å ORIGINAL ARTICLE å Distribution and Clinical Significance of Lupus Anticoagulant and Anticardiolipin Antibody in 349 Patients with

å ORIGINAL ARTICLE å Distribution and Clinical Significance of Lupus Anticoagulant and Anticardiolipin Antibody in 349 Patients with Systemic Lupus Erythematosus Chiho Ninomiya, Osamu Taniguchi, Toshihiko Kato*, Takao Hirano, Hiroshi Hashimoto and Shun-Ichi Hirose Samples from 349 patients with systemic lupus erythematosus (SLE) were tested simul taneously for lupus anticoagulant (LAC) and anticardiolipin antibodies (ACL). LAC was detected in 27.2% of 349 SLE patients by a modified mixing kaolin clotting time. ACL was detected in 34.7% by enzyme-linked immunosorbent assay. Only half of the patients who had LAC or ACL were positive for both of them. In addition, isotypes of ACL in these patients were studied. The IgG isotype was detected in 81.8% of 121 patients, and more than half had only the IgG isotype. When clinical features of patients with LAC or ACL were studied, the incidence of thrombosis, fetal loss, and thrombocytopenia were significantly higher in both groups compared with patients without LAC or ACL. In particular, the patients with both LAC and ACL showed the highest risk of fetal loss (89%) during pregnancy. These results indicate that LAC and ACL are detected in partly different groups of SLE patients, but both of these groups are clinically (Internal Medicine 31: 194-199, 1992) similar. Key words: antiphospholipid syndrome, fetal loss, thrombosis, thrombocytopenia, kaolin clotting time Introduction patients with syphilis, and the antigen bound by reagin was found to be an acid phospholipid named cardiolipin In 1952, Conley and Hartmann (1) first described the (6). Subsequently, a solid phase radioimmunoassay (7) presence of circulating anticoagulants in two systemic and an enzyme-linked immunosorbent assay (ELISA) lupus erythematosus (SLE) patients with hemorrhagic (8) for the detection of ACL have been reported, disorders, and prolonged prothrombin and whole blood Recent studies have revealed a close relationship clotting times. The term "lupus anticoagulant" (LAC) between the presence of LAC and elevated ACL levels was introduced in 1972 by Feinstein and Rapaport (2). (7, 9); the significant association is shown by the similar LAC is an antiphospholipid antibody directed against clinical features, such as thrombosis, recurrent fetal loss, the platelet wall phospholipids (platelet factor III) that and thrombocytopenia (7, 10). In addition, it has been interacts with factor X and factor V in the presence of reported that both LAC and ACL cross-react with nega Ca2+ ions (3). The presence of LAC causes prolonged tively charged phospholipids (3, ll). Thus, all these results prothrombin or partial thromboplastin times which can suggest that both LAC and ACL are antiphospholipid not be corrected by the addition of normal plasma, antibodies, and probably have the same or very similar Kaolin clotting time (4) and the Russell viper venom specificities. But there are few reports in which LAC time (5) have been reported to be methods which more and ACL were measured simultaneously in the same were specifically first reported detect in LAC. 1906 population when Wassermann of SLE patients, described and Here, the identity in an attempt of LAC to and clarify the relationship between On a complement the other hand, fixation anticardiolipin test to detect antibodies reagin in (ACL) the sera ACL of LAC is still and controversial. ACL and their clinical significance in SLE, From Division of Rheumatology, ""Department of Medicine and Department of Clinical Laboratory, Juntendo University School of Med Tokyo. Received for publication October 29, 1990; Accepted for publication August 26, 1991 Reprint request should be addressed to Chiho Ninomiya, MD, Division of Rheumatology, Department of Medicine, 2-1- 1 Hongo, Bunky Tokyo113,Japan 194 Internal Medicine Vol. 31, No. 2 (February 1992)

