Journal of Postgraduate Medicine Medknow Publications and Staff Society of Seth GS Medical College and KEM Hospital, Mumbai, India ISSN: 0022-3859 EISSN: 0972-2823 Vol. 48, Num. 1, 2002, pp. 5-10

Journal of Postgraduate Medicine, Vol. 48, Issue 1, 2002 pp. 5-10

ß₂-Glycoprotein I Dependent Anticardiolipin Antibodies and Lupus Anticoagulant in Patients With Recurrent Pregnancy Loss

Kumar KSD, Jyothy A, Prakash MS,* Rani HS, Reddy PP

Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Begumpet, Hyderabad, India and
*National Institute of Nutrition, ICMR, Tarnaka, Hyderabad, India.
Address for Correspondence: A. Jyothy, PhD Department of Cell Biology, Institute of Genetics, Osmania University, Begumpet, Hyderabad - 500 016, India E-mail: ksdkumar@rediffmail.com

Code Number: jp02002

Abstract:

AIM: The present study was aimed to define the incidence of antiphospholipid antibodies of different types lupus anticoagulant (LAC), venereal disease research laboratory test (VDRL) and ß₂-glycoprotein I dependent anticardiolipin antibodies (ß₂ I aCL) in our cohort of population experiencing recurrent pregnancy loss (RPL) from Andhra Pradesh, South India. SETTING AND DESIGN: A referral case-control study at a tertiary centre over a period of 5 years. PARTICIPANTS: 150 couples experiencing 3 or more recurrent pregnancy losses with similar number of matched controls. MATERIAL AND METHODS: LAC activity was measured by the activated partial thromboplastin time (aPTT) according to the method of Proctor and Rapaport with relevant modifications. VDRL analysis was performed by the kit method supplied by Ranbaxy Diagnostics Limited and ß₂ Glycoprotein I dependant anticardiolipin antibodies were estimated by ELISA kit (ORGen Tech, GmbH, Germany) with human ß₂ Glycoprotein I as co-factor. STATISTICAL ANALYSIS: Statistical analysis was performed using Student's t test. RESULTS: LAC activity was found positive in 11 women (10.28%). The mean ± SE ß₂ I aCL concentration in the study group was 14.53 (u/ml) ± 1.79 (range 0 to 90.4 u/ml) which was higher than the control group with a mean ± SE of 7.26 (u/ml) ± 0.40 (range 0 to 18 u/ml). The binding of the antibodies to the antigen was observed in 40.24% (n=33) of the cases compared to 6.09% (n=5) in controls. VDRL test was positive in 7(2.34%) individuals (3 couples and 1 male partner) and none among controls. CONCLUSIONS: The present study indicates the importance of antiphospholipid antibodies in women experiencing RPL and suggests the usefulness of screening for these antibodies as a mandatory routine for instituting efficient therapeutic regimens for a successful outcome of pregnancy.

Key Words: Antiphospholipid antibodies, Antiphospholipid syndrome, ß₂ glycoprotein I dependent anticardiolipin antibodies, Lupus anticoagulant, Recurrent pregnancy loss.

Recurrent pregnancy loss, three or more consecutive (primary or secondary) pregnancy losses with no more than one pregnancy extending into the third trimester, is the cause of childlessness in 2-5% of couples trying to conceive.^1-2 Some of the common factors incurring risk for recurrent miscarriage include anatomic defects of uterus, chromosomal aberrations, endocrine factors, and sub-clinical infections and immunological disturbances. Even after conventional investigations 40-60% of cases remain idiopathic. Accumulating evidence suggests that there are immunological mechanisms responsible for repeated abortions, with otherwise unknown cause.³

Over the past decade increasing attention has been drawn to the association of antibodies having an apparent specificity for negatively charged phospholipids with thromboembolic episodes such as arterial and venous thrombosis, foetal loss and thrombocytopaenia, widely recognised as the antiphospholipid syndrome (APS).^4,5 Antiphospholipid syndrome is accounted in 5-20% of women with recurrent pregnancy loss.

