Antiphospholipid Antibodies

Antiphospholipid antibodies are measured by solid phase immunoassay (enzyme linked or chemiluminescent) and are a group of autoantibodies directed at phospholipid binding proteins, mainly beta-2 glycoprotein I and prothrombin. The immunoassay panel is anticardiolipin IgG and IgM, and anti-beta-2 glycoprotein I IgG and IgM, and it is read with the lupus anticoagulant. Together they define laboratory criteria for antiphospholipid syndrome, an acquired thrombophilia causing venous, arterial and microvascular thrombosis, and obstetric complications.

Antiphospholipid syndrome is diagnosed when a clinical event (vascular thrombosis, or pregnancy morbidity) occurs together with persistent positive tests on 2 occasions at least 12 weeks apart. Antibodies can be present for years before an event, and many positive individuals never develop thrombosis.

About 1 to 5% of healthy people carry low titre antibodies. About 10 to 15% of people with systemic lupus erythematosus have clinically relevant antiphospholipid antibodies, and a higher proportion carry them. Antiphospholipid syndrome accounts for about 10 to 15% of unexplained recurrent miscarriage and a similar proportion of young patients with unexplained stroke.

Indications. Unexplained venous or arterial thrombosis, stroke or myocardial infarction in a person under 50 years, thrombosis at an unusual site (cerebral venous, hepatic, portal, renal, retinal), recurrent early pregnancy loss, foetal loss after 10 weeks, early severe pre-eclampsia or placental insufficiency, unexplained thrombocytopenia, livedo reticularis, systemic lupus erythematosus, valvular heart disease of the Libman-Sacks type, a prolonged aPTT, and a false positive syphilis test.

Specimen. Serum or plasma. Fasting is not needed. Samples can be stored frozen. The laboratory should be given anticoagulant and clinical details so that lupus anticoagulant testing is timed correctly.

Results are reported in assay specific units. Cut offs are set locally at the 99th percentile of healthy controls, and local ranges take precedence.

Anticardiolipin IgG. Antibody binding to cardiolipin, mostly through beta-2 glycoprotein I, measured in GPL units. Range: under about 20 GPL units (or under the 99th percentile). Raised: antiphospholipid syndrome, systemic lupus erythematosus, other autoimmune disease, infection (syphilis, HIV, hepatitis C, Epstein-Barr virus, malaria, leprosy), malignancy, drugs, healthy carriers. Lowered: not applicable.

A moderate or high titre (over 40 GPL units, or over the 99th percentile) on 2 occasions at least 12 weeks apart satisfies the laboratory criterion. IgG correlates with thrombosis better than IgM.

Anticardiolipin IgM. Measured in MPL units. Range: under about 20 MPL units (or under the 99th percentile). Raised: antiphospholipid syndrome, infection (notably Epstein-Barr virus and other viral illness), autoimmune disease, rheumatoid factor interference, healthy carriers.

Isolated IgM positivity has weaker association with thrombosis, and transient positivity after infection is common.

Anti-beta-2 glycoprotein I IgG. Antibody to the beta-2 glycoprotein I protein. Range: under about 20 units (or under the 99th percentile). Raised: antiphospholipid syndrome, systemic lupus erythematosus, other autoimmune disease.

It is more specific than anticardiolipin, particularly at moderate to high titre, and a positive result is less likely to follow infection. Antibodies to domain I of beta-2 glycoprotein I are the pathogenic subset and add specificity.

Anti-beta-2 glycoprotein I IgM. Range: under about 20 units (or under the 99th percentile). Raised: antiphospholipid syndrome (weaker association), infection, autoimmune disease.

IgA antibodies. Anticardiolipin and anti-beta-2 glycoprotein I IgA are measured in some laboratories when clinical suspicion is high and the usual tests are negative (seronegative antiphospholipid syndrome). Their role is limited, and they are not part of the laboratory criteria.

Additional antibodies. Anti-phosphatidylserine/prothrombin antibodies correlate with lupus anticoagulant and thrombosis, and are used in some centres for seronegative disease. Their use is outside routine criteria.

• Triple positive (lupus anticoagulant, anticardiolipin and anti-beta-2 glycoprotein I, all persistent): highest risk of thrombosis and pregnancy loss, with high rates of recurrence.
• Double positive: intermediate risk.
• Single positive: lower risk, particularly isolated low titre anticardiolipin or IgM.
• Isolated lupus anticoagulant: carries more thrombotic risk than isolated anticardiolipin.
• Persistently negative: antiphospholipid syndrome is unlikely. Seronegative disease is described but uncommon, and a prolonged aPTT with thrombosis should lead to repeat testing and, in specialist centres, non-criteria antibodies.
• Persistence: two positive results at least 12 weeks apart are needed. A single positive result is not diagnostic.

Causes of transient or false positive results. Infection (including syphilis, HIV, hepatitis C, Epstein-Barr virus, cytomegalovirus), drugs (chlorpromazine, hydralazine, procainamide, phenytoin, quinidine), malignancy, intravenous immunoglobulin, acute illness, and rheumatoid factor in IgM assays. The false positive syphilis serology (non-treponemal tests) can indicate the antibody before other tests.

Primary and secondary disease. Primary antiphospholipid syndrome occurs alone. Secondary disease occurs with systemic lupus erythematosus or another autoimmune disease.

Catastrophic antiphospholipid syndrome. Thrombosis in 3 or more organs within a week, microvascular occlusion on biopsy, and positive antibodies, with a mortality of about 30 to 50%.

Critical findings needing immediate action.

• Positive antibodies with multiple new thromboses, falling platelets and organ failure indicate catastrophic antiphospholipid syndrome, and need immediate specialist management.
• A positive result in a pregnant woman with previous loss or thrombosis needs early obstetric and haematology management.
• A triple positive profile in a patient with an arterial or venous event needs specialist decisions about anticoagulation, and direct oral anticoagulants are generally avoided in this group.

Interferences and pitfalls.

• Assays differ between manufacturers and results are not interchangeable, so serial testing uses the same method.
• Rheumatoid factor and high immunoglobulin M can give false positive IgM results.
• Intravenous immunoglobulin can give passive positive results, and recent infusion confuses interpretation.
• A single positive result, particularly at low titre or in IgM, is often clinically unimportant.
• A normal result does not exclude antiphospholipid syndrome if lupus anticoagulant is positive, and the three tests are interpreted together.
• Persistence at 12 weeks is required before diagnosis, and testing in acute thrombosis or infection is repeated later.
• Testing in a patient on anticoagulants is reliable for the immunoassays, which are unaffected by anticoagulation, unlike lupus anticoagulant.
• Treatment decisions are not based on titre alone.

Special groups.

• Pregnancy: positive tests need early specialist care. IgG antibodies cross the placenta, and neonates can show transient positive results with rare neonatal thrombosis.
• Children: transient positivity after infection is frequent, and persistence is needed.
• Older adults: low titre positives are more common without disease.
• Systemic lupus erythematosus: screen at diagnosis, and repeat before pregnancy, surgery or when starting oestrogen therapy.

Follow up tests. Lupus anticoagulant (dilute Russell viper venom time and lupus sensitive aPTT), repeat testing at 12 weeks, antinuclear antibodies, anti-dsDNA, complement, full blood count with platelets, clotting screen, syphilis and HIV serology, hepatitis C testing, direct antiglobulin test, renal function and urine protein, echocardiography for valve disease, imaging of the thrombotic site.