Antiphospholipid Antibodies in SLE LAC and ACL were simultaneously measured in 349 patients with SLE. In addition, the isotype of ACL was studied. Materials and Methods Patients Three hundred and forty-nine SLE patients followed in our hospital were selected at random and tested for LAC and ACL. They fulfilled the 1982 revised criteria for SLE of The American Rheumatism Association (12). One hundred out of 349 patients, who were registered in The Juntendo University Computer Center were chosen at random and their clinical features were analyzed to study the clinical significance of LAC and ACL. Patients with LAC or ACL, with both LAC and ACL, and without LAC or ACL indicated no significant dif ferences with respect to sex, age, disease duration, and therapeutic procedures. Clinical and Laboratory Data Clinical features such as thrombosis, fetal loss, and thrombocytopenia in the 100 patients selected for study were evaluated by their records since the time of SLE diagnosis. Hematological and serological tests were performed at the same time as LAC and ACL determinations. Samp les Sample sera were obtained by collecting blood by venipuncture into plain glass tubes. Platelet-poor plasma (PPP) was obtained by centrifuging the citrated blood (one-tenth volume of 3.8% w/v sodium citrate, ph 6.5) at 2500g for 30min at 4 C. Sera were stored at -20 C and PPP was stored at -80 C until assayed. Modified Mixing Kaolin Clotting Time (KCT) for Lupus Anticoagulant (LAC) A modified mixing kaolin clotting time (KCT) was carried out on a mixture of patient platelet-poor plasmas (PPP) and control PPP in the proportion of 1:1 (4). Control PPP was obtained by mixing PPP from 5 healthy male volunteers without coagulation abnormalities. One hundred microliters of kaolin solution (4mg/ml) was added to the same volume of the mixture of patient PPP and control PPP. After incubation for 3min at 37 C, 100 ja of 0.025 M CaCl2 was added and the time required for clotting was measured. A control was performed by measuring the KCT of a control PPP. Results were expressed as a ratio of patient to control clotting time, and were positive for LAC if the ratio was greater than 1.2 which is the upper limit of the normal range Enzyme-Linked Immunosorbent Assay (ELISA) for Anticardiolipin (mean + 3SD). Antibodies (A CL) ACL was measured using a modification of a pre viously described ELISA (8). Fifty microliters of car diolipin (Sigma Chemical Co., St. Louis, U.S.A.) at a concentration of loo^g/ml in ethanol was added to a microtiter well (Immulon 2 flat bottom plates, Dynatech Laboratories, Inc. Biotechnology Products, Chantilly, U.S.A.) and coated onto the surface of the well. Plates were then blocked with 10% fetal calf serum (FCS) in phosphate-buffered saline (PBS). Fifty microliters of test or standard serum diluted 1:50 in 10% FCS-PBS was added to each well. The plates were incubated for 1h at room temperature and washed. Fifty microliters of peroxidase-conjugated goat anti-human immunoglobulin (Sigma Chemical Co., St Louis, U.S.A.) diluted 1:4000 (IgG) or 1:2000 (IgM or IgA) in 10% FCS-PBS was added to each well and the plates were again incubated for lh at room temperature and washed. Substrate solution (4 mg of o-phenylenediamine dichlorohydride and 4 fa of H2O2 in 10ml of phosphate citrate buffer, ph 5.3) was added to each well and the plates were incubated in the dark at 25 C for 1h. The reaction was stopped by the addition of 2N H2SO4 and the optical absorbance was read at 492nm. Abnormal values were defined as those 2SD above the mean of normal controls. The Results control group consisted of 25 normal individuals. Frequency of LAC and ACL in Patients with SLE Figure 1 shows the frequency of LAC and ACL in 349 SLE patients. One hundred and sixty out of 349 patients (45.8%) had LAC or ACL. The percentage of patients with LAC was 27.2% (95 out of 349) and that with ACL was 34.7% (121 out of 349). Only 16% of the total patients had both LAC and ACL. The percentage of LAC-positive patients with ACL was 59.8% (56 out of 95), while LAC was detected in 46.3% (56 out of 121) of patients with ACL. Therefore, only half of the patients with LAC or ACL had both antibodies. Fig. 1. The incidence of lupus anticoagulant (LAC) and anticar diolipin antibodies (ACL) in patients with systemic lupus erythematosus (SLE). The overall incidence of LAC or ACL is 45.8% (160 out of 349), the incidence of LAC is 27.2% (95 out of 349), that of ACL is 34.7% (121 out of349) and that of both LAC and ACL is 16% (56 out of349). Internal Medicine Vol. 31, No. 2 (February 1992) 195