The two best characterised antiphospholipid antibodies (aPL's), lupus anticoagulant (LAC) and anticardiolipin antibodies (aCL) have been widely recognised as risk factors for pregnancy related complications, including recurrent pregnancy loss, intrauterine growth retardation, pre-eclampsia, antepartum haemorrhage and the failure of in-vitro fertilisation and embryo transfer, thus emerging as a clinical entity of "reproductive autoimmune failure syndrome".⁶

The lupus anticoagulant is an acquired immunoglobulin (IgG, IgM or both) that interferes with phospholipid-dependent coagulation reactions. LAC prolongs phospholipid coagulation tests by binding to epitopes on the phospholipid portion of prothrombinase (factor Xa, factor Va, phospholipid and calcium ions). Anticardiolipin autoantibodies exist in the immunoglobulin classes IgG, IgM and/or IgA. Two test systems have been employed to detect and measure antibodies to cardiolipin-coagulation tests that identify LAC and a standardised enzyme linked immunosorbent assay to detect aCL. Recent studies have implicated that the binding of both LAC and aCL to phospholipids is co-factor dependent. The co-factor for LA is prothrombin and that for aCL is a 50 KDa ß₂-globulin (ß₂-glycoprotein I).⁷

This study has been undertaken to define the incidence of aPL of different types (LAC, VDRL and ß₂-glycoprotein I dependent aCL) in a cohort of population from South India.

Antiphospholipid antibodies are associated with pregnancy related complications including recurrent pregnancy loss, intrauterine growth retardation, pre-eclampsia, antepartum haemorrhage and failure of in-vitro fertilisation and

Subjects and Methods

The study was carried out at the Institute of Genetics and Hospital for Genetic Diseases, over a period of 5 years (January 1997 to December 2001). A total of 150 couples, with a history of three or more consecutive first and/or second trimester pregnancy losses in (<22 weeks gestation), were studied (mean number of abortions 3.42, range 3-8). Detailed clinical histories and information pertaining to age, region, religion, habits, number of previous spontaneous abortions, number of live born children, pedigree, past medical histories were recorded in special case proforma. All the patients underwent chromosomal investigations in both partners, ultrasonography, hormonal screening tests and tests to rule out infections. Among 150 subjects, 107 had no conventional causes for abortions and were selected for aPL screening tests. 107 age and sex matched healthy women who had experienced at least one successful pregnancy and no previous histories of abortions were taken as controls.

Blood for LAC was obtained by clean venepuncture with minimal stasis using a single plastic syringe and was collected into 105 mM trisodium citrate tubes. Plate poor plasma (PPP) was obtained by double centrifugation at 3000 rpm for 20 minutes at room temperature. All samples for LAC assay from patients and controls were processed within 2 hours of venepuncture. LAC activity was measured by the activated partial thromboplastin time (aPTT) according to the method of Proctor and Rapaport⁸ with relevant modifications. For each batch of test samples, plasma collected from normal healthy women (pooled) was included to arrive at normal reference control clotting time. The test was carried out in 3 stages. Brain cephalin (aPTT, Tulip Diagnostics, Mumbai, India) was used as a phospholipid reagent. For determination of aPTT, cephalin was diluted 1 in 10 dilutions in 0.9% saline. 100µl of patients' plasma and 100µl of diluted cephalin were mixed and incubated exactly for 2 minutes at 37⁰C. After incubation 100µl of 25 mM calcium chloride (CaCl₂) was added and the clotting time was measured using automated stopwatch. Clotting times were measured in duplicates and are expressed in seconds.

The general guidelines for the diagnosis of LAC suggested by the Kingston antiphospholipid antibody study group was followed.⁹ A patient's plasma was considered LAC positive when the following criteria was fulfilled: 1) Prolongation of coagulation time in stage I; 2) Consistent prolongation after mixing patient's plasma with equal volumes of normal control plasma in stage II and 3) Inhibition of anticoagulant affect, thereby reversal to normal aPTT, when direct cephalin reagent (excess phospholipid) was added.

Blood samples for ß₂-glycoprotein I dependent anticardiolipin antibodies and VDRL analysis were collected in plain glass tubes and the serum obtained was stored at - 20⁰C until used. VDRL analysis was performed by a commercial kit (Ranbaxy Diagnostics Limited, New Delhi, India) and aCL screen with human ß₂I as cofactor by ELISA (ORGen Tech, GmbH, Germany) as per manufacturer's instructions. The optical density (OD) was measured at 450 nm. The amount of aCL was determined based on the standard curve produced using standard antibodies provided with the kit.

Differences between the study groups were analysed for significance by Students t test.

