By Luis Lopez, MD, Ken Dier, and Ann Steinbarger

 Thrombosis is a significant cause of morbidity and mortality. Hypercoagulability (or thrombophilia) refers to a broad group of inherited or acquired molecular defects that may result in hyperactivity of the coagulation system leading to the development of arterial and/or venous thrombosis. The underlying cause(s) of hypercoagulability can now be identified in 40% to 60% of patients with thrombosis. Recently, immunologic abnormalities have been recognized as active participants in the pathogenesis of thrombosis. Due to the multifactorial nature of the disease, the identification of the exact mechanism(s) promoting thrombosis provides valuable information not only for the immediate clinical management (type and duration of anticoagulation) but also for the development of programs directed to prevent the recurrence of thrombotic episodes.

Antiphospholipid syndrome (APS) is probably the most common cause of acquired hypercoagulability. Thrombotic events of both venous and arterial vasculatures are reported in over 30% of patients with elevated serum levels of antiphospholipid antibodies. The recurrence of thrombosis is also high, estimated to be greater than 35% with mortality up to 10% over a 10-year follow-up period. Deep venous thrombosis (DVT) of the legs and/or pulmonary embolism (PE) account for about two thirds of thrombotic events, and cerebral thrombosis (stroke) is the most common arterial complication. Long-term anticoagulation is the only treatment proven to reduce the risk of thrombosis. Therefore, an accurate diagnosis of APS is critical.

Clinical Features of APS
The preliminary classification criteria for definite APS was formulated by international consensus in Sapporo, Japan in 1998, and subsequently validated by several independent groups worldwide. Clinical and experimental evidence reviewed indicated a significant association (and a possible causative role) of antiphospholipid antibodies with vascular thrombosis and pregnancy morbidity, which represent the major clinical features of APS. APS may be present in the context of a systemic autoimmune disorder and referred to as secondary APS, or in the absence of an obvious underlying disease (primary APS). There could be one or more vascular thrombotic episodes in arterial, venous, or small vessels in any tissue or organ. With the exception of superficial vein thrombosis, thrombosis must be confirmed by imaging or Doppler procedures or histopathology. For histopathology confirmation, thrombosis without vasculitis should be present. The presence of at least one of these major clinical features, along with the presence of antiphospholipid antibodies, is required to establish the diagnosis of APS. Other features that may be seen in patients with APS include thrombocytopenia, autoimmune hemolytic anemia, livedo reticularis, transient ischemic attacks, cardiac valve disease, transverse myelopathy, chorea, and migraine headaches. Two or more of these minor clinical features, along with antiphospholipid antibodies may classify a patient as “possible” APS. Although rare, a condition characterized by generalized vascular occlusions has been reported and referred to as catastrophic APS.

Laboratory Features of APS
Laboratory criteria for the classification of APS include anticardiolipin antibodies (IgG and/or IgM) in medium or high tiers demonstrated on two or more occasions at least 6 weeks apart. Anticardiolipin antibodies should be measured by a standardized ELISA capable of detecting Beta-2 Glycoprotein I (B2GPI)-dependent antibodies. Serologic criteria suggest an important role of B2GPI in APS. Lupus anticoagulants (LA) are antiphospholipid antibodies measured by coagulation assays (such as aPTT, dRVVT, KCT) and their presence may establish the diagnosis of APS. LA should also be demonstrated on two or more occasions at least 6 weeks apart. The LA coagulation assays should follow the guidelines of the International Society on Thrombosis and Hemostasis (Scientific Subcommittee on Lupus Anticoagulants/Phospholipid-Dependent Antibodies). Most LAs require B2GPI and have been shown to be more specific for APS than anticardiolipin (aCL) antibodies. Although valuable, technical limitations with LA assays (antibody isotype identification, quantification, and standardization issues) have limited the use of LAs in research and routine clinical laboratories.

