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Indian J Hematol Blood Transfus

DOI 10.1007/s12288-015-0535-0

ORIGINAL ARTICLE

A Study of Anti Beta-2 Glycoprotein I and Anti-Prothrombin


Antibodies in Patients with Unexplained Recurrent Pregnancy
Losses
Angad Singh1 Anita Nangia1 Sunita Sharma1 Manju Puri2

Received: 2 December 2014 / Accepted: 24 March 2015


Indian Society of Haematology & Transfusion Medicine 2015

Abstract To compare the levels of IgG and IgM anti


beta-2 glycoprotein I antibodies and IgG and IgM anti
prothrombin antibodies among women with unexplained
recurrent pregnancy losses and women with at least 2 live
issues. To compare the prevalence of newer anti beta-2
glycoprotein I & anti prothrombin antibodies with conventional Lupus anticoagulant & anticardiolipin antibodies.
50 women with recurrent pregnancy losses & 50 matched
controls were evaluated for the presence of: Lupus anticoagulantscreened by LA sensitive aPTT& DRVV and
confirmatory Staclot Assay. ELISA kits were used for detecting IgG & IgM anticardiolipin, anti beta-2 glycoprotein
I & anti prothrombin antibodies. 11/50 (22 %) women in
study group and none in control group had circulating
antiphospholipid antibodies. 2 cases (4 %) had lupus anticoagulant. 1 case (2 %) had anticardiolipin antibody & 6
cases (12 %) were positive for anti beta-2 Glycoprotein I
antibody (p value = 0.027). 3 cases (6 %) had anti prothrombin antibody. All were mutually exclusive except for
one. Women with recurrent pregnancy losses should be
tested for anti beta-2 Glycoprotein I antibodies & anti
prothrombin antibodies in addition to conventional lupus
anticoagulant and anticardiolipin antibodies. This approach
can decrease the incidence of SNAP (seronegative antiphospholipid syndrome) cases while establishing the true
prevalence of antiphospholipid syndrome.

& Angad Singh


drangad@gmail.com
1

Department of Pathology, Lady Hardinge Medical College


and Associated Hospitals, New Delhi 110001, India

Department of Obstetrics & Gynaecology, Lady Hardinge


Medical College and Associated Hospitals,
New Delhi 110001, India

Keywords Anticardiolipin  Antiphospholipid


syndrome  Lupus coagulation inhibitor  Pregnancy 
Prothrombin  Beta 2-glycoprotein I

Introduction
Recurrent pregnancy losses (RPL) are defined as three or more
consecutive pregnancy losses with not more than one pregnancy successfully reaching into the third trimester. 12 % of
women in reproductive age group experience 3 or more
pregnancy losses [1, 2]. Pregnancy losses can be attributed to a
wide variety of causes but after evaluating patients for the
etiology of pregnancy loss and performing all routine investigations, about 50 % of the cases still remain undiagnosed [3].
Immunologic causes are a diagnosis of exclusion. Foremost
among them is antiphospholipid syndrome which comprises
about 520 % cases of recurrent pregnancy loss [4].
Antiphospholipid syndrome is characterised by thromboembolic events, pregnancy losses along with presence of
circulating antiphospholipid antibodies. The antiphospholipid antibodies are a heterogeneous group of autoantibodies that bind to different protein epitopes (beta-2
glycoprotein I, prothrombin, annexin V etc.) complexed to
phospholipids. The two best known antiphospholipid antibodies are lupus anticoagulant and anticardiolipin antibodies. It is diagnosed according to modified Sapporo
criteria [5]. It requires presence of any one of three antibodies viz. lupus anticoagulant (LA), anti cardiolipin (aCL)
and anti beta-2 glycoprotein I (anti beta-2 GP I) antibody
on 2 occasions 12 weeks apart. At present anti prothrombin
antibody is not a part of the modified Sapporo criteria. This
study aims to compare the presence of anti beta-2 GP I, anti
prothrombin antibodies with classical antiphospholipid
antibodies (LA, aCL).

