What is the clinical significance when samples test positive for the anti-cardiolipin antibodies and are negative for anti-b2GPI?

The aCL assay can detect antibodies of differing specificities. This includes antibodies specific for the cardiolipin molecule itself, and antibodies that are directed against either a cofactor molecule such as b2GPI, or a special binding site created by the interaction of a cofactor with CL. Antibodies directed against CL may be associated with infectious disease, or may be specific for a different cofactor, such as prothrombin. The clinical significance of these antibodies must be assessed in conjunction with the patient’s symptoms, clinical history, and other laboratory findings. Follow-up testing of these patients is recommended in 3-6 months to confirm reactivity. Only b2GPI cofactor dependent antibodies react in the anti-b2GPI assay; these antibodies show a higher correlation with thrombosis and are more specific for the antiphospholipid syndrome.

What is the advantage of an assay for anti-phospholipid antibody cofactors over an assay for the antibody itself?

Binding of b2GPI to the microwell surface in an ELISA assay may produce a neoepitope similar to that when combined with a phospholipid; assay results with this system show a good correlation with the APS. The serologic detection of anti-b2GPI antibodies provides enhanced clinical sensitivity for thrombosis.

Summarize the b2-Glycoprotein 1 test kit. What is the normal range?

Patient serum is diluted with sample diluent and incubated in microwells coated with human b2GPI. Antibodies to b2GPI present in the sample will bind to the coated wells. After washing, enzyme conjugated anti-human IgG, IgM, or IgA immunoglobulin is added, the wells are washed again, substrate added, and color development measured in a spectrophotometer at 450 nm. With a simple calculation, semi-quantitative results in G, M, or A units are available in less than 1 hour. The normal range is less than 20 units for each isotype (IgG, IgM, or IgA).

What is the role of Beta 2 Glycoprotein 1 (B2GP1) in anti-phospholipid antibody binding?

b2GPI, also called apolipoprotein H, is an antiphospholipid protein cofactor with natural anticoagulant properties and an affinity for negatively-charged phospholipids. Antibodies directed against b2GPI have been shown to be specific markers for thrombosis in individuals with systemic lupus erythematosus (SLE) and lupus-like disorders (APS). Most autoimmune anti-phospholipid antibodies require the serum cofactor b2GPI for optimal binding. It has been shown that many anti-phospholipid antibodies may react to a neoepitope formed on the b2GPI molecule by the interaction between the phospholipid and b2GPI. The physiological role of b2GPI may additionally be to participate in apoptosis.

Summarize the IgG/IgM and IgA REAADS anti-cardiolipin test. What is the normal range?

The test is an indirect ELISA. Diluted serum samples, calibrator sera, and controls are incubated in cardiolipin coated microwells, allowing aCL antibodies present in the samples to react with the immobilized antigen. After their removal of unbound serum proteins by washing, antibodies specific for human IgG, IgM, or IgA labeled with HRP are added forming complexes with the CL bound antibodies. Following another washing step, the bound enzyme-antibody conjugate is assayed by the addition of TMB and H2O2 as the chromogenic substrate. Color develops in the wells at an intensity proportional to the serum concentration of aCL antibodies. The OD is read at 450 nm.

Normal ranges are less than 23 GPL (IgG per liter), less than 11 MPL (IgM per liter), and 22 APL (IgA per liter).

What are autoantibodies?

Autoantibodies are antibodies to self components produced when immunologic tolerance is broken.

What are some phospholipid cofactors?

  • beta2 Glycoprotein 1
  • Prothrombin
  • Protein C
  • Protein S
  • Annexin V
  • Kininogen

Is administering one Anti-Phospholipid test sufficient for diagnosis?

No, it has been decided that more than one test is should be performed. For example, a coagulation assay for the detection of LA and an ELISA for the detection of anti-phospholipid antibodies or cofactors like anti-CL or anti-b2GPI are recommended. Future testing may require a panel of tests. Physicians should interpret the results of the APS tests in light of the patient’s history, physical findings, and other diagnostic procedures.

Describe the location and function of the various phospholipids.

Cardiolipin (CL), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidylinsitol (PI), Cardiolipin (diphosphatidylglycerol) (CL) – located in the inner mitochondrial membrane. It has an anionic charge and the function is unknown, as cardiolipin is not involved in the immune or coagulation systems.

Phosphatidylserine (PS) – located in the inner platelet membrane and, during activation, the exterior platelet membrane. It carries an anionic charge, and is the primary phospholipid which promotes the anticoagulant protein C pathway, providing feedback inhibition of thrombin formation.

Phosphatidylethanolamine (PE) – located in both the interior and exterior of cell membranes. It carries a neutral (zwitterionic) charge, and promotes the anticoagulant protein C pathway, but to a lesser degree than PS.

Phosphatidylcholine (PC) – located in the interior and exterior of cell membranes. It carries a neutral charge, and promotes the anticoagulant protein C pathway, but to a lesser degree than PS.

Phosphatidylinsitol (PI) – located in the interior and exterior of cell membranes. It carries a cationic charge, and promotes the anticoagulant protein C pathway, but to a lesser degree than PS.

What are anti-phospholipid antibodies and anti-phospholipid antibody cofactors?

Antiphospholipid antibodies are a heterogeneous group of auto-antibodies (IgG, IgM, and IgA) initially thought to be specific only to negatively charged phospholipids. It is now well recognized that many antiphospholipid antibodies are directed to phospholipid-protein complexes and /or to proteins in the absence of phospholipids.

Antiphospholipid antibody cofactors are plasma proteins mostly with function in coagulation and strong phospholipid binding activities. Cofactors increase the binding of antiphospholipid antibodies in vitro and may help the development of thrombosis in vivo.