Results of recent clinical studies suggest that IgA antiphospholipid antibodies may be relevant in APS. In addition, antibodies to other phospholipids (for

example, to phosphatidylserine (aPS), phosphatidylethanolamine (aPE), and the like) or to other proteins (such as B2GPI and prothrombin) measured in the absence of exogenosus phospholipids, have been shown to be associated with clinical manifestations of APS. Pending further studies, testing for these antibodies has not been included in the Sapporo criteria for APS. Anti-B2GPI antibodies should soon be adopted as a major serologic feature of APS, as B2GPI has been shown to be a common and clinically relevant antigenic target for antiphospholipid antibodies.

 Corgenix Anti-Phospholipid ELISA kits are easily automated and offer rapid turnaround time and open reagent stability for the life of the kit.

Immunology of Antiphospholipid Antibodies
Antiphospholipid antibodies, anticardiolipin antibodies, or lupus anticoagulants are a heterogeneous group of autoantibodies with a possible pathogenic role in the development of the clinical manifestations of APS. The heterogeneity of these antibodies may be the reflection of their reactivity to various negatively charged phospholipids, phospholipid/protein complexes, and/or certain plasma proteins. Several plasma proteins that participate in coagulation and interact with phospholipids have been described to function as antiphospholipid antibody cofactors (for example, B2GPI, prothrombin, annexin V, etc).

The participation of protein cofactors (eg, B2GPI) in the binding of antiphospholipid antibodies has been used to further assess their role in thrombosis. Antiphospholipid antibodies may be found in low levels for short periods of time with no thrombotic disease association. Because these antibodies are frequently B2GPI-independent and seen in association with various infections, they have been referred to as infectious antiphospholipid antibodies. On the contrary, high and persistent serum levels of antiphospholipid antibodies (mostly B2GPI-dependent) are frequently found in patients with a systemic autoimmune disorder and usually associated with thrombotic events or with increased risk for thrombosis and are referred to as “autoimmune” antiphospholipid antibodies. These are the types of antibodies seen in patients with APS. The search for laboratory assays specific for pathogenic (prothrombotic) antiphospholipid antibodies continues.

Antibodies Detected by Classic aCL ELISA
ELISAs for aCL antibodies are the most commonly used assays for the laboratory evaluation of APS. However, significant variability of aCL results from laboratory to laboratory, and even within a single laboratory, have been reported. Intense standardization efforts by several groups worldwide have not been successful. Possible explanations for aCL variability include: 1) the heterogeneous nature of antiphospholipid antibodies related to both antibody isotypes (IgG, IgM, and IgA) and antigen specificity (cardiolipin, phosphatidylserine, B2GPI, etc); 2) the interaction of the phospholipids with B2GPI may create a variety of epitopes depending on the conditions for this interaction (eg, buffers, blocking, and surface); 3) cardiolipin (diphosphatidylglycerol), the most common antigen used, does not have a role in coagulation, nor is it found in cell membranes. Unlike cardiolipin, phosphatidylserine is the major phospholipid of the prothrombinase complex, and is found almost exclusively in the interior membrane layer of resting cells, exposed on procoagulant cell membranes upon cell activation. Thus, phosphatidylserine represents a more clinically relevant antigen for the laboratory evaluation of antiphospholipid antibodies than cardiolipin.

ELISAs for aCL antibodies have shown low specificity for APS, as they can be present in patients with various infectious diseases. Bovine serum containing B2GPI is used as a cardiolipin blocking agent on coated microwell plates and/or in the sample diluent in most ELISA tests for aCL antibodies. Thus aCL ELISAs may detect both B2GPI-independent (or infectious) and B2GPI-dependent (or autoimmune) antibodies.

Antibodies to Beta-2 Glycoprotein I (anti-B2GPI)
The most commonly studied serum protein cofactor is B2GPI. This protein appears to be the most relevant cofactor for the development of thrombosis in vivo. B2GPI is a 50 kd plasma protein present at a concentration of approximately 200 µg/mL, which binds strongly to anionic molecules, such as phospholipids, heparin, lipoproteins, etc. It also binds to activated platelets and apoptotic cell membranes with exposed phosphatidylserine. In addition, B2GPI inhibits the intrinsic coagulation pathway, prothrombinase activity, and ADP-dependent platelet aggregation, and has been reported to interact with several elements of the protein C and protein S anticoagulant system. The peptide sequence of B2GPI reveals a single peptide chain of 326 amino acid residues arranged in five homologous domains belonging to the short consensus repeats of the complement control superfamily. B2GPI’s fifth domain contains a patch of positively charged amino acids that likely represent the binding region for phospholipids.

