Value of autoantibodies to ß2-glycoprotein 1 in the diagnosis of antiphospholipid syndrome

M. A. P. Audrain, D. El Kouri1, M. A. Hamidou1, L. Mioche, A. Ibara, M.-L. Langlois and J.-Y. Muller

Laboratoire d'Immunologie and
1 Service de Médecine Interne, Centre Hospitalier Universitaire, 9 quai Moncousu, 44093 Nantes cedex 1, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives. To define the specificity and positive predictive value of anti-ß2-glycoprotein 1 (anti-ß2GP1) antibodies for the diagnosis of antiphospholipid syndrome (APS).

Methods. We determined the presence of anticardiolipin (aCL) antibodies and anti-ß2-glycoprotein 1 (anti-ß2GP1) immunoglobulin (Ig) G and IgM in 191 consecutive sera from 191 patients and reviewed clinical data separately. aCL IgG and IgM were detected separately using commercial ELISA kits. Anti-ß2GP1 antibodies were detected with an in-house ELISA using ß2GP1.

Results. Seven patients were diagnosed as having APS and 184 as having other diseases. Thirty-six patients were aCL-positive and 12 were anti-ß2GP1-positive, seven of these 12 were APS patients. The specificity for anti-ß2GP1 in our population was 97%, with a positive predictive value (PPV) of 58%. Among the aCL-positive patients, specificity was 90% and PPV 70–87%.

Conclusions. This study shows that anti-ß2GP1 antibodies have a higher specificity and PPV than aCL for APS. The PPV of anti-ß2GP1 was greater in aCL-positive than in all patients. We conclude that screening for anti-ß2GP1 antibodies in aCL-positive patients increases the specificity and the PPV of aCL testing. In addition, we show that there is no need to screen for anti-ß2GP1 antibodies in the absence of aCL antibodies and in the absence of strong clinical suspicion of APS.

KEY WORDS: APS, Anti-ß2GP1 antibodies, aCL antibodies.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antiphospholipid antibodies are a heterogeneous family of autoantibodies and include antibodies directed against cardiolipin (aCL) or other phospholipids, detected by enzyme-linked immunosorbent assay (ELISA), and lupus anticoagulant (LAC), detected by clotting tests. These antibodies are associated with the clinical manifestations of venous or arterial thrombosis and recurrent foetal losses. The association of one clinical criterion with one biological criterion (aCL and/or LAC on two or more occasions at least 6 weeks apart) defines the antiphospholipid syndrome (APS) [1]. APS can be primary or secondary, when it occurs in patients with other autoimmune diseases, particularly systemic lupus erythematosus. aPL antibodies can also be found in other clinical circumstances, such as infections, malignancies and sometimes in healthy subjects.

It is now known that aPL antibodies are directed not only against phospholipids but also against a complex of a phospholipid with one of a group of phospholipid-binding plasma proteins (cofactors), such as ß2-glycoprotein 1 (ß2GP1) [2, 3] and prothrombin [4], and that antibodies directed against these cofactors can be detected by ELISA in the absence of phospholipids [5, 6]. Many reports demonstrate the association of anti-ß2GP1 antibodies with the APS [6, 711] and some reports show that anti-ß2-GP1 antibodies can be found alone without aPL antibodies [12, 13].

The aim of our study was (i) to define the specificity and positive predictive value (PPV) of anti-ß2GP1 antibodies for the diagnosis of APS in a large population without prior knowledge of aCL status and to compare them with the specificity and PPV of aCL antibodies, and (ii) to investigate whether anti-ß2GP1 antibodies may be found in the absence of aCL and establish whether searching for them in the absence of aCL is helpful in the diagnosis of APS.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
We selected sera from 191 consecutive patients (from the departments of internal medicine and gynaecology and the thrombosis unit) that had been sent to the laboratory for routine aPL determination. We searched for aCL immunoglobulin (Ig) G and IgM and anti-ß2GP1 IgG and IgM. Sera were collected by venipuncture and stored at -20°C in aliquots until use. Medical records of the study subjects were reviewed retrospectively by one physician without prior knowledge of the biological data. The clinical criteria for the diagnosis of APS were as follows: vascular thrombosis (venous or arterial) or pregnancy morbidity (one or more unexplained deaths beyond the 10th week of gestation; one or more premature births because of severe pre-eclampsia or severe placental insufficiency; three or more unexplained consecutive spontaneous abortions before the 10th week of gestation).