IgGy IgM, and IgA Isotypes ofacl in SLE The isotype of ACL in 121 patients with ACL was analyzed as shown in Table 1. The IgG isotype was the most frequent and was detected in 81.8% of ACL-positive patients. The frequencies of IgM and IgA isotypes were 33.9% and 9.9%, respectively. The pattern of the isotype combination was also studied. More than half of the patients (59.5%) had only the IgG isotype and 16.5% of The patients percentages had the of IgM other isotype patterns in addition were relatively to IgG. The low. frequency of patients with only the IgM isotype was 14%. Clinical and Laboratory Findings of Patients with LAC and ACL As mentioned in Materials and Methods, 100 patients with SLE were selected from 349 patients at random and the clinical significance of LAC and ACL was studied as shown in Table 2. Out of those 100 patients, 27 patients Table 1. Isotypes of Anticardiolipin Antibodies (ACL) in Patients with Systemic Lupus Erythematosus (SLE) No. of isotypes patients (%) IgG IgM IgA IgG only IgM only IgA only IgG + IgM IgG + IgA IgM + IgA IgG + IgM + IgA total 99 (81.8) 41 (33.9) 12( 9.9) 72 (59.5) 17 (14.0) 4( 3.3) 20 (16.5) 4( 3.3) 1( 0.8) 3( 2.5) 121 (100.0) Mokunoet al had LAC, 41 patients had ACL and 21 of the patients with LAC or ACL had both antibodies. The incidence of thrombosis, fetal loss, or thrombocytopenia was signifi cantly higher in the LAC-positive group than in the LAC-negative group, and it was also significantly higher in the ACL-positive group than in the ACL-negative group. In particular, the patients with both LAC and ACL showed the highest risk of fetal loss (89%) and thrombosis (48%). The PLT counts and the serum level of CH50 were significantly lower in the LAC-positive group than in the LAC-negative group and they were also lower in the ACL-positive group than in the ACL negative group. There were no significant differences in the PLT counts or the serum level of CH50 among LAC positive, ACL-positive, and both LAC and ACL-positive groups. The frequency of high titer of ANA was signifi cantly higher in patients with LAC or ACL compared with that of patients without LAC or ACL. The titer of anti-dna was also significantly higher in the LAC positive group and the ACL-positive group. In addition, the mean titer of anti-dna in the LAC-positive group was about two times as high as that of the ACL-positive or the LAC and ACL-positive group. Even though it was not statistically significant, the frequency of BFP-STS (biological false positive of serological tests for syphilis) was higher in the LAC-positive and the ACL-positive groups than in the LAC-negative and ACL-negative group. The frequency of positive direct Coombs' test was significantly higher in the LAC-positive and the ACL positive groups than in the LAC-negative and ACL Table 2. Correlation of Lupus Anticoagulant (LAC) or Anticardiolipin negative (ACL) groups. Antibody When the with clinical Clinical features and Laboratory of patientsfindings in Patien Systemic Lupus Erythematosus (SLE)* with only LAC were compared with those of patients with only ACL in the above-mentioned 100 patients, there were no statistically significant differences between them (data not shown). * Values for WBC, PLT, CH50 and Anti-DNA are the mean ± SD; the other values are number/total (%) of patients with the finding. PLT: platelets, ANA: antinuclear antibody, BFP-STS: biological false positive of serological tests for syphilis. ** PLT< l x ltfvmm3. # LAC-positive group vs LAC-negative group. ## ACL antibody-positive group vs ACL antibody-negative group. NS: not significant 196 Internal Medicine Vol. 31, No. 2 (February 1992)

