Background

Antiphospholipid antibodies or APL Abs are a set of diverse heterogeneous antibodies. When plasma proteins bind to the anionic phospholipids, they are exposed, and these antibodies targets the epitopes on the proteins on the plasma membrane. Along with APL antibodies, anticardiolipin antibodies (ACL), anti-β2 -glycoprotein I antibodies, and lupus anticoagulant (LAC) can also be detected.

LAC test is a functional test. This test detects APL antibodies with anticoagulant activity in vitro. LAC is a misleading or inaccurate because clinically it is linked to the clotting rather than the anticoagulant activity. About 50% of the cases with LAC is linked to systemic lupus erythematosus (SLE).

The clinical importance of these antibodies is their relationship with thrombosis. Antiphospholipid antibody syndrome (APS) is detected when individuals have one of the antibodies with the pregnancy morbidity or vascular thrombosis. APS occurs as a primary disease or associated with the other disease like SLE, known as secondary APS.

In the study of Pablo et al., they found that hypertension, smoking, triple APL antibody positive, and thrombocytopenia are the distinct risk factors for thrombosis development who have APL antibodies but do not have clinical significance for APS.

In addition to the APL antibodies, other ALP antibodies are also present, like annexin V, prothrombin, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine. The experience is confined, and clinical significance remains exclusive. They are not part of the APS diagnosis.

Indications/Applications

APL antibodies test is most useful for individuals with suspected APS like young individuals who have spontaneous arterial or venous thrombosis, pregnancy morbidity, early pregnancy loss, with disease like SLE.

It is likely appropriate to test for APL antibodies in young individuals with repeated early pregnancy loss or induced venous thrombosis. It is also helpful to test in asymptomatic patients who have an unexplained extended aPTT detected during typical testing. Although the patients are asymptomatic, APL antibodies are present, yet it is not confirmed of the APS diagnosis. Though patients may develop the symptoms later and can be diagnose with APS.

Individuals who have APL antibodies are at risk of APS development later. Since the antibodies presence are temporary without any clinical sign. Other conditions like nephropathy, livedo reticularis, heart valvular disease, and thrombocytopenia may be linked with individuals with APS. Still this conditions are not part of the diagnosis. Patients with these conditions and particularly with pregnancy morbidity or thrombosis can suspect with APS and should test for the APL antibodies.

Clinical Significance:

APS have a clinical and pathological relation with pregnancy morbidity or venous or arterial thrombosis and certain APL antibodies with positive result in blood like IgG Cardiolipin, IgA or b2GPI antibodies with more than 99% or a LAC. As per the international guidelines, it is necessary to wait for 12 weeks before the retest to ensure the antibodies are persistence. Low level of APL antibodies can be detected in infection, aging, or drug therapy.

Some target specific anticoagulant treatment like direct thrombin inhibitors, anti-Xa medications may lead to false positive test result for LAC test but does not affect the B2GPI, Cardiolipin, and phosphatidylserine-Prothrombin antibody test.

By testing at least 40 young individual’s healthy donors whose age is below 50 years, the local cutoff levels are determined.

The test is performed when the patient is stable and not in the stage of an acute thromboembolic event. It is because of 2 reasons: one is during the acute stage, it may trigger to the temporary anticardiolipin antibodies production, second is acute stage can increase the acute stage reactants like factor VIII and fibrinogen and this can alter the result.

LAC detection test is performed after the sufficient time when the anticoagulant therapy is stopped or before the therapy starts. Anti-β2 -glycoprotein I antibody or anticardiolipin test is not affected by the anticoagulant therapy. LAC detection test result is affected by the phospholipids concentration. Phospholipids can react to the APL antibody in screening test and avoid the extension of the test. Platelet free plasma is used in the tests because it can avoid the false negative test result as platelets are rich in phospholipids. Excessive phospholipids are introduced to ensure the blocker is phospholipid dependent.

These tests have different specificity and sensitivity. The intra laboratory variation is considerable. Standardization for these tests is currently ongoing. Patients with current or past syphilis may lead to the false positive test result without the risk of thrombosis.