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Indian J Hematol Blood Transfus

Materials and Methods


Place of Study
The study was conducted in the Department of Pathology
and Department of Obstetrics and Gynaecology, Lady
Hardinge Medical College and associated Shrimati Sucheta
Kriplani Hospital, New Delhi-110001.
Study Subjects
The case group was formed by 50 women with recurrent
pregnancy losses after excluding known patients with Rh/
ABO incompatibility, blighted ovum, diabetes mellitus,
thyroid disease, positive TORCH/Hepatitis/HIV serology,
anatomical defects of uterus on USG, chromosomal
anomaly in the participant, spouse or previous abortus/fetus. The control group comprised 50 women with 2 live
births and no history of pregnancy loss.
Laboratory Tests
All cases were tested for (a) Routine tests: Complete blood
count with peripheral blood smear. (b) Screening tests for coagulation: PT (Prothrombin time) & APTT (Activated partial
thromboplastin time) (c) Tests for antiphospholipid antibodies:
Lupus anticoagulant (PTT-LA- STACLOT assay & Dilute
Russell Viper Venom time screen ? confirm STACLOT assay), Anticardiolipin antibody IgG/IgM (ELISA, Asserachrom
Diagnostics), Anti Beta-2 Glycoprotein I antibody IgG/IgM
(ELISA, Asserachrom Diagnostics), Anti-prothrombin antibody IgG/IgM (ELISA, DRG Diagnostics). A positive test was
confirmed by repeat testing after a period of 12 weeks and was
considered positive only when both samples were positive.

control group. In the study group all 50/50 (100 %) cases


experienced first trimester pregnancy losses while 14/50
(28 %) cases also experienced second trimester pregnancy
losses. Only 3/50 (6 %) cases experienced third trimester
pregnancy losses while 1 patient had pregnancy losses in
all trimesters.
There was no statistically significant difference in the
CBC parameters, PT & APTT between study and control
groups.
In the study group, overall 11/50 cases were positive for
circulating antiphospholipid antibodies (Fig. 1). Of these 2
cases (4 %) were positive for LA & 1 case (2 %) was
positive for anticardiolipin antibody. There was no statistically significant difference between study and control groups
with respect to LA and anticardiolipin antibodies (Table 1).
In the study group 6 cases (12 %) tested positive for anti
beta-2 GP I antibody, all controls were negative and there
was a statistically significant difference (p value \ 0.05)
between study and control groups (Table 2). A total of 3
cases (6 %) were positive for anti prothrombin antibody
while all controls were negative, however p value was not
significant (p value [ 0.05) (Table 2). A single case
showed positivity for both anti beta-2 GP I and anti prothrombin antibody. In the control group all the cases tested
negative for lupus anticoagulant, anticardiolipin, anti beta2 GP I & antiprothrombin antibodies (Table 1, 2).

Discussion
Antibodies against beta 2 GP I were identified in 1990 by 3
different groups when they found that among the patients
of antiphospholipid syndrome, anticardioipin antibodies
are directed against a plasma protein cofactor beta 2

Statistical Analysis

Anphospholipid Anbody Posive Cases


15
13

9
7
5
3

Observations and Results

12

11

% age

The data was expressed as mean 1 standard deviation.


Comparisons between two group frequencies were made
using Chi Square Test. For the comparison of two group
means, Student t test was applied. When comparing means
of more than two parameters, ANOVA (A one-way analysis of variance) test was applied. The statistical software
used was SPSS & SS.

6
4
2

1
-1

All cases and controls were age matched. The age group
ranged from 19 to 30 years with a mean of 24.84
3.026 years for cases and 25.28 2.214 years for controls.
A total of 177 pregnancy losses occurred in 50 Cases of
the study group. There was no pregnancy loss among the

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Lupus
Ancardiolipin An beta-2 GP
An
Ancoagulant
anbody
I
prothrombin
anbody
Percentage posivity

Fig. 1 Antiphospholipid antibody positive cases (all figures in


percentage)

Indian J Hematol Blood Transfus


Table 1 Cases testing positive
for Lupus anticoagulant &
anticardiolipin antibody

Table 2 Cases testing positive


for anti Beta-2 GP I &
antiprothrombin antibodies

Cases (n = 50) (Mean SD)