An ELISA for ACL allowed the detection of isotypes of ACL so that the relationship between the isotype of ACL and clinical features could be studied further (data not shown). Out of the above-mentioned 41 patients with ACL, 26 (63%) had only the IgG isotype, 10 (24%) had both the IgG and IgM isotypes and 4 (10%) had only the IgM isotype. The patients with only the IgG ACL had significantly higher incidences of thrombosis (9/26, 35%, p<0.01), fetal loss (6/8, 75%, p<0.05), and thrombocytopenia (7/26, 27%, p < 0.02), compared with ACL-negative patients. In contrast, only the incidence of thrombocytopenia was significantly higher in patients with IgG and IgM ACL compared to that of ACL negative patients (40% vs 7%, p<0.01). In patients with only IgM ACL, there were no significant differences in the incidences of these clinical features compared to Discussion patients without ACL. In the present study, the frequencies of LAC- and ACL-positive patients were 27.2% and 34.7%, respect ively. Reports of the prevalence of LAC in SLE patients vary from 5 to 73% (10, 13, 14), and that ofacl from 21 to 63% (10, 14, 15). According to a previous report (13), the average prevalence of LAC is 28% and that of ACL is 42%. The prevalence of LAC obtained by KCT in the present study was compatible with this reported average. The prevalence of ACL reported here was reasonable but appreciably lower than the reported average. This may be due to the specificity of the method used to detect ACL or a different population of patients; some of these earlier studies were done before the intro duction Several clinical of the 1982 features revised such criteria as venous for SLE. and arterial thrombosis, fetal loss, thrombocytopenia, low serum level of complement, and high titer of ANA including anti-dna antibodies were associated with LAC and ACL in our patients with SLE. Although many previous reports have shown a significant association of LAC or ACL with the clinical manifestations considered by our study (10, 13, 14), there are few reports in which the clinical features associated with LAC and ACL have been Both studied LAC using and same ACL series have been of patients considered as in this to bestudy. associated with venous or arterial thrombosis (7, 16). According to accumulated studies, a history of throm boembolic events was seen in 42% of LAC-positive patients and in 40% of ACL-positive patients with SLE. These percentages are higher than those in the present study (13), but it is noteworthy that the incidence of thrombosis was the highest in our patients who had both LAC and ACL; almost half of these patients had a history of thrombosis. Many studies show a strong association between anti phospholipid antibodies and fetal loss in SLE and other Antiphospholipid Antibodies in SLE autoimmune disorders (10, 13). Previous studies indicate that the percentage of patients who have had fetal loss ranged from 13 to 68% in LAC or ACL-positive patients. In our lupus patients with a history of pregnancy, over 60% of patients with LAC or ACL had a history of fetal loss. Moreover, 8 out of 9 patients with both LAC and ACL had fetal loss. It is noteworthy that the coexistence of LAC and ACL indicated a high risk for fetal loss. The occurrence of thrombocytopenia was statistically associated with LAC and ACL in our patients as pre viously reported (10, 13, 17). The presence of LAC or ACL was also significantly related to a low level of platelets, when these were evaluated simultaneously. The binding of these antibodies to the platelet cell mem brane is a likely mechanism, but it has not been proven in vivo. In addition, a positive direct Coombs' test was signifi cantly associated with the presence of LAC and ACL in the present patients. This may be due to the reactivity of the antibodies with the red cell membrane phospholipids The as with present the platelets study demonstrated (18), but this a significant has not been associ proven. ation between a low serum level of complement and LAC and/or ACL-positive SLE patients. The previous study had noted an association between a low serum level of C4 or C3 and ACL, and a low serum level of C4 and LAC in SLE (18). While the association of these antibodies with hypocomplementemia may simply reflect the underlying disease activity, the mechanism of A hypocomplementemia significant association is between unclear. the high titer of ANA, and LAC and ACL was observed in the present patients. The frequency of anti-dna antibody was also correlated with both LAC and ACL. These correlations have been shown in many studies and there have been several reports concerning the