Because autoimmune antiphospholipid antibodies are cofactor (B2GPI) dependent, it has been proposed that antibodies to B2GPI should be more specific for thrombosis than classic aCL antibodies. Recent clinical studies have confirmed this assumption. The attachment of purified human B2GPI to a plastic surface (microwell plate) may mimic the interaction of this protein with phospholipid membranes in vivo. The interaction of B2GPI with a plastic surface may produce the neoepitope(s) or a high-density antigenic surface to favor the bivalent antibody binding.

 Significance of aCL, aPS, and anti-B2GPI Antibodies Measured by ELISA
One problem with diagnosing patients with thrombosis and/or APS is the low specificity of aCL antibodies measured by ELISA, which might lead to misdiagnosis and unnecessary anticoagulation. Antiphospholipid antibodies directed to other phospholipids more relevant than cardiolipin have been explored (eg, antiphosphatidylserine, aPS) as well as antibodies to protein cofactors (eg, anti-B2GPI). Due to the recognized heterogeneous nature of the antiphospholipid antibodies, it may be assumed that some of these antibodies may coexist in patients with APS. It has been suggested that different antiphospholipid antibodies might have different clinical association or significance, and that the combination of these antibodies in patients would confer a stronger risk for thrombosis. In addition, several groups have explored the usefulness of measuring IgA antiphospholipid antibodies reporting their possible pathogenic role and diagnostic value.

Studies have shown that a single test may not be sufficient, as the prevalence of any of the antibodies never reached 100%, even in selected APS patients. The addition of IgA determinations to IgG and IgM aCL antibodies in a group of SLE patients with history of antiphospholipid antibodies increased the prevalence of positive reactors by 36%. Several groups have recently reported that not only are anti-B2GPI antibodies superior to aCL antibodies for diagnosis of APS, but also that IgA anti-B2GPI antibodies appeared to be the most important isotype detected.

SLE patients with APS had not only higher antiphospholipid antibody levels compared to SLE without APS, but more than 70% had two or more antiphospholipid antibodies present in their serum. In contrast, SLE patients without history of APS usually presented only one antibody. These findings are consistent with the polyclonal nature of the autoimmune response in APS. The common coexistence of various antibodies leads to increased pathologic risk, and adds diagnostic value when at least two antibodies are present in a patient suspected of having APS. It has been recently reported that aPS and anti-B2GPI antibodies have a stronger predictive value and association with thrombosis and APS compared to aCL and aPT antibodies in a selected population of patients with APS. The presence of two or more antiphospholipid antibodies increased the predictive value for APS to about 100%. Further, these antibodies demonstrated the strongest association with arterial thrombosis compared to venous thrombosis and pregnancy morbidity.

Screening for APS
Test results should always be interpreted in the context of clinical manifestations. Thrombosis is a multifactorial disease and due to the heterogeneous nature of antiphospholipid antibodies frequently requires various diagnostic procedures (eg, different assays and technologies, such as LA). Significant cost may be incurred, so careful selection of assays is important for accurate diagnosis. The presence of LA has been shown to be more specific for thrombosis than antiphospholipid antibodies detected by ELISA and adds valuable information to the serologic diagnosis of APS.

ELISAs for aCL and aPS antibodies may detect different populations of antiphospholipid antibodies, including some not associated with thrombosis or APS (eg, infectious antibodies), or other bovine proteins present in blocking reagents or sample diluent. Thus, these assays may be used to screen for a wide range of antibodies. A negative screen in the absence of clinical finding of thrombosis would obviously not require further testing. However, a negative screen with clinical findings would require additional testing with a more specific assay (eg, anti-B2GPI). If the anti-B2GPI assay is negative, testing for other thrombotic risk markers is recommended (protein C, protein S, antithrombin, APC resistance, etc), as well as repeat testing for serconversion. A positive anti-B2GPI result would strongly point toward APS. Repeat testing is also recommended for the serologic diagnosis of APS, which requires the demonstration of persistently high serum levels of antiphospholipid antibodies.

