Journal of Autoimmunity (1989) 2,843-849

IgG Class Anti-cardiolipin Antibody as a Possible Marker for Evaluating Fetal Risk in Patients with

Systemic Lupus Erythematosus

Kaoru Shimada, Takao Koike, Kenji Ichikawa, Akito Tsutsumi, Katsuhiko Takabayashi, Hisao Tomioka and Sho Yoshida

Second Department of Internal Medicine, Chiba University School of Medicine,

Chiba,Japan

To identify the correlation between incidence of anti-phospholipid anti- bodies and fetal prognosis in pregnant SLE patients, we measured the amount of anti-cardiolipin antibody in their sera, using solid-phase enzyme immunoassay (EIA) methods. Findings in the group having poor obstetric results (fetal loss group) and in those with a history of full-term births (live birth group) were compared with regard to other anti- phospholipid antibodies. The incidence of IgG class anti-cardiolipin antibody was 60% in the fetal loss group and 19% in the live birth group, (P~0.05). The .incidence of the other anti-phospholipid antibodies, including lupus anticoagulant and biological false-positive serological test for syphilis (BFP-STS), did not differ significantly between the two groups. Therefore, the presence of IgG class anti-cardiolipin antibody may prove to be a useful marker for evaluating fetal risk in SLE patients.

Introduction

Since systemic lupus erythematosus (SLE) is predominantly a disease of young women; pregnancy and delivery in these patients require close clinical follow-up. In SLE patients, spontaneous abortion or intrauterine fetal deaths may occur. Wallace

and Dubois reported that 28% of pregnancies in SLE women resulted in miscarriage [l]. It has been suggested that fetal loss in SLE patients is related to the presence of anti-phospholipid antibodies, including lupus anticoagulant and anti-cardiolipin antibody [2-81.

Correspondence and reprint requests to Takao Koike, MD, Second Department of Internal Medicine, Chiba University School of Medicine, 1-8-1 Inohana, Chiba 280, Japan.

843

0896-841 l/89/060843 + 07 $03.00/O 0 1989 Academic Press Limited

844 K. Shimada et al.

Table 1. History of pregnancy in 31 SLEpatients

Patient Pregnancy Full term Fetal Artificial number number birth loss abortion

Live birth group Fetal loss group

21 40 29 0 11 10 31 2 25 4

We examined the level of anti-cardiolipin antibody in sera from SLE patients with a history of pregnancies, using solid-phase enzyme immunoassay, and investigated the correlation between anti-phospholipid antibodies, in particular anti-cardiolipin antibody in addition to the biological false-positive serological test for syphilis (BPF-STS), lupus anticoagulant and fetal outcome in these patients.

Materials and methods

Patients

Thirty-one patients with history of pregnancies after the diagnosis of SLE were studied. Those with a history of artificial abortion alone were excluded. All patients fulfilled the diagnostic criteria of the American Rheumatism Association for SLE [9]. They were all in inactive state with 10 mg/d or less of oral prednisolone at the time of pregnancy. There was a total of 71 deliveries, including 31 full-term births, no premature births, 14 spontaneous abortions, 11 intrauterine fetal deaths and 15 artificial abortions. Spontaneous abortions and intrauterine fetal deaths were considered together as fetal losses, making a total of 25 fetal losses (Table 1).

The patients were divided into two groups: group 1, history of full-term live births alone and no fetal losses, designated the live birth group and including 21 cases; group 2, history of one or more fetal losses, designated the fetal loss group and including 10 cases.

Sera were obtained during pregnancy or after delivery when SLE was inactive. The sera were stored at - 20°C until use.

Anti-cardiolipin antibody

Anti-cardiolipin antibody was measured by a solid-phase enzyme immunoassay (EIA), as described by Koike et al [lo]. Briefly, each well of polyvinyl chloride microtiterplates (Dynatech Laboratories, Alexandria, Virginia, USA) was first coated with 50~1 of cardiolipin (Sigma Chemical Company, St Louis, Missouri, USA) solution (1 mg/ml in 0.01 M PBS) by incubation overnight at 4°C. After blocking procedures, 50 ul of sample seradiluted with 10% fetal calf serum/phosphate buffered saline (FCS-PBS) were added to each well in triplicate and the plates incubated for 2 h at room temperature. Alkaline phosphatase (Sigma) labelled mouse anti-human IgG or anti-human IgM (Cappel Laboratories, West Chester, Pennsylvania, USA) were used to detect the autoantibodies bound to cardiolipin. Optical absorbance was measured at 405 nm by a Microelisa Auto Reader, MR 580 (Dynatech).

