follow-up. The aim of this study was to investigate these unanswered questions.
We therefore extracted the first available stored serum sample for 501 patients with SLE, tested these samples for IgG aCL, IgG anti-b2GPI and IgG anti-DI, and compared these serological profiles to clinical outcomes of death or VE during the subsequent follow-up period.

Methods
At University College London Hospital (UCLH), the specialist SLE clinic has been running since 1979 and has followed over 600 patients. Stored serum samples from these patients have been kept at À80 C. All patients entered into the lupus cohort at UCLH fulfil the American College of Rheumatology criteria for SLE [11]. The earliest available serum samples from 501 patients were retrieved from storage and used in the ELISAs. For over 90% of patients, this earliest sample was obtained within two years of diagnosis.
Clinical information on VE and LA positivity was obtained from patient records and from interviews with patients (for those still alive and under follow-up in the clinic) with informed consent and with ethics approval from the London Hampstead Research Ethics Committee (Reference Number 12/LO/0373).
VE were defined as including deep venous thrombosis, pulmonary embolism, catastrophic APS, myocardial infarction, stroke and coronary heart disease (the last two required proof by appropriate imaging).

LA assay
LA is assessed at UCLH in accordance with national and international guidelines [12,13]. Samples are processed within four h of collection and platelet-poor plasma is prepared from blood withdrawn by venepuncture in 0.109 M sodium citrate 9 : 1, then double-centrifuged at 1500 g for 15 min and stored at À80 C immediately after preparation. LA activity is confirmed in non-anticoagulated samples by the DRVVT, using Siemens Healthcare (Marburg, Germany) LA1 (screening) and LA2 (confirmation) reagents (Siemens Healthcare (Marburg, Germany). For patients receiving warfarin, the DRVVT includes testing with screen and confirm reagents on equal volume mixtures of patient/normal plasma (which, if positive, confirms the presence of an inhibitor and phospholipid dependence) and a Taipan venom time (TVT)/Ecarin clotting time (Diagnostic Reagents Ltd, Thame, UK) ratio is also performed. The normalized ratio cut-off value for the DRVVT is 1.20 and for the Taipan venom time/Ecarin clotting time 1.12.

ELISA
The methods for the IgG aCL, IgG anti-b2GPI and IgG anti-DI ELISAs have been detailed fully in previous papers [9,14,15]. For this study, we had established positive/negative cut-offs in all three of these ELISAs as the 99th percentile of 40 healthy controls. These levels are 21 GPLU for aCL, 22 GBU for anti-b2GPI and 22 GDIU for anti-DI.
In this paper we use the term single-ELISA-positive for any patient positive for either IgG aCL or IgG anti-b2GPI or IgG anti-DI, double-ELISA-positive for any patient positive in any two of these ELISAs and triple-ELISA-positive for any patient positive in all three ELISAs. These terms are used to make a clear distinction with the terms double and triple-positive that are generally used in APS to refer to aCL, anti-b2GPI and LA [3,4].

Statistical analysis
For the KaplanMeier analysis of mortality, patients were divided on the basis of serology of the initial sample into aPL-single-ELISA-positive, aPL double/triple-ELISA positive and aPL-negative. Patients were censored at the time of loss to follow-up or at the end of the study period. For the KaplanMeier analysis of VE, patients were censored at the time of death, loss to follow-up or the end of the study period.

Results
For the 501 patients with SLE included in the study, mean age at the time of sample was 30 years (S.D. 12.2) and mean follow-up was 12.1 years, with a maximum of 36 years. There were 457 (91%) women and 307 (67%) Caucasians. For the 40 healthy controls, mean age was 31.3 years (S.D. 7.1), 26 (65%) were women and 33 (83%) Caucasian. Table 1 shows demographic, clinical and serological features of these patients.

Prevalence of IgG aCL, IgG anti-b2GPI and IgG anti-DI positivity at baseline
Of the 501 samples from early disease, 190 (38%) were positive for one or more of IgG aCL, IgG anti-b2GPI and IgG anti-DI. A total of 39 patients were double-ELISApositive for any of the three aPL, or triple-ELISA positive. The Venn diagram in Fig. 1 shows how the populations seropositive for these antibodies overlapped. IgG anti-DI was the most common antibody found (146/501 patients, 29%) whereas IgG anti-b2GPI was the least common (24/ 501, 5%). In fact, 112 patients (22%) were single-positive for anti-DI alone. Correlation was stronger between aCL and anti-b2GPI (correlation coefficient = 0.60) than between anti-DI and anti-b2GPI (0.19) or anti-DI and aCL (0.12). The mean IgG anti-DI level was higher in those who were positive for both IgG anti-DI and IgG anti-b2GPI than in those who were positive for IgG anti-DI alone (49.5 vs 24.2 GDIU, P = 0.04).

Vascular events
We had both aPL serology in the earliest sample and complete history of VE for a sub-group of 276 patients (25 men). Of these patients, 112 (41%) were positive for at least one of IgG aCL, IgG anti-b2GPI and IgG anti-DI in the original sample.
Eighty-three of these 276 patients had suffered a VE, including 43/112 (38%) in the aPL-positive group, 12/21 (57%) in the double/triple-ELISA-positive group and 40/ 164 (24%) in the aPL-negative group. The types of VE in each group are shown in Table 2.
Twenty-five (10 aPL-positive, four double/triple-ELISApositive) patients suffered the VE before the date of the earliest sample and were censored at a value of 0.1 years in the KaplanMeier analysis. The numbers remaining   We also compared aPL-positive (any) to aPL-negative, anti-DI-positive to anti-DI-negative and high anti-DI positive (>40 GDIU) to low-anti-DI positive and anti-DI negative and no differences between groups were seen.

