1 Liverpool Women's Hospital, Crown Street, Liverpool L8 7SS, 2 Department of Obstetrics and Gynaecology, University of Liverpool, Liverpool, UK, 3 Department of Haematology, University of Sheffield, Sheffield, UK and 4 Department of Medicine and Therapeutics, University of Aberdeen, Aberdeen, UK
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Abstract |
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Key words: antibodies/antiphospholipid/pregnancy
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Introduction |
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There are diagnostic difficulties in APS which relate principally to the indirect nature of the tests used and problems in their performance and interpretation (the Lupus Anticoagulant Working Party, 1990; Jennings et al., 1998
). In relation to anticardiolipin, there is controversy over the clinical significance of low titre positivity, and it has been suggested that intervention using potentially toxic drugs such as heparin and corticosteroids should be restricted to those women with strongly positive tests (Kutteh, 1996
; Silver et al., 1996
). In contrast, women with low titre antibodies have apparently benefited from treatment with heparin (Rai et al., 1997
). There are also problems associated with the assays for lupus anticoagulant (LA), as they are essentially non-quantitative. Furthermore, the physiological haemostatic changes which occur during healthy pregnancy result in a shortening of clotting times (Wright et al., 1988
). The complexity of the situation is compounded by the large intra-patient variation in test results over time, one-third to one-half of APA-positive subjects testing negative when reassessed (Creagh et al., 1991a
; Rai et al., 1995
). Because of these observations, it has been recommended that APS be diagnosed only when tests for APA are positive on two occasions at least 8 weeks apart in a subject with a consistent clinical history. Whether the recent introduction of tests for antibodies to the phospholipid binding proteins which carry the target antigens for APA will improve specificity, remains to be determined.
Little is known of variations in APA during pregnancy. In our recurrent miscarriage cases, ~20% of women have persistent APA pre-conceptually (Quenby and Farquharson, 1993), when we employ comprehensive laboratory testing. In some cases we have noted a rise in the anticardiolipin titre in early pregnancy. In view of this, and the recent potentially important suggestion that rising titres of anticardiolipin herald pregnancy loss (Kwak et al., 1994
), we have monitored APA serially through pregnancy in women with APS and in a control group of women with recurrent miscarriage but negative results of tests for APA pre-conceptually.
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Materials and methods |
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IgG and IgM anticardiolipin were quantified in serum using a previously validated ELISA calibrated against an international standard (Hill et al., 1995). The upper limit of the normal range was determined from the log transformed mean plus 2 SD of results in 50 healthy adults [9 IgG phospholipid units (GPU)/ml IgG and 4 IgM phospholipid units (MPU)/ml IgM anticardiolipin]. Screening for lupus anticoagulant was by the Kaolin Cephalin Clotting Time (KCCT) utilizing sensitive reagents and by the Dilute Russell's Viper Venom Time (DRVVT) with a neutralization procedure using frozenthawed platelets. Based on results in 50 healthy non-pregnant subjects a positive result was considered to be a DRVVT ratio to normal of
1.1 with
20% correction in the platelet neutralization step.
The data were analysed by trimester, where the first trimester was defined as 012 weeks, second trimester 1224 weeks and third trimester after 24 weeks. The mean of the two results for each patient in each trimester was used for analysis. Statistical analysis was by the Wilcoxon signed ranks tests and was performed using the Arcus software package for personal computers.
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Results |
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There was considerable intra-individual variation in test results throughout pregnancy. Amongst the controls none was positive for APA on pre-pregnancy testing, by definition. However, five control women had positive tests in the first trimester (three were positive for lupus anticoagulant and two had a raised titre of IgG anticardiolipin) (Table II). These increases in assay results for women negative for APS pre-pregnancy, were surprisingly large (for the DRVVT ratio: from 0.9, 1.0 and 1.09 to 1.36, 1.15, and 1.19 respectively, and for the IgG ACA: from 2 and 4.4 to 41.7 and 15.8 respectively). Among the women in the APS group, highest ACA and DRVVT results were also found in the first trimester (Figures 1 and 2
). The highest titres of IgG anticardiolipin were seen in the first trimester in 24 out of the total 32 patients studied longitudinally. The highest ratios in the DRVVT were also seen at this time, in 23 out of 32 patients. In the entire study cohort of 32 women a statistically significant difference in both the DRVVT and IgG anticardiolipin titres was demonstrated between pre-pregnancy and the first trimester samples (Tables III and IV
). In the control group, the change did not persist later in pregnancy; APA positivity was noted in the second and third trimesters in one subject only (Table II
). The APS group had a progressive decline in the level of positivity as pregnancy progressed (Figures 1 and 2
).