Controls (n = 50) (Mean SD)

p value

LA positive

2/50 (4 %)

0/50

0.495

aCL positive

1/50 (2 %)

0/50

1.000

Cases (n = 50) (Mean SD)

Controls (n = 50) (Mean SD)

p value

Anti Beta-2 GP I positive

6/50 (12 %)

0/50

0.027

Anti prothrombin positive

3/50 (6 %)

0/50

0.242

glycoprotein I [68]. It has been found that anticardiolipin


antibodies which correlate with thrombotic complications
and pregnancy losses are actually antibodies which
recognise beta 2 GP I and have an affinity for cardiolipin
(anticardiolipin dependent beta 2 GP I antibodies) [9].
Antigen/antibody complexes of prothrombin and beta-2
glycoprotein I are largely responsible for the lupus anticoagulant activity detected in in vitro tests for coagulation.
These antibodies are responsible for the pathogenic
manifestations of antiphospholipid syndrome, namely
thrombotic events and pregnancy loss [1013].
In our study among 11 antiphospholipid antibody positive women, 9 were negative for commonly used screening
tests for the presence of antiphospholipid antibodies i.e. LA
(using LA sensitive APTT & Dilute Russell viper venom
time) and aCL. Among these 9 cases, 6/9 showed the
presence of anti beta-2 GP I antibodies & 3/9 had circulating anti prothrombin antibodies with 1 case showing
presence of both anti beta-2 GP I and anti prothrombin
antibodies (Fig. 1). Hence 9 LA & aCL negative cases
showed the presence of newer antiphospholipid antibodies.
In contrast to other studies, in our study all control subjects
tested negative for antiphospholipid antibodies. A review
of literature shows a widely varying prevalence of antiphospholipid antibodies in patients with recurrent pregnancy loss. There is paucity of literature from the Indian
subcontinent regarding anti beta-2 GP I & antiprothrombin
antibodies. Vora et al. [14] studied 198 women with RPL &
found LA positivity in 10.11 % patients, aCL IgM in
19.6 % patients, aCL IgG in 22.2 % cases. Anti Beta-2 GP
I IgM was positive in 6.3 % cases, anti beta-2 GP I IgG
was positive in 9.7 % cases. Bizzaro et al. [15] studied 77
women with RPL of whom 6 % cases & 4 % controls were
positive for anti beta-2 GP I & aCL IgG/IgM antibodies.
16 % cases & 8 % controls were positive for anti prothrombin IgG/IgM antibodies. Zammiti et al. [16] studied
200 women with RPL and found anti beta-2 GP I IgG to be
positive in 4.5 % cases, anti beta-2 GP I IgM to be elevated
in 1.5 % cases. To the best of our knowledge antiprothrombin antibodies have previously not been studied in
Indian females with recurrent pregnancy losses. Sater et al.
[17] studied 277 recurrent pregnancy loss cases and

compared them with 288 controls. They found 12 %


positivity for anti-prothrombin antibodies among cases and
also 6.1 % in controls. Marozio et al. [18] studied 56
women with late fetal death ([26 weeks) who were
negative for lupus anticoagulants and anticardiolipin.
28.6 % patients tested positive for antiprothrombin antibodies as compared to 3.6 % controls. In patients with
seronegative antiphospholipid syndrome with pregnancy
loss, Conti et al. [19] found antiprothrombin antibody to be
positive in 2 of 8 patients. Recent studies also indicate that
Domain I of anti beta-2 glycoprotein I antibodies to be
highly associated with antiphospholipid syndrome [20].
Therefore women with recurrent pregnancy losses
should be routinely tested for all antiphospholipid antibodies viz. anti beta-2 Glycoprotein I antibodies, anti
prothrombin antibodies, lupus anticoagulant and anticardiolipin antibodies. By doing so, we can reduce the diagnosis of seronegative antiphospholipid syndrome. A larger
cohort study should be done to find out the true prevalence
of anti beta-2 glycoprotein I & antiprothrombin antibodies
in patients with recurrent pregnancy losses.

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