Discussion
The strengths of this analysis are the large number of individual patients studied and the long-term follow-up of mortality and VE, as shown in the KaplanMeier analysis. The major findings were the high proportion of patients positive for IgG anti-DI in early disease and a trend towards increased risk of developing VE in patients who were either double or triple-ELISA-positive in early disease. The role of testing for anti-DI in diagnosis of APS has been tested in a number of studies. In a meta-analysis of 11 studies including 1218 patients with APS, 318 patients with SLE, 49 asymptomatic aPL-positive individuals and 1859 healthy controls, Radin et al. [16] reported that 45.4% of patients with APS were positive for anti-DI and that four of the five studies that looked at association between anti-DI-positivity and risk of thrombosis found such an association, with odds ratios ranging between 2.5 and 4. Other studies, however, have noted inconsistency of results obtained using different techniques for measuring anti-DI [17] and concluded that the anti-DI assay does not add to the predictive value for thrombosis of the combination of three assays used in current clinical practice [1720].
A large number of patients in our study tested positive for IgG anti-DI in samples taken very early in disease. There are two surprising things about this result. Firstly, several other papers have shown that IgG anti-DI positivity is less common than IgG aCL or anti-b2GPI positivity in patients with SLE [9, 18,19,21], leading to the argument that anti-DI is more specific for APS but less sensitive [1720]. A different result was obtained by Wahezi et al. [22] in a population of 183 children with SLE, of whom 25.1% were IgG anti-DI positive, though that paper did not report on presence of anti-b2GPI antibodies and only a minority of patients were tested for IgG aCL.
Secondly, 112 patients in our study were positive for IgG anti-DI but negative for aCL and anti-b2GPI. There was very low correlation between IgG anti-b2GPI and IgG anti-DI titres. This is very different to results obtained in previous studies in which these antibody specificities are strongly correlated [18,19] as would be expected from the fact that DI is part of b2GPI. For example, De Craemer et al. [19] tested 426 subjects and found that of 60 who were IgG anti-b2GPI-positive, 55 were also IgG anti-DIpositive. Iwaniec et al. [18] tested 103 patients with APS, 99 with SLE and 102 healthy control subjects and found that of 109 who were IgG anti-b2GPI-positive, 78 were also IgG anti-DI-positive. Only one patient was IgG anti-DI-positive but IgG antib 2GPI-negative.
One possible explanation for this apparently high level of isolated anti-DI positivity in our study is that it arises for methodological reasons. As described by Yin et al. [17] in a recent review, both IgG anti-b2GPI and IgG anti-DI assays give variable results depending on factors such as source of antigen, coating density of antigen and exposure of the immunogenic G40-R43 epitope on DI. Thus, it is possible that our anti-b2GPI ELISA gives falsely low results because that epitope is not fully exposed. Alternatively, if the positive cut-off for IgG anti-DI in this study (established in a relatively small number of healthy control subjects) is too low, there might be false-positive readings. It is impossible to exclude either of these possibilities completely, although in our paper comparing results from 111 patients with APS, 119 patients with SLE and 200 healthy controls, the specificity of the IgG anti-DI ELISA for APS was 96% [9].
The majority (100/146) of the IgG anti-DI-positive patients have low positive titres (between 21 and 40 GDIU) suggesting antibodies of low affinity. Arbuckle et al. [23] studied samples from 130 American military personnel that had been stored many years before they were diagnosed with SLE and found that autoantibodies were often present before clinical manifestations of the disease had developed. aPL were among the earliest specificities detected. It is possible that early in the disease course of our 501 patients, low affinity anti-DI antibodies are present. These antibodies could potentially bind to DI coated on an ELISA plate much better than whole b2GPI, where the key epitope on DI is not accessible in all molecules. As the disease progresses, some patients might lose these low affinity antibodies altogether, while others develop higher affinity anti-DI. However, because we do not have IgG anti-DI data from later time points, this idea is speculative. Although this study shows that 38% (190/501) of patients had at least one of these three aPL very early in disease, the analysis does not suggest that aPL-positivity per se predicts clinical outcomes. KaplanMeier analysis of mortality or vascular mortality did not show significant differences between aPL-positive and aPL-negative patients and causes of death in aPL-positive patients were not skewed towards VE.
The one subgroup of patients identified as having a potentially worse long-term outlook in this study were those who tested positive for either two or three of IgG aCL, anti-b2GPI and anti-DI early in disease. There were 39 such patients, but if anti-DI had not been tested, only 14 of these would have been identified (i.e. aCL plus anti-b2GPI positive). The KaplanMeier curves suggest that this double/triple-ELISA positive group do worse in terms of vascular outcomes, but this result cannot be over-interpreted because even in this large group of patients, the results did not reach statistical significance. There was, however, a strong association between double/triple-ELISA positivity and LA. Pengo et al. [24] noted that while there are some patients who test LApositive but anti-b2GPI negative (due to presence of IgM anti-phosphatidylserine/prothrombin antibodies), those positive in both assays have a much higher risk of thrombosis.
Limitations include the fact that this study is cross-sectional, with ELISA data for a single time-point for each patient, and that VE were recorded retrospectively from patients' charts and interviews. It is not possible to test LA in stored serum samples because of the method used in the LA assay, which requires the blood to clot.
Only IgG isotype antibodies were tested. In future studies it would be of interest to obtain samples taken from these same serological groups of patients at later dates to assess the extent to which single, double and triple-ELISA positivity remain stable over time.
In summary, IgG anti-DI antibodies were found in early serum samples from 29% of 501 patients with SLE and were more commonly detected than IgG anti-b2GPI and aCL. There was no strong evidence that testing for IgG anti-DI in early disease would alter management of patients with SLE.