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Discussion |
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In support of the apparent change in clotting times however, the IgG anticardiolipin titre was also highest in the first trimester in women with APS, with a subsequent fall in some women, often into the normal range. This apparent increase in anticardiolipin titre in the first trimester is of interest. The serum concentration of total IgG falls during pregnancy (El-Roiey et al., 1990). Although levels of autoantibodies appear to increase in healthy pregnancy they remain within the range of normal, including anticardiolipin (Patton et al., 1987
). It is therefore possible to speculate that maternal exposure to antigenic stimuli may result in an increase in the autoimmune response in early pregnancy in women with APS. Alternatively, deficiency in the cellular adhesion mechanism of the maternalconceptus interface caused by antiphospholipid antibodies, may lead to increased exposure of fetal cells to the maternal circulation (Lim et al., 1996
). Further evidence for such autoimmune aetiologies of recurrent pregnancy loss has been discussed in detail by Christiansen (1996).
It is unlikely that the treatment given was responsible for the changes in APA noted through pregnancy. Aspirin is not known to have any immunomodulatory effect and does not influence the fluid phase of coagulation. Heparin can result in prolongation of the KCCT and DRVVT, but does not do so when administered in prophylactic doses. Also, low molecular weight heparin has limited antithrombin activity and does not influence the clotting time in the doses used. Heparin has been shown to reduce APA binding to cardiolipin in vitro (Ermel et al., 1995). However, in this study, the low molecular weight heparin treatment was initiated prior to the first trimester test which was generally higher than the pre-pregnancy test (Figures 1 and 2
), and therefore the change in DRVVT was independent of the administered low molecular weight heparin.
It is noteworthy that a decrease in the anticardiolipin titre was a feature in all of those pregnancies with successful outcomes including the control patients, who although tested negative preconceptually had the highest levels in the first trimester that decreased progressively until delivery. Because we had no late pregnancy complications, we could not demonstrate whether a persistently high level would have predicted an adverse event, e.g. pre-eclampsia, abruption or sudden intrauterine death. Our findings are consistent with the observations of Kwak et al. (1994), who suggested that a down-regulation of maternal antiphospholipid antibodies is a good prognostic feature, although no attempt was made to assay for lupus anticoagulant in pregnancy in that study. Some data from studies in experimental animals suggest a possible causal role for anticardiolipin in pregnancy loss (Branch et al., 1990), although this is disputed and no such evidence exists in humans. Despite this, serial measurement of anticardiolipin titre in pregnancy may serve as a surrogate marker for the pathogenic process which leads to miscarriage and permit better discrimination of those at particular risk. As evidence on the efficacy of various therapeutic regimens accrues (Rai et al., 1997
) such a facility could be of benefit, especially as treatment with heparin is not without risk.
The findings in the control group were unexpected, but consistent with the known occurrence of transient antiphospholipid antibodies. These have frequently been associated with intercurrent and chronic infections (Al-Saeed et al., 1994), although there is no reason to suspect an infectious pathogenesis in these women with recurrent miscarriage. Persistently positive tests for APA were found in ~3% of 501 unselected women in the first trimester of apparently healthy pregnancies and a similar proportion had transiently positive tests (Creagh et al., 1991a
). There is, therefore, a low prevalence of often transient APA positivity in apparently healthy pregnancies and the data presented here indicates that this may be increased in pregnancies in women with a history of recurrent miscarriage. The transience of the test abnormality and the uniformly good outcome of the pregnancies distinguishes this situation from that in women with APS, where persistence of APA is found pre-conceptually and maintained through pregnancy, especially in those who miscarry. The occurrence of transient APA in the control group may reflect the rise in titres seen in the APS group. It is unknown whether the development of APA during pregnancy has pathological significance.
The results of this study emphasize the need for a comprehensive clinical and laboratory approach to the diagnosis of APS. Future studies, in larger cohorts of women, should further address the prognostic value of the changes in APA reactivity which occur through pregnancy in women with APS; ideally, these studies should include women on no treatment.
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Notes |
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References |
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Submitted on April 23, 1998; accepted on October 20, 1998.