A positive aCL and/or aPS result would also require follow-up testing for anti-B2GPI antibodies. A negative anti-B2GPI result may suggest the presence of “infectious” antibodies, which may be transient and present in low titers. Repeat testing would help to clarify the significance of this antibody. A positive anti-B2GPI result would strongly suggest APS and/or increased risk of thrombosis.

For additional information, contact Luis Lopez, MD, Ken Dier, or Ann Steinbarger at Corgenix Inc, Westminster, CO (800) 729-5661.


Suggested Reading
Bick RL, Baker WF. Antiphospholipid syndrome and thrombosis. Semin Thromb Hemost. 1999;25(3):333-350.

Brandt JT, Triplett DA, Alving B, Scharrer I. Criteria for the diagnosis of lupus anticoagulants: an update. Thromb Haemost. 1995;74(4):1185-1190.

Finazzi G, Brancaccio V, Moia M, Ciaverella N, Mazzucconi MG, Schinco PC, et al. Natural history and risk factors for thrombosis in 360 patients with antiphospholipid antibodies: a four-year prospective study from the Italian Registry. Am J Med. 1996;100(5):530-536.
Francis JL. Laboratory investigation of hypercoagulability. Semin Thromb Hemost. 1998;24(2):
111-126.

Greco TP, Amos MD, Conti-Kelly AM, Naranjo JD, Ijdo JW. Testing for the antiphospholipid syndrome: importance of IgA anti-beta2-glycoprotein I. Lupus. 2000;9(1):33-41.
Guerin J, Feigherty C, Sim RB, Jackson J. Antibodies to beta2-glycoprotein I—a specific marker for the antiphospholipid syndrome. Clin Exp Immunol. 1997;109(2):304-309.

Harris EN, Gharavi AE, Hughes GR. Antiphospholipid antibodies. Clin Rheum Dis. 1985;11(3):591-609.

Lopez LR, Dier KJ, Lopez D, Merrill JT, Fink CA. Anti-beta 2-glycoprotein I and antiphosphatidylserine antibodies are predictors of arterial thrombosis in patients with antiphospholipid syndrome. Am J Clin Pathol. 2004;121(1):142-149.

Lopez LR, Santos ME, Espinoza LR, LaRosa FG. Clinical significance of immunoglobulin A versus immunoglobulins G and M anticardiolipin antibodies in patients with systemic lupus erythematosus. Correlation with thrombosis, thrombocytopenia and recurrrent abortion. Am J Clin Pathol. 1992;98(4):449-454.

Matsuura E, Igarashi Y, Fujimoto M, Ichikawa K, Suzuki T, Sumida T, et al. Heterogeneity of anticardiolipin antibodies defined by the anticardiolipin cofactor. J Immunol. 1992;148(12):3885-3891.

Reddel SW, Krilis SA. Testing for and clinical significance of anticardiolipin antibodies. Clin Diagn Lab Immunol. 1999;6(6):775-782.

Roubey RA. Immunology of the antiphospholipid antibody syndrome. Arthritis Rheum. 1996;39(9):
1444-1454.
Tsutsumi A, Matsuura E, Ichikawa K, Fujisaku A, Mukai M, Kobayashi S. Antibodies to beta2-glycoprotein I and clinical manifestations in patients with systemic lupus erythematosus. Arthritis Rheum. 1996;39(9):1466-1474.

Wilson WA, Gharavi AE, Koike T, Lockshin MD, Branch DW, Piette JC et al. International consensus statement on preliminary classification criteria for definite antiphospholipid syndrome. report of an international workshop. Arthritis Rheum. 1999;42(7):1309-1311.