Anti-phospholipid antibody and SLE 845

The amount of anti-cardiolipin antibody was expressed in units, using one sample

as standard which was stated to include 100 units of the antibody. Sera from 17

healthy females with no history of habitual abortion were used as normal controls. A titre greater than mean + 2 SD of control samples was regarded as positive for the antibody.

Lupus anticoagulant

Lupus anticoagulant activity was measured by two methods, kaolin-clotting time

and the tissue thromboplastin inhibition test, described by Proctor and Rapaport

[Ill.

Serological test for syphilis

Serological test for syphilis was measured by a modified flocculation test (IATRON

Laboratories, Tokyo, Japan) and TPHA test (Fujirebio Inc., Tokyo, Japan), respectively.

Statistical analysis

Statistical analysis was performed by Chi-square test.

Results

Anti-cardiolipin antibody in pregnant SLE patients

The values of IgG and IgM anti-cardiolipin antibodies for the live birth group and

the fetal loss group are shown in Figure 1. Sera from four of 21(19.0%) patients in the live birth group and six of 10 (60%) patients in the fetal loss group were positive for IgG class anti-cardiolipin antibody. The magnitude of the IgG class anti-cardiolipin antibody was significantly different between the live birth group (10.6 + 10.9 units) and the fetal loss group (24.7 f 30.6 units, I’< 0.05). IgM class anti-cardiolipin anti- body was detected in sera from eight of 21(38.1x) patients in the live birth group and four of 10 (40%) patients in the fetal loss group. There was no significant difference in antibody levels between the live birth group (20.4 + 32.8 units) and the fetal loss group (15.8-t 14.5 units).

Other anti-phospholipid antibodies in pregnant SLE

Lupus anticoagulant activity was detected in sera from 12% of the live birth group and 33% ofthefetal loss group. BFP-STS was observedin 14% ofthelive birthgroup

and 10% of the fetal loss group. There were, however, no statistically significant differences in these findings between the groups.

Relationship between anti-cardiolipin antibody and other anti-phospholipid antibodies

The correlation between the incidence of anti-cardiolipin antibody and lupus anti- coagulant is shown in Figure 2. Forty-three percent of the IgG anti-cardiolipin

846 K. Shimada et al.

(A)

Normal

in= 17)

Live birth

(/7=21)

Fetal loss (n = IO)

(8)

Normal (n=211

Live birth

(n=Zll

Fetal lot% (n-IO)

unit

16 50 100 ........................ I I ............ ............ ............ ............ ............ .~_~,~,‘.‘.~,~_~.~.-.~.~

. . ............ ............ ............ ::::: ‘.~_~_~.‘.‘.’ ,‘,‘.~.‘.‘.‘.~_~_‘_~,~,’ ,~.~.~_~.~.~,~.~.-.~.~.~ ............ ............ ............ ............ ............ ............ _‘_~_~_‘.‘.‘.~_~ ::: .‘_~.‘.‘.‘.‘_‘_~ ::: _~_~_‘.‘.‘.~_~_~ ::: fML+ .;:::: . . ‘ . ......

t ___.,.,.,_____:. 1.:::: _~_‘_~.‘.‘.~_~_~ ::: ....................... .‘_~.‘.‘.‘.‘_~_~_~_‘.‘.~ ............ ............ ............ ............ ............ .......... ............ .:,I :.:.:.:.:.:.:.:.:.: ............ ............ ............ ............ ............ ............ ........................ ............ .~.~.~,‘,‘.‘.~.‘.-_-.~.’ ...... .............. ..ww ............ ............ ............ ............ ............ ............ ............ I

. ............ ............ ............ _‘_~,‘.‘.‘.~_~.~.‘.‘.~.~ .~.~,~_~.~_~.-.-.-.-.-.- ............ ............ t

unit

21 50 100

Figure 1. Levels of anti-cardiolipin antibody in sera from 31 pregnant patients with SLE (21 cases with history of full-term births: live birth group, and 10 cases with history of fetal losses: fetal loss group). The values of anti-cardiolipin antibody of the IgG (A) or IgM (B) class are expressed as a unit of the standard sera. The shaded area represents the mean + 2 SD for the control group. The vertical bars indicate the means.

antibody-positive patients and 56% of the IgM anti-cardiolipin antibody-positive patients had lupus anticoagulant. Conversely, all patients with lupus anticoagulant had IgG class and/or IgM class anti-cardiolipin antibody. Similarly, 44% of the IgG class anti-cardiolipin antibody-positive patients and 33% of the IgM class anti- cardiolipin antibody-positive patients had BFP-STS. Conversely, all patients with BFP-STS had both IgG and IgM class anti-cardiolipin antibodies. Fifty percent of the patients with BFP-STS also had lupus anticoagulant.