cross-reactivity with DNA and phospholipids. Monoclonal anti-dna antibodies react with cardiolipin (19, 20), monoclonal ACL reacts with DNA (21), and monoclonal antibodies obtained by hybridoma derived from SLE patients with LAC react with dsdna (22, 23). In addition, it has been shown that monoclonal LAC derived from SLE patients can react with negatively charged phospholipids (22, 23), and the specificity of ACL measured by ELISA has been shown to be not only for cardiolipin but also for negatively charged phospholipids in general (24). These studies suggest that anti-dna antibodies, LAC and ACL are closely related antibodies that recognize the same or similar antigenic determinants. In addition to the close relationship between anti-dna antibodies, and LAC and ACL, a significant association between LAC or ACL and BFP-STS has also been reported (13). In the present study, the LAC-positive and ACL-positive Internal Medicine Vol. 31, No. 2 (February 1992) groups had a higher frequency of BFP-STS than their 197 respective antibody-negative groups. This may be related

serological test for syphilis, which consists of cardiolipin cholesterol-lecithin mixtures. It has been shown that ACL and LAC can be sig nificantly associated with the clinical features known as antiphospholipid syndrome. We further studied the clinical significance of ACL in relation to its isotype. Pre vious reports have shown a significant association of IgG ACL and thrombosis, fetal loss, and thrombocytopenia (25), and these observations were confirmed by the present results. More than half of the ACL-positive patients had only the IgG isotype, and they showed a significantly high incidence of thrombosis, fetal loss, and thrombocytopenia. The coexistence of IgG and IgM isotypes however, was associated with only a history of thrombocytopenia, and patients only with IgM ACL did not show any significant correlation with any of these clinical features. These results indicate that the isotype As analysis shown is in important the present in the results, determination LAC and ACL of the clinical features were associated that are with associated exactly with the ACL. same clinical features, suggesting that LAC and ACL have identical specificity. Several of the above-mentioned reports concerning the cross-reactivity between anti-dna antibody, LAC and ACL may support this observation. However there was a discrepancy between the distributions of LAC and ACL in the present lupus patients, and only half of the patients with LAC or ACL had both activities. One possible explanation for this descrepancy is the difference between methods (KCT and ELISA) used to detect these anti bodies. Recent studies identified the plasma protein The co factor other to possibility which ACL is that binds LAC directly and ACL in ELISA are essentially (26). different antibodies, although both are directed against phospholipids. The identity of LAC and ACL is still con troversial and further studies are needed for clarification. The present study revealed that the distribution of patients with LAC and patients with ACL was not exactly the same, and the simultaneous detection of both LAC and ACL affords an advantage in the management of SLE patients with higher sensitivity for antiphospholipid syndrome. In addition, it was shown to be very important for the management of lupus patients during pregnancy to detect both LAC and ACL, owing to the extremely high risk for fetal loss in patients with both LAC and ACL. Recently, there have been several reports con cerning a successful treatment which allowed normal delivery in patients who had a history of recurrent fetal loss and LAC (27). The present results suggest that preventive treatment should be considered in the case of patients who have both LAC and ACL during preg nancy. In addition, isotype analysis of ACL may also help in the evaluation of ACL-positive lupus patients, since the IgG isotype shows a strong correlation with antiphospholipid syndrome. Mokunoet al Acknowledgments: The authors gratefully acknowledge the useful comments and suggestions of Drs. Y. Takasaki, S. Kobayashi and M. Noguchi. References 1) Conley CL, Hartmann RC. A hemorrhagic disorder caused by circulating anticoagulant in patients with disseminated lupus erythematosus. J Clin Invest 31: 621, 1952. 