Clinical and biological data were then recorded. A definitive diagnosis of APS was made when at least one clinical and one biological criterion was present. A positive biological criterion was confirmed at least 6 weeks later.

Methods
aCL IgG and IgM were detected separately using commercial kits (Kallestad; Chaska, MN, USA). The negative cut-off was established at <10 U GPL and <10 U MPL; values >15 were considered positive and values between 10 and 15 were considered doubtful.

Anti-ß2GP1 antibodies were detected with an in-house ELISA using ß2GP1 from Diagnostica Stago (Asnières, France). The assay was validated with sera proposed for the European APL Forum for ß2GP1 ELISA Standardization [14]. Briefly, microplates (Maxisorp; Nunc, Roskilde, Denmark) were coated overnight with 5 µg/ml of ß2GP1. The plates were then blocked with gelatin. After washing, 1:50 diluted sera [serum samples from the patients, a pool of normal human sera as negative control, an aliquot of a positive serum as positive control (from one positive patient) and a pool of five positive sera (made by mixing several positive sera) used to produce a standard curve)] were incubated on coated and uncoated wells in duplicate for 1 h at 20°C. After washing, human serum antibodies were detected using an alkaline phosphatase-conjugated goat anti-human IgG (heavy and light chain) (Bioatlantic, Nantes, France), and if a positive result was obtained, the serum was also tested with an alkaline phosphatase conjugated goat anti-human IgG and IgM separately ({gamma} or µ specific) (Sigma, St Louis, MO). After addition of pNPP (Sigma), optical density (OD) was measured at 405 nm. Results were expressed in semiquantitative units (negative, doubtful, low positive, positive) with respect to the standard curve after subtracting the OD of uncoated wells from the OD of coated wells. The cut-off value was defined as the mean +2 S.D. of the OD for a panel of 20 blood donors. Results were low positive above 5 S.D. and positive above 10 S.D. Between 2 and 5 S.D., results were classed as doubtful and were confirmed by heavy-chain-specific anti-Ig ELISA.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We studied 191 sera (from 87 patients from the department of internal medicine, 27 from the department of gynaecology and 77 from the thrombosis unit). Seven patients were diagnosed as having primary or secondary APS and the 184 remaining patients were diagnosed as having other diseases.

aCL antibodies
Of the 191 patients, 36 were aCL-positive (cut-off 10 U GPL or U MPL) (Table 1AGo). Thirteen were confirmed in tests on a second serum. Seven were the patients diagnosed as having APS (aCL positivity was confirmed for all seven patients) and 29 were diagnosed as having other diseases. The specificity of aCL for the diagnosis of APS was 84% and the PPV was 19% (Table 2AGo). If we chose a cut-off of 15 U GPL or U MPL, 22 sera were aCL-positive (Table 1BGo). Eleven were confirmed in tests on a second serum. The seven APS patients were still positive. Fifteen were diagnosed as having other diseases, giving a specificity for diagnosis of APS of 92% and a PPV of 32% (Table 2BGo).


View this table:
[in this window]
[in a new window]
 
TABLE 1.  aCL and anti-ß2GP1 results

 

View this table:
[in this window]
[in a new window]
 
TABLE 2.  Specificity and PPV of aCL and anti-ß2GP1 antibodies

 

Anti-ß2GP1 antibodies
Twelve patients were anti-ß2GP1-positive (doubtful, low positive or positive). All except one were confirmed by heavy-chain specific anti-Ig ELISA. Seven were aCL-positive and had clinical manifestations of APS. Five were in the group that had other diseases; three of these were aCL-positive (cut-off 10 U GPL or U MPL) (Table 1Go) and one was aCL-positive when the cut-off value was 15 U GPL or U MPL. Two of these five patients were aCL-negative and anti-ß2GP1-positive; none of the five had clinical manifestations related to APS. The patient who was doubtful for anti-ß2GP1 and had a high level of aCL antibodies was a 25-yr-old woman. Doubtful anti-ß2GP1 antibodies were found in this patient on several occasions. She had an aPL determination after her first pregnancy, which stopped spontaneously after 5 weeks of gestation. She had no lupus anticoagulant and no history of thrombosis. Her next pregnancy was conducted under aspirin (100 mg) treatment from conception until 35 weeks of gestation and was successful.

