blood vessels could never- theless not be demonstrated these antibodies should

As published in CLI November 2005
A utoimmunity
Autoantibodies in the diagnosis of APS
by Dr Rainer Woehrle and Prof. Dr Klaus Helmke
Some patients with clinical symptoms suggestive of antiphospholipid
syndrome (APS) nevertheless test negative with the well standardised
assays that detect antibodies against cardiolipin/ß2-glycoprotein-I
complex. In such cases we recommend testing for antibodies against
the human prothrombinphosphatidylserine complex, phosphatidylethanolamine, and against phosphatidylinositol with human
ß2-GPI present as a co-factor in the assay. In patients who are still
negative for these phospholipid antibodies, testing for antibodies
against phosphatidylcholine, sphingomyelin, thrombin and annexin
V may be helpful to identify individuals who would benefit from anticoagulant therapy. Tests should always detect IgG and IgM isotypes.
The clinical manifestations of antiphospholipid syndrome (APS), which are mainly
arterial and venous thrombosis and obstetric complications, are generally closely correlated with laboratory results. Historically the first indications of the existence of APS
were false positive tests for syphilis: patients with Systemic Lupus Erythematosus
(SLE) as well as some healthy subjects had positive venereal disease research laboratory (VDRL) tests in the absence of syphilis. Furthermore, many of these individuals
showed prolonged coagulation times in in vitro assays. In 1952 a coagulation inhibitor
was identified, the so called lupus anticoagulant (LAC), which in contrast enhances
coagulation in vivo.
Cardiolipin (CL), the negatively charged phospholipid present in the mitochondrial
membrane, is a major component of the VDRL test. For this reason, an assay was
developed in the early 1980s for the detection of anti-Cardiolipin (aCL) antibodies in
plasma. Further investigations in 1990 showed that aCL-antibodies require a co-factor to bind to the cardiolipin targets; this co-factor is the plasma protein ß2-glycoprotein-I (ß2-GPI). In the meantime ELISAs for the detection of antibodies against the
CL/ß2-GPI complex have become well standardised and accepted. These tests form
the backbone of the laboratory diagnosis of APS [Table 1].
In addition to CL, antibodies against other phospholipids and proteins have been identified in the plasma of patients with clinical manifestations of APS [Table 2]. The aim
of this review is to evaluate the diagnostic role of these antibodies against various phospholipid/protein complexes.
Antibodies against cardiolipin and ß2-glycoprotein-I
These antibodies bind to a cryptic epitope on the fifth domain of ß2-glycoprotein-I.
When this serum protein binds to a surface containing cardiolipin (e.g. biological cell
membranes or coated ELISA plates), the hidden epitope is exposed, allowing any aCLantibodies to bind [1]. Test systems for the detection of aCL-antibodies should therefore always contain sufficient amounts of ß2-GPI as a co-factor. These well standardised ELISAs have a reasonably high sensitivity (80-90%) for the diagnosis of APS
patients. IgG antibodies are found most frequently, but a number of APS patients are
only positive for the IgM isotypes. Testing for both IgG and IgM isotypes is therefore
necessary. To facilitate the comparability of results between different test systems and
laboratories, the test results should be reported by range of positivity in addition to
the standardised G phospholipid (GPL) and M phospholipid (MPL) units [2].
Because the antibodies bind to the CL/ß2-GPI complex, tests detecting antibodies
against ß2-GPI alone have a lower sensitivity (40-90%), especially if they use bovine
ß2-GPI. On the other hand, conventional aCL antibody assays often also detect "ß2GPI-independent aCL" that are not associated with thromboembolic complications.
Utilising anti-ß2-GPI antibody tests increases the specificity for patients with APS
from 60% to 80%. As ß2-GPI antibodies of some patients are species-specific, the
assay should use human ß2-GPI [3].
Antibodies against phosphatidylserine and prothrombin
As with antibodies against CL and ß2-GPI, the binding of antibodies to prothrombin
is improved if the prothrombin is itself bound with anionic phospholipids, especially
phosphatidylserine (PS). While the levels of these antibodies showed a strong association with pregnancy loss [4], a significant correlation with thrombotic occlusions of
blood vessels could nevertheless not be demonstrated
in all studies. Testing for
these antibodies should
therefore be particularly
carried out in women with
pregnancy
losses
of
unknown origin who are
negative for aCL-antibodies
and LAC. Since a high percentage of these antibodies
have species-specificity for
human protein, the assays
should contain human prothrombin [5].