Results

Of the 107 women (age ranged from 18-36 years with a mean ± SD of 26 ± 4.25 years) included in the study, 11 (10.28%) revealed the presence of LAC (Table-1). These women registered a significant prolongation of activated partial thromboplastin time of 86.63 ± 12.0 seconds in stage I and 93.77 ± 11.65 seconds in stage II, which later returned to a clotting time of 52.85 ± 3.85 seconds in stage III similar to that of normal controls whose clotting time was 51.77 ± 3.71 seconds. The difference in the prolongation of clotting time in 11 subjects in stage I and stage II was very high compared to the mean clotting time of 51.77 ± 3.71 seconds in controls (P<0.005, highly significant). The remaining 96 women did not show any prolongation of aPTT in stage I and were corresponding to the normal controls.

The ß₂-glycoprotein I dependent aCL levels were estimated in serum samples of 82 women with RPL and in equal number of controls by ELISA kit method (Table 2). The amount of ß₂I aCL >10 units/ml with this kit was considered to be positive for the presence of the sum of IgG/IgM/IgA autoimmune antibodies and <10 u/ml was considered to be negative. The mean ± SE aCL concentration in the study group was 14.53 (u/ml) ± 1.79 (range 0 to 90.4 u/ml) which was higher than the control group with a mean ± SE of 7.26 (u/ml) ± 0.40 (range 0 to 18 u/ml). The difference in the mean values between the two groups was found to be highly significant (P<0.001). Of the 11 women who showed the presence of LAC, two of them also tested positive for ß₂I aCL autoantibodies.

VDRL test was positive in 7(2.34%) individuals (3 couples and 1 male partner). However, among control subjects none tested positive for VDRL antibodies. Of the 11 women who showed the presence of LAC, one woman also showed VDRL reactivity.

Discussion

Antiphospholipid antibodies (aPL) constitute a family of immunoglobulins that appear to react with negatively charged phospholipids. Included in this family are lupus anticoagulant (LAC) and anticardiolipin antibodies (aCL). Even though antiphospholipid antibodies were initially identified in patients with systemic lupus erythemat-osous, they were later commonly found in persons who do not have a classical autoimmune disease. Therefore, the antiphospholipid antibody syndrome (APS), has been described as a discrete clinical entity.⁴

The APS, first described in 1983, by Hughes is now recognised as an important prothrombic disease, refers to the association between aPL (LAC and aCL) and recurrent miscarriage, thrombosis and thrombocytopaenia.⁴ Preliminary criteria for the classification of definite APS are presented in Table 3. Definite APS is considered to be present in a given patient when at least one of the clinical criteria and at least one of the laboratory criteria are met.⁵ LAC and aCL are the two autoantibodies, which represent the serologic criteria for the diagnosis of APS. The binding of both LAC and aCL to phospholipids is co-factor dependent. The co-factor for LAC is prothrombin and that for aCL is ß₂-glycoprotein I, a naturally occurring anticoagulant.⁷ Approximately 20 to 60% of women with RPL harbour aPL antibodies. The prospective foetal loss rate in these women without any pharmacological intervention was reported to be between 50 and 90%.^10,11,12

LAC is an acquired immunoglobulin usually IgG or IgM or both and rarely IgA.¹³ LAC prolongs phospholipid coagulation tests by binding to epitopes on the phospholipid portion of prothrombinase. It was first identified in systemic lupus erythematosous patients.¹⁴ It was in this group of patients that an increased rate of abortions and foetal deaths has been observed.¹⁵ This led to the investigation of LAC in women without any apparent cause for RPL. Various laboratory tests have been proposed to identify LAC, but activated partial thromboplastin time (aPTT) is the most commonly used test for its detection.¹¹

The distinguishing feature of LAC associated pregnancy loss is the high incidence of early foetal deaths. An adequate explanation for this association is still lacking and the most striking clinical implication is its association with systemic and placental vascular thrombosis with decidual vasculopathy leading to placental infarction.¹⁶ The most commonly proposed mechanisms include decreased prostacycline production by endothelial cells, increased thromboxane production by platelets and decreased protein-C activation.¹⁷

The reported prevalence of LAC associated with RPL is between 4 to 20%.^18,19 But, majority of the studies reported the prevalence between 7 to 11%.^13,20 In the present study, LAC was detected in 11 (10.28%) out of 107 women experiencing RPL which is in accordance with the earlier reports.