Discussion

We examined the correlation between fetal loss and occurrence of anti-phospholipid antibodies in pregnant patients with SLE. The incidence of IgG class anti-cardiolipin

Anti-phospholipid antibody and SLE 847

(At ACA

LA positive

(0) ACA

(+I t-1

BFP poritivr

Figure 2. Schematic diagram showing the relationship between the incidence of anti-cardiolipin antibody and the presence of lupus anticoagulant (LA) (A) or the presence of biological false-positive serological test for syphilis (BFP-STS) (B).

antibody differed significantly between the live birth group and the fetal loss group. On the other hand, the incidence of IgM class anti-cardiolipin antibody differed little between the two groups. A tendency toward positive lupus anticoagulant was seen in the fetal loss group, but without statistical significance. Therefore, measurement of IgG class anti-cardiolipin antibody was considered to yield pertinent results for determining the fetal risk in pregnant SLE patients.

We also examined other autoantibodies, including anti-ssDNA antibody, anti- dsDNA antibody and anti-SS-A antibody, but found no significant difference between the occurrence of these antibodies and fetal prognosis (data not shown).

Anti-phospholipid antibodies (anti-cardiolipin antibody, lupus anticoagulant and BFP-STS) were most often simultaneously present in a single specimen. It is unclear whether this is due to the cross-reactivity of a single antibody or to coexistence

848 K. Shimada et UC.

of different antibodies. Colaco et al. [12] described the cross-reactivity of anti- cardiolipin antibody to ssDNA and Branch et al. [ 131 reported that the lupus anti- coagulant was assumed to be an anti-phosphatidylserine antibody. We also observed that some of the monoclonal anti-cardiolipin antibodies established from SLE prone MRLjZpr mice cross-reacted to ssDNA and acted as lupus anticoagulant [ 141.

It is suggested that fetal loss in SLE patients occurs as a result of placental infarc- tion due to thrombosis [15]. Carreras et al. [16] and De Wolf et al. [17] described anti-phospholipid antibody inhibition of prostacyclin production in the endothelium of blood vessels and suggested that this may play an important role in causing thrombosis. There is also a report that lupus anticoagulant has an inhibitory effect on prekallikrein and on the plasminogen system [ 181. Whether anti-phospholipid antibodies, especially IgG class anti-cardiolipin antibody, is responsible for the occurrence of these vascular manifestations is not yet determined.

Lubbe et al. [19] reported that five of six patients with poor obstetric histories and lupus anticoagulant had a successful delivery following administration of 40- 60 mg/d of prednisolone and 75 mg/d of aspirin. Branch et al. [20] reported that oral administration of 40-50 mg/d of prednisolone and 80 mg/d of aspirin decreased the rate of fetal death in SLE patients with lupus anticoagulant from 96.8% to 37.5%. They stressed the importance of treating SLE patients with lupus anticoagulant to prevent fetal loss. But successful pregnancies without specific therapies have been reported [21], and worldwide prospective studies must be made to seek suitable therapeutic procedures.

In conclusion, detection of the IgG class anti-cardiolipin antibody in sera from pregnant SLE patients is expected to be a useful tool for determining the risk of fetal loss.

Acknowledgements

This work was supported by a grant from the Ministry of Health and Welfare, Japan. We thank M. Ohara for critical comments.

1.

2.

3.

4.

5.

6.

References

Wallace, D. J. and E. D. Dubois. 1987. Lupus pregnancy and neonatal lupus. In Dubois’ Lupus Erythematosus. D. J. Wallace and E. D. Dubois. Lea and Febiger, Philadelphia. pp. 565-579 Drue, G. J., H. J. Enlert, E. N. Harris, A. E. Gharavi, S. H. Morgan, R. G. Hull, M. G. Elder, D. F. Hawkins, and G. R. V. Hughes. 1985. Fetal loss in systemic lupus: association with anticardiolipin antibodies. J. Obstet. Gynaecol. 5: 207-209 Lockshin, M. D., M. L. Druzin, S. Goei, T. Quman, M. S. Magid, L. Jovanoric, and M. Fereng. 1985. Antibody to cardiolipin as a predictor of fetal distress or death in pregnant patients with lupus erythmatosus. N. Engl. J. Med. 313: 152-156 Derksen, R. H. W. M. and L. Kater. 1985. Lupus anticoagulant: revival of an old phenomenon. Clin. Exp. Rheumatol. 3: 349-357 Lockshin, M. D., P. C. Harpel, M. L. Druzin, C. G. Becker, R. F. Klein, R. M. Watson, K. B. Elkon, and E. Reinitz. 1985. Lupus pregnancy. II. Unusual pattern of hypo- complementemia and thrombocytopenia in the pregnant patient. Arthritis Rheum. 28: 58-66 Mintz, G., J. Niz, G. Guitierrez, A. Garcia-Alonso, and S. Karchmer. 1986. Prospective study of pregnancy in systemic lupus erythematosus. Results of a multidisciplinary approach. J. Rheumatol. 13: 732-739

