Hexagonal Lupus Anticoagulant Test
What is the Hexagonal Lupus Anticoagulant Test?
The hexagonal lupus anticoagulant test is a confirmatory assay that uses hexagonal (II) phase phospholipids—specifically egg phosphatidylethanolamine—to neutralize lupus anticoagulant antibodies and confirm their presence when screening tests are prolonged. 1, 2
The test exploits a unique property: lupus anticoagulant antibodies are specifically inhibited by hexagonal (II) phase phospholipids but not by lamellar phase phospholipids. 2 This phospholipid-dependent neutralization distinguishes true lupus anticoagulant from other causes of prolonged clotting times such as factor deficiencies or heparin contamination. 1, 2
How Lupus Anticoagulant Testing Works
The complete lupus anticoagulant detection algorithm requires three sequential steps, not just the hexagonal phase confirmatory test alone:
Step 1: Screening Tests
- Two parallel screening tests must be performed using different principles: dilute Russell's viper venom time (dRVVT) and an LA-sensitive activated partial thromboplastin time (APTT). 1
- Omitting either test increases the risk of missing lupus anticoagulant in up to 55% of triple-positive antiphospholipid antibody samples and 31% of antiphospholipid syndrome patients. 1
- A prolonged screening test triggers the next steps. 1
Step 2: Mixing Test
- Mix patient plasma 1:1 with pooled normal plasma without preincubation. 1
- If the prolonged clotting time fails to correct (normalize), this suggests an inhibitor rather than a factor deficiency. 1
Step 3: Confirmatory Test with Hexagonal Phase Phospholipids
- Add high concentrations of phospholipids, specifically hexagonal (II) phase phospholipids or bilayer phospholipids. 1, 2
- If the clotting time normalizes with excess phospholipids, this confirms phospholipid-dependent inhibition characteristic of lupus anticoagulant. 1, 2
- The hexagonal phase phospholipids specifically neutralize lupus anticoagulant antibodies with high specificity. 2
What Does a Positive Result Signify?
A positive hexagonal lupus anticoagulant test signifies the presence of antiphospholipid antibodies that increase thrombotic risk, but a single positive test is insufficient for diagnosis and must be repeated after 12 weeks to confirm persistence. 1, 3, 4
Clinical Significance of Positive Results:
- Thrombotic Risk: Lupus anticoagulant positivity is mandatory to identify high-risk triple antiphospholipid antibody-positive patients and informs prognosis and management of thrombotic and obstetric antiphospholipid syndrome. 1
- Risk Stratification: Persistent single-positive lupus anticoagulant has lower thrombotic risk than triple antiphospholipid antibody positivity (lupus anticoagulant plus anticardiolipin plus anti-β2-glycoprotein I antibodies). 1
- Transient vs. Persistent: Transient positivity is common and does not warrant long-term anticoagulation—only persistent positivity confirmed on repeat testing after >12 weeks has clinical significance. 3, 4
Complete Antiphospholipid Antibody Profile Required:
A lupus anticoagulant result must always be interpreted alongside a complete antiphospholipid antibody profile including:
- Anticardiolipin antibodies (IgG and IgM) by ELISA 3
- Anti-β2-glycoprotein I antibodies (IgG and IgM) by ELISA 3
- The presence of medium-high titers of anticardiolipin and anti-β2-glycoprotein I of the same isotype (most often IgG) confirms high thrombotic risk when combined with positive lupus anticoagulant. 1
Recommended Management Steps
Immediate Actions After Initial Positive Test:
Do NOT initiate long-term anticoagulation based on a single positive test—transient positivity is common and does not warrant treatment. 4
Schedule repeat testing after >12 weeks to confirm persistent positivity, which is mandatory before making any treatment decisions. 1, 3, 4
Complete the full antiphospholipid antibody profile with anticardiolipin and anti-β2-glycoprotein I antibodies by ELISA to assess thrombotic risk stratification. 4