Antibodies against phosphatidylethanloamine
In contrast to CL and the
other anionic phospholipids,
phosphatidylethanolamine (PE) is a zwitterionic phospholipid which
is normally present in the
outer layer of cell membranes. Antibodies against
this phospholipid do not
bind
to
phosphatidylethanolamine itself,
but rather to a complex of
PE and certain proteins in
bovine serum, e.g. kininogens and kininogen-binding
proteins; ß2-GPI is not
involved.
Table 1. Sapporo criteria for the diagnosis of APS, revised
during the 11th International Congress on Antiphospholipid Antibodies in Sydney in 2004. The definite diagnosis
of APS requires the presence of at least one clinical manifestation and one positive laboratory test result.
Table 2. Phospholipid/Protein complexes as targets of
antiphospholipid antibodies.
To date studies on larger patient cohorts have not demonstrated the clinical utility of
testing for PE antibodies, but many anecdotal case reports and antibody studies support the suggestion that there is a correlation between anti-PE antibodies and thromboembolic events [6]. Our own investigations also show this. It is reasonable therefore
to consider anti-PE-testing in patients suspected of having APS, but who are negative
with the standard aCL-ELISA.
Antibodies against phophatidylinositol
Since the late 1990s the significant association between the presence of antibodies to
another anionic phospholipid, namely phosphatidyl-inositol (PI), and cerebral
ischaemia especially in young patients, has been well documented [7]. In a cohort of
162 patients with autoimmune disease we also found anti-PI-antibodies to be significantly elevated in individuals with a history of cerebral ischaemia. Multivariate analyses showed that these antibodies were the only ones that were independently associated with stroke. Other groups have also demonstrated the association between anti-PI
antibodies and clinical manifestations of APS.
As with anti-PE antibodies, testing for anti-PI antibodies should be considered when
patients are suspected of APS, as well as when there are negative test results with the
standard aCL-ELISA, especially in young patients with cerebral ischaemia of undetermined cause. Since the binding of anti-PI antibodies is improved by the presence of
ß2-GPI as a co-factor in the assay, test systems should contain human ß2-GPI.
Antibodies against phosphatidylcholine or sphingomyelin
In a study of 156 patients with autoimmune disease who were tested for antibodies
against different phospholipids, 61 individuals were found to be negative for IgG antibodies
against
CL,
ß2-GPI
A utoimmunity
or PE. In the latter group, IgG-antibodies against the anionic phospholipids phosphatidyl-choline (PC) and sphingomyelin (SM) were significantly elevated in patients
with typical clinical symptoms of APS [8] using test systems that did not contain ß2-GPI
as a co-factor.
We therefore recommend testing for antibodies against PC and SM in individuals suspected of having APS who are negative for the tests discussed above. The test systems
should not contain ß2-GPI as a co-factor. Data in the literature on the clinical utility of
testing for anti-PC antibodies are contradictory. So far, anti-SM-antibodies have rarely
been tested.
Antibodies against thrombin and annexin V
During recent years, antibodies specific for the coagulation factor thrombin have
come under increased scrutiny. These antibodies are present in a number of APS
patients and are associated with thromboembolic complications, mainly arterial
thrombosis [9]. We also found 7 out of 8 women with unexplained pregnancy losses
to be positive for antibodies against thrombin [10]. These results are preliminary and
have to be verified in larger patient cohorts. Nevertheless in research projects, testing
for thrombin antibodies should be considered in patients with suspected APS and
whose results with the tests mentioned above are negative.
The plasma protein annexin V (A5) binds to PS-rich surfaces such as the membrane
of endothelial cells and placental cells forming an anticoagulant layer. Theoretically, it
could be expected that disruption of this "anticoagulant shield" by antibodies could
lead to an elevated risk of thromboembolic events. Clinical studies have verified that
this theory is correct, with a significantly higher risk of thrombotic events being present in patients with systemic autoimmune disease.