For anti-ß2GP1 antibodies, specificity for the diagnosis of APS among the population tested regardless of aCL status was 97% and PPV was 58% (Table 2Go). In the population of aCL-positive patients (cut-off at 10 U GPL or U MPL), specificity was 90% and PPV 70%.

With a cut-off of 15 U GPL or U MPL, one patient had anti-ß2GP1 antibodies with aCL and no clinical manifestations. The PPV was 87% in aCL-positive patients and specificity 93% (Table 2Go).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous reports have shown that anti-ß2GP1 antibodies are closely correlated with aCL in APS and that they are not usually present in other circumstances in which aCL can be found (e.g. infections). Different authors have reported various associations: anti-ß2GP1 IgG and thrombosis [6, 15], anti-ß2GP1 IgM [16] or IgG [17] and obstetric complications. In the present study, all seven APS patients were positive for anti-ß2GP1. Nevertheless, the sensitivity and negative predictive value of anti-ß2GP1 antibodies for the diagnosis of APS cannot be calculated because of the small number of APS patients in our series. This study shows that anti-ß2GP1 antibodies have a higher specificity for the diagnosis of APS than aCL and, more importantly, a better PPV. We conclude that screening for anti-ß2GP1 antibodies in aCL-positive patients increases the specificity and the PPV of testing for aCL. The sensitivity of anti-ß2GP1 antibodies in APS needs to be determined before we can substitute this test for aCL testing. Testing for anti-ß2GP1 in aCL-positive patients could identify high-risk patients and allow the physician to propose prophylactic treatment with regard to vascular thrombosis or obstetric complications.

Some reports have described patients with antibodies directed against ß2GP1 but no detectable antibodies against phospholipids in standard assays, raising the question of testing systematically for anti-ß2GP1 antibodies when APS is suspected. A recent study in patients with recurrent abortion or failure of implantation after in vitro fertilization concluded that screening for anti-ß2GP1 antibodies without aPL was not warranted [18]. In another recent study, testing for anti-ß2GP1 did not identify additional patients who were negative for aCL with either recurrent spontaneous abortion or unexplained foetal death. The authors concluded that there were no data to support routine testing for anti-ß2GP1 in addition to testing for aPL antibodies in these patients [19].

We tested 191 sera from 191 patients for both antibodies (aCL and anti-ß2GP1 antibodies) and found 12 anti-ß2GP1-positive sera. Seven were aCL-positive and had clinical features of APS. Four were anti-ß2GP1-positive with no or very little aCL detectable. All four patients were free of clinical signs of APS. One patient had doubtful anti-ß2GP1 antibodies and a high level of aCL antibodies. She was placed in the category of other diseases because she had no clinical criteria of APS [1]. However, to prevent obstetric complications she was treated with aspirin during her second pregnancy. APS might have developed in the absence of prophylactic treatment (e.g. pre-eclampsia or foetal growth retardation). We conclude that there is no need to screen for anti-ß2GP1 antibodies in the absence of aCL antibodies and in the absence of a strong clinical suspicion of APS.


    Acknowledgments
 
We thank all physicians from the departments of internal medicine and gynaecology and the thrombosis unit (CHU Nantes) for allowing us to consult the clinical data for their patients.