2) Feinstein DI, Rapaport SI. Acquired inhibitors of blood coagu lation. Prog Hemostasis Thromb 1: 75, 1972. 3) Thiagarajan P, Shapiro SS, De Marco L. Monoclonal immunog lobulin M coagulation inhibitor with phospholipid specificity. J Clin Invest 66: 397, 1980. 4) Exner T, Rickard KA, Kronenberg H. A sensitive test demon strating lupus anticoagulant and its behavioral patterns. Br J Haematol 40: 143, 1978. 5) Thiagarajan P, Pengo V, Shapiro SS. The use of the dilute Russell viper venom time for the diagnosis of lupus anticoagulants. Blood 68: 869, 1986. 6) Pangborn MC. A new serologically active phospholipid from beef heart. Proc Soc Exp Biol Med 48: 484, 1941. 7) Harris EN, Gharavi AE, Boey ML, et al. Anticardiolipin anti bodies: detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet ii: 1088, 1983. 8) Loizou S, McQrea JD, Rudge AC, et al. Measurement of anti cardiolipin antibodies by an enzyme-linked immunosorbent assay (ELISA): standardization and quantitation of results. Clin Exp Immunol 62: 738, 1985. 9) Harris EN, Loizou S, Englert H, et al. Anticardiolipin antibodies and lupus anticoagulant. Lancet Hi: 1099, 1984. 10) Harris EN, Gharavi AE, Hughes GRV. Anti-phospholipid anti bodies. Clin Rheum Dis ll: 591, 1985. ll) Harris EN, Gharavi AE, Tincani A, et al. Affinity purified anticardiolipin and anti-dna antibodies. J Clin Lab Immunol 17: 155, 1985. 12) Tan EM, Cohen AS, Fries JF, et al. The 1982 revised criteria for the classification of SLE. Arthritis Rheum 25: 1271, 1982. 13) Love PE, Santoro SA. Antiphospholipid antibodies: Anticar diolipin and the lupus anticoagulant in systemic lupus erythema tosus (SLE) and in non-sle disorders. Ann Intern Med 112: 682, 1990. 14) Espinosa LR, Hartmann RC. Significance of the lupus anticoagu lant. Am J Hematol 22: 331, 1986. 15) Fort JG, Cowchock FS, Abruzzo JL, et al. Anticardiolipin anti bodies in patients with rheumatic diseases. Arthritis Rheum 30: 752, 1987. 16) Boey ML, Colaco CB, Gharavi AE, et al. Thrombosis in SLE: striking association with the presence of circulating 'lupus antico agulant'. Br Med J 287: 1021, 1983. 17) Harris EN, Asherson RA, Gharavi AE, et al. Thrombocytopenia in SLE and related autoimmune disorders: association with anti cardiolipin antibodies. Br J Haematol 59: 227, 1985. 18) Hazeltine M, Rauch J, Danoff D, et al. Antiphospholipid anti bodies in systemic lupus erythematosus: evidence of an association with positive Coombs' and hypocomplementemia. J Rheumatol 15: 80, 1988. 19) Koike T, Maruyama N, Funaki H, et al. Specificity of mouse hybridoma antibodies to DNA. Phospholipid reactivity and bio logical false positive serological test for syphilis. Clin Exp Immunol 57: 345, 1984. 20) Shoenfeld Y, Rauch J, Massicotte H, et al. Polyspecificity of monoclonal lupus autoantibodies produced by human-human hybridomas. N Engl J Med 308: 414, 1983. 21) Rauch J, Tannenbaum H, Stollar BD, et al. Monoclonal anti cardiolipin antibodies bind to DNA. Eur J Immunol 14: 529, 1984. 22) Rauch J, Tannenbaum M, Tannenbaum H, et al. Human hybri- 198 Internal Medicine Vol. 31, No. 2 (February 1992)

Antiphospholipid Antibodies in SLE doma lupus anticoagulants distinguish between lamellar of and anticardiolipin antibodies in systemic lupus erythematosus: hexagonal prospective phase analysis lipid of systems. a series J of Biol 100 Chem patients. 261: Ann 9672, Rheum 1986. Dis Janoff 49:109,1990. AS, Rauch J. The structural specificity of anti-phospholipid antibodies 26) Galli M, in Comfurius autoimmune P, disease. Maassen Chem C, et Phys al. Anticardiolipin Lipids 40: anti 315,1986. bodies (ACA) directed not to cardiolipin but to a plasma protein Gharavi co factor. AE, Lancet Harris 335: EN, 1544, Asherson 1990. RA, et al. Anticardiolipin antibodies: 27) Kobayashi isotype S, Tamura distribution N, Tsuda and phospholipid H, et al. Immunoadsorbent specificity. Ann Rheum plasmapheresis Dis 46: 1, for 1987. a patient with antiphospholipid syndrome Cevera during R, pregnancy. Font J, Lopez-Soto Ann Rheum A, Dis, et al. (in Isotype press). distribution Internal Medicine Vol. 31, No. 2 (February 1992) 199