Antiphospholipid (APL) antibodies are group of antibodies directed against epitopes on plasma proteins that are uncovered by binding of these proteins to anionic phospholipids on plasma membranes. The most commonly used tests to detect APL include lupus anticoagulant (LAC), anticardiolipin (ACL) antibodies, and anti-β2-glycoprotein I antibodies.
The International Society of Thrombosis and Haemostasis (ISTH) criteria for lupus anticoagulant detection include 4 mandatory steps, in the following sequence:
1. Prolongation of a phospholipid-dependent clotting assay (activated partial thromboplastin time [aPTT], kaolin clotting time [KCT], diluted Russell’s viper venom test [dRVVT], diluted prothrombin time [dPT]); two tests that have different assay principles should be used, usually the dRVVT and aPTT; this is the screening step
2. Mixing study with 1:1 proportion of patient’s plasma and a normal pooled plasma without preincubation and reassessment of the clotting assay used in step one; if it remains prolonged, an inhibitor is present, either LAC or a specific factor inhibitor; if it corrects, then LAC is excluded and the cause is likely a specific factor deficiency
3. Confirmation that the inhibitory activity is phospholipid-dependent by relative correction of the abnormal clotting time when the concentration of phospholipid is increased in the screening test(s) that yielded abnormal results; this step confirms that the cause of abnormal mixing study is LAC (phospholipid-dependent inhibitor), not a specific factor inhibitor; knowing the clinical history helps in diagnosis—thrombosis in case of LAC or hemorrhage in the case of factor deficiency
4. Exclusion of other coagulopathies that may yield similar results or accompany the LAC presence; specific factor assay might be necessary
The results from the above tests are considered positive when they are above the local cutoff value. For tests in steps 1 and 2, the cutoff value is the 99th percentile of the distribution of the tests performed on plasmas from at least 40 healthy donors younger than 50 years. In the confirmatory tests in step 3, the cutoff value is equal to the mean of the individual percentage of corrections, calculated with the following equation:
[(Screen – confirm)/screen] × 100†
Enzyme-linked immunoassay (ELISA) is currently the test of choice. The reference range findings are as follows:
Less than 15 immunoglobulin G (IgG) phospholipids units (GPL): Absent or none detected
Less than 12 immunoglobulin M (IgM) phospholipids units (MPL): Absent or none detected
Less than 12 immunoglobulin A (IgA) phospholipids units (APL): Absent or none detected
If the test is obtained to diagnose antiphospholipid syndrome, only medium to high titers of IgG or IgM antibodies (defined as >40 GPL or MPL, or >99th percentile
) are considered a positive test result.
Anti-β2 glycoprotein I
ELISA is the test used to detect these antibodies, IgM and IgG isotypes. Per antiphospholipid syndrome diagnostic criteria, these anti-β2 -glycoprotein I antibodies of IgG and/or IgM isotype should be present in the in plasma, in a titer greater than the 99th percentile to be considered a positive test result.