    Notes
 
Correspondence to: M. A. P. Audrain. Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Wilson WA, Gharavi AE, Koike T et al. International consensus statement on preliminary classification criteria for definite antiphospholipid syndrome. Arthritis Rheum1999;42:1309–11.[ISI][Medline]
  2. Matsuura E, Igasrashi Y, Fujimoto M et al. Heterogeneity of anticardiolipin antibodies defined by the anticardiolipin cofactor. J Immunol1992;148:3885–91.[Abstract/Free Full Text]
  3. Forastiero RR, Martinuzzo ME, Kordich LC, Carreras LO. Reactivity to ß2-glycoprotein I clearly differentiates anticardiolipin antibodies from anti-phospholipid syndrome and syphilis. Thromb Haemost1996;75:717–20.[ISI][Medline]
  4. Arvieux J, Darnige L, Caron C, Reber G, Bensa JC, Colomb MG. Development of an ELISA for autoantibodies to prothrombin showing their prevalence in patients with lupus anticoagulants. Thromb Haemost1995;74:1120–5.[ISI][Medline]
  5. Arvieux J, Roussel B, Jacob MC, Colomb MG. Measurement of anti-phospholipid antibodies by ELISA using ß2-glycoprotein I as an antigen. J Immunol Methods1991;143:223–9.[ISI][Medline]
  6. Viard JP, Amoura Z, Bach JF. Association of anti-ß2-glycoprotein I antibodies with lupus-type circulating anticoagulant and thrombosis in systemic lupus erythematosus. Am J Med1992;92:181–6.
  7. Cabiedes J, Cabral A, Alarcon-Segovia D. Clinical manifestations of the antiphospholipid syndrome in patients with systemic lupus erythematosus associate more strongly with anti-ß2-glycoprotein I than with antiphospholipid antibodies. J Rheumatol1995;22:1899–906.[ISI][Medline]
  8. Cabral AR, Cabiedes J, Alarcon-Segovia D. Antibodies to phospholipid-free ß2-glycoprotein I in the serum of patients with primary antiphospholipid syndrome. J Rheumatol1995;22:1894–8.[ISI][Medline]
  9. El-Kadi HS, Keil LB, DeBari VA. Analytical and clinical relationships between human IgG autoantibodies to ß2-glycoprotein I and anticardiolipin antibodies. J Rheumatol1995;22:2233–7.[ISI][Medline]
  10. Tsutsumi A, Matsuura E, Ichikawa K et al. Antibodies to ß2-glycoprotein I and clinical manifestations in patients with systemic lupus erythematosus. Arthritis Rheum1996;39:1466–74.[ISI][Medline]
  11. Roubey RAS. Immunology of the antiphospholipid antibody syndrome. Arthritis Rheum1996;39:1606–7.[ISI][Medline]
  12. Cabral AR, Amigo MC, Cabiedes J, Alarcon-Segovia D. The antiphospholipid/cofactor syndromes: a primary variant with antibodies to ß2-glycoprotein I but no antibodies detectable in standard antiphospholipid assays. Am J Med1996;101:472–81.[ISI][Medline]
  13. Alarcon-Segovia D, Mestanza M, Cabiedes J, Cabral AR. The antiphospholipid/cofactor syndromes. II. A variant in patients with systemic lupus erythematosus with antibodies to ß2-glycoprotein I but no antibodies detectable in standard antiphospholipid assays. J Rheumatol1997;24:1545–51.[ISI][Medline]
  14. Reber G, Arvieux J, Schousboe I, Boffa MC. Multicenter evaluation of the ELISA for anti-ß2GP1 antibodies [abstract]. J Autoimmun2000;15:PD21.
  15. Martinuzzo ME, Forastiero RR, Carreras LO. Anti-ß2-glycoprotein I: Detection and association with thrombosis. Br J Haematol1995;89:397–402.[ISI][Medline]
  16. Forastiero RR, Martinuzzo ME, Cerrato GS, Kordich LC, Carreras LO. Relationship of anti-Xa and anti-prothrombin antibodies to thrombosis and pregnancy loss in patients with antiphospholipid antibodies. Thromb Haemost1997;78:1008–14.[ISI][Medline]
  17. Balestrieri G, Tincani A, Spatola L et al. Anti-ß2-glycoprotein I antibodies: a marker of antiphospolipid syndrome? Lupus1995;4:122–30.[ISI][Medline]
  18. Balasch J, Reverter JC, Creus M et al. Human reproductive failure is not a clinical feature associated with ß2-glycoprotein I antibodies in anticardiolipin and lupus anticoagulant seronegative patients (the antiphospholipid/cofactor syndrome). Human Reprod1999;14:1956–9.[Abstract/Free Full Text]
  19. Lee RM, Emlen W, Scott JR, Branch W, Silver RM. Anti-ß2-glycoprotein I antibodies in women with recurrent spontaneous abortion, unexplained fetal death, and antiphospholipid syndrome. Am J Obstet Gynecol1999;181:642–8.[ISI][Medline]
Submitted 14 September 2000; Accepted 9 November 2001