Efthymiou et al study found that 28.7% od samples with LAC results were unclear or inconsistent between basic or local/hospital laboratory. The research suggested that incorrect result have been got from the local/hospital laboratories. About 24.7% of non-anticoagulant and 23% anticoagulant samples results are incorrect. They stated that the unclear laboratory results may be caused by the different laboratory data. The investigators suggested that excellent agreement between the laboratories can be obtained in LAC test by using same methods, reagents, and analyzer type.

Reference Range

The other tests which are used to detect APL like anticardiolipin (ACL) antibodies, anti-β2-glycoprotein I antibodies, and LAC.

Lupus anticoagulant (LAC)

The International Society of Thrombosis and Haemostasis has included the main steps to detect the lupus anticoagulant.

1. Prolongation of phospholipid-dependent clotting test: This step is a screening test. This includes kaolin clotting time (KCT), activated partial thromboplastin time (aPTT), diluted prothrombin time (dPT), and diluted Russell’s viper venom test (dRVVT). Any 2 tests with different principles should be performed. More often aPTT and dRVVT is used.
2. Mixing trail with 1:1 ratio of plasma of patient and a normal pooled plasma before the incubation and retest the clotting test. If it is remains continuous, there is a presence of inhibitor, either specific factor inhibitor or LAC inhibitor. If it improves, LAC is eliminated, and the reason is deficiency in a specific factor.
3. The abnormal clotting time in screening tests is observed because of the LAC phospholipid-dependent inhibitor nor a particular inhibitor. It ensures that the root cause of the abnormal mixing studies is LAC.
4. To exclude the other coagulant that can lead to similar results or LAC presence, particular factor test is necessary.

The above test results are positive if the values are greater than the local cutoff value. For steps 1 and 2, the cutoff level is 99% of the at least 40 young individual’s healthy donors whose age is below 50 years. In step 3 the confirmatory test, the cutoff level is equal to the mean of the patient percentage of corrections. The question is below:

[(Screen – confirm)/screen] × 100^†

Anticardiolipin (ACL) antibodies

ELISA (Enzyme-linked immunoassay) is a recommended test. The normal findings are below:

Below 15 IgG phospholipids units (GPL): No or absent detected

Below 12 IgM phospholipids unit (MPL): No or absent detected

Below 12 IgA phospholipids unit (APL): No or absent detected

IF the test is used to diagnose APS, only high or medium titer of IgM or IgG antibodies are used. Greater than 40 GPL or MPL or greater than 99% are suggested as a positive test result.

Anti-β2 glycoprotein I (AB2GPI)

To detect the antibodies isotypes IgG and IgM, ELISA is used. As per the APS criteria, AB2GPI antibodies isotypes IgG or IgM must be present in the plasma and the titer value should be more than 99%, then the test result is positive.

Interpretation

APL test is used to detect the APL antibodies in the blood. The presence of phospholipid antibody is an indicator of an autoimmune disease like multiple sclerosis, systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA). This test is also used to check the unusual blood clot, thrombocytopenia, and repeated miscarriage.

Collection And Panels

Lupus anticoagulant (LAC) test

Sample type: Blood plasma

Sample collection container: Blue top tube which contain 0.109 M sodium citrate.

Sample storage requirement: If the test is delayed, frozen plasma is needed for testing. They should be thawed at 37°C.

Considerations: Blood must be collected before the anticoagulant therapy started or after it stopped after the particular time of interval. Platelet free plasma less than 10000/ μL is prepared by centrifugation with precautions. As the fragmentation of platelet and presence of phospholipids can lead to negative false test results.

Anticardiolipin (ACL) antibodies and anti-β2-GPI (AB2GPI) antibodies test

Sample type: Blood serum

Sample collection container: Red top tube

Sample volume: 7 mL

Considerations: Serum is considered over plasma to test. Running duplicate samples is suggested. High-sensitivity plates or polystyrene ELISA plates are recommended. Cardiolipin must be diluted in ethanol.

Control samples should be obtained from at least 40 young individual’s healthy donors whose age is below 50 years to determine the local cutoff levels.

References

https://link.springer.com/article/10.1007/s11926-020-00916-5

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4848066/