Anti-phospholipid antibody and SLE 849

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

Harris, E. N., J. K. H. Chan, R. A. Asherson, V. R. Aber, A. E. Gharavi, and G. R. V. Hughes. 1986. Thrombosis, recurrent fetal loss and thrombocytopenia. Predictive value of the anticardiolipin test. Arch. Intern. Med. 146: 2153-2156 Lockshin, M. D., T. Qamar, M. L. Druzin, and S. Goei. 1987. Antibody to cardiolipin lupus anticoagulant and fetal death. J. Rheumatol. 14: 259-262 Tan, E. M., A. S. Cohen, J. F. Fries, A. T. Masi, D. J. Mcshane, N. F. Rothfield, J. G. Schaller, N. Talal, and R. J. Winfield. 1982. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 25: 1271-1277 Koike, T., M. Sueishi, H. Funaki, H. Tomioka, and S. Yoshida. 1984. Anti-phospholipid antibodies and biological false positive serological tests for syphilis in patients with systemic lupus erythematosus. Clin. Exp. Zmmunol. 56: 193-199 Proctor, R. R. and S. I. Rapaport. 1961. The partial thromboplastin time with kaolin. A simple screening test for first stage plasma clotting factor deficiencies. Am.J. Cl&. Pathol. 36: 212-219 Colaco, C. B., G. K. Scadding, and S. Lockhart. 1987. Cross-reaction with anti-single stranded DNA activity. Clin. Exp. Zmmunol. 68: 313-319 Branch, D. W., N. S. Rote, D. A. Dostal, and J. R. Scott. 1987. Association of lupus anticoagulant with antibody against phosphatidyl serine. Clin. Zmmunol. Zmmunopathol. 42: 63-75 Ichikawa, K., K. Shimada, Y. Nawata, T. Ishii, H. Tomioka, S. Yoshida, and T. Koike. 1988. Monoclonal hybridoma anti-cardiolipin antibodies from SLE mice. C&n. Exp. Zmmunol. 74: 110-l 14 Carreras, L. O., G. Defreyn, S. J. Machin, J. Vermylen, R. Deman, B. Spitz, and A. van Assche. 1981. Arterial thrombosis intrauterine death and lupus anticoagulant: detection of immunoglobulin interfering with prostacyclin formation. Lancet 1: 244-246 Carreras, L. 0. and J. G. Vermylen. 1982. ‘Lupus’ anticoagulant and thrombosis-possible role of inhibition of prostacyclin formation. Thromb. Haematstas. 48: 38-40 De Wolf, F., L. 0. Carreras, P. Moerman, J. Vermylen, A. Van Assche, and M. Renaer. 1982. Decidual vasculopathy and extensive placental infarction in a patient with repeated thrombotic accidents, recurrent fetal loss and a lupus anticoagulant. Am. J. Obstet. GynecoE. 77: 829-834 Sanfelippo, M. J. and C. J. Drayne. 1982. Prekallikrein inhibition associated with the lupus anticoagulant, a mechanism of thrombosis. Am. 3. CZin. Pathol. 77: 275-279 Lubbe, W. F., S. J. Palmer, W. S. Butler, and G. C. Liggins. 1983. Fetal survival after prednisolone suppression of maternal lupus-anticoagulant. Lancet 1: 1361-1363 Branch, D. W., J. R. Scott, N. K. Kochenour, and E. Hershgold. 1985. Obstetric compli- cations associated with lupus anticoagulant. N. Engl. 3. Med. 313: 1322-1326 Stafford-Brady, F. J., D. D. Gladman, and M. B. Urowitz. 1988. Successful pregnancy in systemic lupus erythematosus with an untreated lupus anticoagulant. Arch. Intern. Med. 148: 1647-1648