Assess clinical context using the appropriateness criteria:
- High appropriateness: Unprovoked venous thromboembolism or arterial thrombosis in patients <50 years, thrombosis at unusual sites, late pregnancy loss, any thrombosis or pregnancy morbidity in patients with autoimmune diseases. 1
- Moderate appropriateness: Accidentally found prolonged APTT in asymptomatic subjects, recurrent spontaneous early pregnancy loss, provoked venous thromboembolism in young patients. 1
- Low appropriateness: Venous or arterial thromboembolism in elderly patients. 1
After Confirmed Persistent Positivity (>12 weeks):
For Patients WITHOUT Prior Thrombosis (Primary Prophylaxis):
- Start low-dose aspirin if triple-positive (lupus anticoagulant + anticardiolipin + anti-β2-glycoprotein I) or high-titer isolated lupus anticoagulant with systemic lupus erythematosus. 4
- Monitor for development of thrombotic events. 4
For Patients WITH Prior Thrombosis (Secondary Prevention):
- Initiate warfarin with target INR 2.0-3.0 for indefinite anticoagulation. 4
- Avoid direct oral anticoagulants (DOACs)—evidence shows increased thrombotic events with rivaroxaban and other DOACs compared to warfarin in triple-positive antiphospholipid syndrome. 4
Critical Pre-Analytical Requirements
Blood collection and sample handling are crucial to avoid false results:
- Timing: Collect blood before starting any anticoagulant therapy or after sufficient discontinuation period. 1, 3
- Anticoagulant: Use fresh venous blood in 0.109 M sodium citrate at 9:1 ratio (blood:anticoagulant). 1, 3
- Processing: Perform double centrifugation to obtain platelet-poor plasma. 1, 3
- Storage: If testing is delayed, freeze plasma rapidly and thaw at 37°C before testing. 1, 3
Testing During Anticoagulation: Major Pitfalls
Testing for lupus anticoagulant during anticoagulation is strongly discouraged because it results in false positives or false negatives. 1, 5
If Testing Cannot Be Avoided:
- Vitamin K antagonists (warfarin): Test when INR <1.5, ideally 1-2 weeks after discontinuation with low-molecular-weight heparin bridging (last dose >12 hours before blood draw). 1
- Direct oral anticoagulants: Use commercial DOAC removal agents before testing, though availability may be limited and removal may be incomplete. 1, 5
- Low-molecular-weight heparin: Screening is possible with reagents containing heparin neutralizers up to 0.8 U/mL, though effects vary by preparation. 1
- Unfractionated heparin: Perform thrombin time to identify heparin contamination; lupus anticoagulant screening is not possible if plasma is unclottable or heparin exceeds reagent neutralization capacity. 1
Interpretation Caveats:
- Lupus anticoagulant should be interpreted as absent with a comment on repeat testing and potential false negatives when tested during vitamin K antagonist therapy or with coagulation factor deficiency. 1
- Testing close to acute thromboembolic events requires caution due to anticoagulation and elevated acute-phase reactants like factor VIII. 1
Common Pitfalls to Avoid
Never diagnose antiphospholipid syndrome on a single positive test—repeat testing after >12 weeks is mandatory as false positives and transient positivity are frequent. 3, 4
Do not perform generalized screening in asymptomatic individuals—this leads to false positives due to poor test specificity. 1, 3
Avoid testing during anticoagulation whenever possible—all anticoagulants interfere with lupus anticoagulant assays. 1, 5
Do not use direct oral anticoagulants in triple-positive antiphospholipid syndrome—warfarin is superior for preventing thrombotic events. 4
Recognize borderline results—samples showing phospholipid dependence (positive test ratio) but without prolonged screening tests may still represent true lupus anticoagulant and should be repeated. 6
Do not rely on a single screening test—using only dRVVT or only APTT misses a substantial proportion of lupus anticoagulant cases. 1