In recent years several pregnancy related disorders including the failure of in-vitro- fertilisation and embryo transfer, recurrent pregnancy loss, intrauterine death and pre-eclampsia constituting "reproductive autoimmune syndrome" have been linked to the presence of autoimmune aCL.⁶

aCL constitute a group of aPL which exist as three immunoglobulin classes IgG, IgM and/or IgA. aCL has a clearly documented association with recurrent foetal wastage in patients with or without autoimmune disorders.^16,21,22 In a comparative study on LAC and aCL in pregnant patients with systemic lupus erythematosous, it was found that the presence of either assay indicated the probability of foetal loss; however, aCL was found to be more sensitive and more specific for foetal loss.²³

According to previous reports the frequency of aCL in recurrent pregnancy loss ranged from 11% to 42%.^22,24,25 This wide distribution in part may be accounted by variations in assay protocols of various laboratories used to detect aCL.²⁶ A new aspect relating to antigen specificity demonstrated the necessity of a co-factor, namely ß₂-glycoprotein I, for the detection of autoimmune aCL in ELISA test systems, to selectively eliminate aCL associated with syphilis. Present study on ß₂-glycoprotein-I dependent aCL in women experiencing RPL is the first one to be reported from India.

ß₂-glycoprotein-I (ß₂I), is a 50 KDa ß₂-globulin, which occurs in plasma at a level of 200 mg/ml.²⁷ This protein enhances the cardiolipin binding with autoimmune aCL but inhibits the binding of aCL associated with syphilis.²⁸ In vivo ß₂I is associated with lipoproteins, anionic phospholipids, platelets and mitochondria. In vitro it has been found that ß₂I inhibits the prothrombinase activity of platelets and ADP-mediated platelet aggregation, suggesting a possible role of ß₂I in the regulation of blood coagulation.²⁹

In recent years^28,30-32 it has been found that ß₂-glycoprotein-I dependent aCL as superior in predicting adverse pregnancies involving autoimmunity, than co-factor independent aCL. In the present study we found ß₂-glycoprotein-I dependent anticardiolipin antibodies in 40.24% (n=33/82) of the cases investigated. In controls low serum concentrations of aCL were found in 5 (6.09%) women. Yap et al³³ suggested that the significance of aCL in general population in the absence of clinical features is uncertain. Autoimmune aCL may induce pregnancy failure either by impairing embryonic implantation³⁴ and/or binding directly to placenta.³⁵ Hence it is clear that the presence of autoimmune aCL may induce pregnancy loss at any trimester. Data from the present study with increase in the mean activity of ß₂I dependent aCL in women with first and second trimester idiopathic pregnancy losses seems to be involved significantly in the pathogenesis of the pregnancy failure.

In the present study VDRL test revealed reactivity in 7 (2.34%) individuals. Laurell and Nilsson³⁶ were the first to suspect a relationship between biological false positive test for syphilis and lupus anticoagulant. In a study by Henriksen et al³⁷ on 9 VDRL positive women, only one woman with RPL revealed the presence of LAC. They suggested that VDRL test should not be used as a marker to test the presence of LAC antibodies. In a study by Balasch et al³⁸ none of the seven LAC positive subjects revealed VDRL positivity. In the present study, only one woman having LAC revealed reactivity with VDRL antigen and none showed VDRL reactivity in ß₂-glycoprotein-I dependent aCL positive group. This may be due to the presence of a specific co-factor ß₂I in our ELISA test system, which was selected to eliminate aCL associated with venereal diseases.

Thus in light of multifactorial aetiologies incurring risk for RPL, aPL antibodies might offer an explanation for most of the unexplained pregnancy failures. In the present study, ß₂I aCL was the most frequently detected aPL 40.24%, followed by LAC 10.28% and VDRL 2%. In this study only 2 (1.86%) women who were positive for LAC showed cross-reactivity with ß₂I aCL. This low incidence of cross-reaction emphasizes the fact that LAC and aCL are distinct autoantibodies and that it is important to screen for both. Therefore, the present study indicates the importance of these antibodies in women experiencing RPL and suggests the usefulness of screening for these antibodies as a mandatory for instituting effective therapy regimens for a successful outcome of pregnancy.

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