Antiphospholipid Antibodies

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.

Lupus anticoagulant

The International Society of Thrombosis and Haemostasis (ISTH) criteria for lupus anticoagulant detection include 4 mandatory steps, in the following sequence: ^[1]

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 99^th 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^†

Anticardiolipin antibodies

Enzyme-linked immunoassay (ELISA) is currently the test of choice. The reference range findings are as follows: ^[2]

• 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 ^[3] ) 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. ^[3]

Lupus anticoagulant

A blood sample is collected in a blue-top tube containing 0.109 M sodium citrate. Blood should be collected before the start of anticoagulation therapy or after a sufficient period following discontinuation.

Platelet-free (< 10,000/μL) plasma preparation via centrifugation must be done with special care, as it may cause platelet fragmentation and the release of phospholipids, which may lead to false-negative results.

Frozen plasma is required if testing is delayed, and frozen plasma must be thawed at 37°C. ^{[1, 4]}

Anticardiolipin antibodies and anti-β2-GPI antibodies

Obtain a 7-mL blood serum sample in a red-top tube.

Serum is preferred over plasma for testing.

Running samples in duplicate is recommended.

Polystyrene ELISA plates or high-sensitivity plates are preferred.

Cardiolipin should be diluted in ethanol. ^[5]

Control samples should be collected from at least 40 healthy young (< 50 years) people to determine the local cutoff for the above tests.

Description

Antiphospholipid (APL) antibodies are group of heterogeneous antibodies directed against epitopes on plasma proteins that are uncovered by binding of these proteins to anionic phospholipids on plasma membranes. These antibodies can be detected with lupus anticoagulant (LAC), anticardiolipin antibodies (ACL), and anti-β₂ -glycoprotein I antibodies. ^[6]

LAC is a functional assay that is used to detect APL antibodies that have anticoagulation activity in vitro. LAC is a misnomer, since it is clinically associated with clotting tendency rather than anticoagulation activity, and only 50% of people with LAC meet the criteria for systemic lupus erythematosus (SLE).

The clinical significance of the above antibodies is their association with thrombosis; once an individual is found to have one of the above antibodies with vascular thrombosis and/or pregnancy morbidity, antiphospholipid antibody syndrome (APS) is diagnosed. ^{[7, 8, 9, 10]} APS occurs either as a primary disease or in the setting of another disease such as SLE (so-called secondary APS).

A study by Pablo et al indicated that in persons who carry APL antibodies but do not meet the clinical criteria for APS, independent risk factors for the development of thrombosis include smoking, hypertension, thrombocytopenia, and triple APL antibody positivity. ^[11]

In addition of the above APL antibodies, other APL antibodies exist, such as prothrombin, annexin V, phosphatidylserine, phosphatidylinositol, and phosphatidylethanolamine, but their clinical significance remains elusive and experience is limited; thus, none is yet part of the diagnostic criteria of APS.

Indications/Applications

Testing for APL antibodies is most appropriate in patients with suspected APS, such as young people with unprovoked venous or arterial thrombosis, early pregnancy loss, or other pregnancy morbidity, especially those with SLE.

Conversely, it is probably reasonable to test for APL antibodies in young people with provoked venous thrombosis and/or recurrent early pregnancy loss. It may also be prudent to check for these antibodies in asymptomatic patients with unexplained prolonged aPTT discovered incidentally during routine testing, although the presence of APL antibodies in this scenario does not confirm the diagnosis of APS, since the patient is asymptomatic. However, the patient may develop symptoms later, and only then can APS be diagnosed. ^[1]

Persons with APL antibodies are at risk of developing APS, although the presence of these antibodies might be transient, without any clinical significance. Other manifestations, such as thrombocytopenia, livedo reticularis, heart valvular disease, and nephropathy, may be seen in individuals with APS but are still not part of its diagnostic criteria; the presence of these manifestations, especially in people with thrombosis and/or pregnancy morbidity, should alert the physician to suspect APS and to evaluate for the presence of APL antibodies. ^[12]

Considerations

It is recommended that patients suspected of having APS undergo testing for all 3 antibodies (LAC, ACL, anti-β₂ -GPI), although the presence of only one type of these antibodies is sufficient for diagnosis. Nonetheless, patients who are positive for all 3 antibodies have the highest rate of APS-related complications. ^[13] If only one test is to be chosen, LAC is the assay of choice since it has the strongest correlation with APS manifestation.

To meet the diagnostic criteria for APS, the test results should be positive on two occasions, 12 weeks apart. This is so that persistent pathologic APL antibodies can be differentiated from transient nonpathologic APL antibodies.

Local cutoff values are calculated by performing the test on at least 40 young (< 50 years) healthy donors.

Tests should be performed when the patient is clinically stable, not during an acute thromboembolic event, for 2 reasons: first, an acute event may trigger the production of transient anticardiolipin antibodies; second, acute events increase the acute-phase reactants such as fibrinogen and factor VIII, which can alter the results of coagulation tests. ^[14]

LAC detection tests should be performed before anticoagulation therapy is started or after it has been stopped for a sufficient period. Anticardiolipin or anti-β₂ -glycoprotein I antibody testing is generally not affected by anticoagulation therapy.

The concentration of phospholipids affects the LAC detection results. Phospholipids can react with the APL antibodies in the screening tests (step 1; see Lupus anticoagulant) and prevent prolongation of the these tests, and, since platelets are phospholipid-rich, platelet-free plasma is used in these tests to avoid false-negative results, whereas excess phospholipids are added in step 3 (see Lupus anticoagulant) to confirm that the inhibitor is phospholipid-dependent.

These assays differ in their sensitivity and specificity, and their intralaboratory variability remains high. Efforts to standardize these tests are in process.

People with past or current syphilis may have false positive results without being at risk for thrombosis, as is the case with other infection-, medication-, or neoplasm-induced transient antibodies.

A study by Efthymiou et al of laboratories contributing to the Antiphospholipid Syndrome Alliance for Clinical Trials and International Networking (APS ACTION) registry found that in 28.7% of samples, lupus anticoagulant (LAC) results were equivocal or were discordant between core and local/hospital laboratories. The study suggested that unreliable results may have been obtained from the local/hospital laboratories in 24.7% and 23% of non-anticoagulated and anticoagulated samples, respectively. They cautioned, however, that the differences may also have partially stemmed from equivocal core laboratory results. The investigators stated that in LAC analysis, good agreement between laboratories can be attained through employment of the same reagents, analyzer type, and protocols. ^[15]