As published in CLI November 2005
Antiphospholipid Antibody
study (KAPS) group. Am J Clin
Pathol 1994; 94: 476-484.
3. Roubey RAS. Species-specific Autoantibodies to Human
ß2GPI. In: Khamashta MA
(Editor). Hughes Syndrome:
Antiphospholipid Syndrome.
Springer, 2000; 256-257.
4. Matthias T, von Landenberg
P, Zaech J, Blank M, Shoenfeld
Y. Anti- prothrombin antibodies are highly associated with
pregnancy loss in patients with
antiphospholipid syndrome. Table 3. Indications for anti-phospholipid-antibody testing.
Annals of Hematology 2003;
82: S60.
5. Bertolaccini ML, Amengual O, Atsumi T. Antiprothrombin Antibodies. In: Khamashta MA
(Editor). Hughes Syndrome: Antiphospholipid Syndrome. Springer, 2000; 245-252.
6. Roubey RAS. Antibodies Detected in Immunoassays Using Phosphatidylethanolamine. In:
Khamashta MA (Editor). Hughes Syndrome: Antiphospholipid Syndrome. Springer, 2000; 256.
7. Toschi V, Motta A, Castelli C, Paracchini ML, Zerbi, Gibelli A. High prevalence of antiphosphatidylinositol antibodies in young patients with cerebral ischemia of undetermined cause.
In an in vitro experiment, monoclonal anti-A5-antibodies also prevented the formation
of the syncytiotrophoblast, which is a possible cause of early foetal loss. However in the
above-mentioned study [10], only 4 out of 8 women with unexplained pregnancy losses tested positive for anti-A5 antibodies. Another study has also failed to demonstrate a
significant correlation between these antibodies and pregnancy loss. Testing for anti-A5antibodies should therefore only be recommended in patients suffering from thromboembolic events when tests for phospholipid antibodies are negative.
Summary
Testing for antibodies associated with APS is only indicated if the test results are likely to lead to beneficial therapeutic consequences. As long as the benefit of prophylactic therapy in individuals with antibodies but without clinical symptoms remains
unproven, tests should only be performed in patients whose symptoms are typical of
APS or whose symptoms cause the condition to be suspected [Table 3].
As an initial strategy the established and well standardised tests for lupus anticoagulants and ß2-GI-dependent cardiolipin antibodies should be performed. If
these tests are negative in individuals with clinical APS symptoms, we recommend testing for antibodies against other phospholipids and proteins as shown
in Table 4.
When interpreting the results from the tests described above, it should always be
remembered that these test systems are so far not standardised and that the clinical utility of the tests has not yet been proven in large patient cohorts. In the
individual patient, however, the detection of one of these antibodies can be very
helpful in deciding the optimal course of further therapy.
References
1. Tsatsumi A, Koike T. Measurement of Anti-ß2-glycoprotein I Antibodies. In: Khamashta MA
(Editor). Hughes Syndrome: Antiphospholipid Syndrome. Springer 2000; 238-244.
2. Harris EN. The second international anticardiolipin standardization workshop/the Kingston
Table 4. Recommended antibody testing in individuals suspected of having APS.
Stroke 1998; 29: 1759-1764.
8. Woehrle R, Oppermann M, Matthias T, Helmke K. Clinical relevance of antibodies against
different phospholipids. Abstract book of the Third European Forum on Antiphospholipid
Antibodies held in Utrecht, April 20th - 21st, 2001; 70.
9. Miesbach W, Matthias T, Scharrer I. Identification of thrombin antibodies in patients with
antiphospholipid syndrome. Ann N Y Acad Sci 2005; 1050: 250-256.
10. Woehrle R, Oppermann M, Matthias T, Helmke K. Pregnancy losses are stronger correlated with antibodies against thrombin than with other phospholipid- or protein-antibodies.
Autoimmunity reviews 2004; 3 Supplement 2: 44.
The authors
Dr Rainer Woehrle and Prof. Dr Klaus Helmke
Dept. for Rheumatology and Clinical Immunology,
Hospital München-Bogenhausen,
Englschalkinger Str. 77, D-81925 München, Germany