What is the ISTH‑recommended protocol for detecting lupus anticoagulant, including screening, mixing, confirmatory steps, and management of anticoagulant interference?

ISTH Protocol for Lupus Anticoagulant Testing

The ISTH guidelines mandate a systematic three-step approach: screening with two phospholipid-dependent tests (dRVVT and sensitive aPTT), followed by 1:1 mixing studies, and confirmation with high-phospholipid reagents—all performed on properly prepared platelet-poor plasma obtained before anticoagulation or after adequate washout. 1

Pre-Analytical Requirements: Blood Collection and Processing

Critical timing: Blood must be collected before starting any anticoagulant therapy, or after sufficient discontinuation period to avoid false results 1

Sample preparation steps:

• Collect fresh venous blood in 0.109 M sodium citrate at 9:1 ratio (blood:anticoagulant) 1
• Double centrifugation is mandatory to ensure platelet-poor plasma: first spin at 2000g for 15 minutes at room temperature, then transfer plasma carefully (avoiding residual platelets at tube bottom) to non-activating plastic tube and re-centrifuge at >2500g for 10 minutes 1
• Plasma filtration is not recommended due to variable results, von Willebrand factor loss, and added costs 1
• If testing is delayed, freeze plasma quickly at -70°C or below to prevent coagulation factor loss 1
• Thaw frozen plasma by complete immersion in 37°C water bath for 5 minutes, then mix thoroughly before testing to avoid cryoprecipitate formation 1

Step 1: Screening Tests

Two tests based on different principles are required:

• dRVVT (dilute Russell viper venom time) should be the first test 1
• Sensitive aPTT (with low phospholipids and silica as activator) should be the second test 1, 2
• LA is considered positive if either test gives a positive result 1

Cut-off determination:

• Establish local cut-offs using plasma from healthy donors 1
• Set cut-off at the 99th percentile of the normal distribution 1
• Results are potentially suggestive of LA when clotting times exceed the local cut-off 1

Express all results as patient-to-pooled normal plasma (PNP) ratio 1

Step 2: Mixing Studies

When to perform: Required for all positive screening tests to distinguish inhibitors from coagulation factor deficiencies 1, 3

Mixing protocol:

• Use 1:1 proportion of patient plasma to PNP 1
• PNP should ideally be prepared in-house; adequate commercial lyophilized or frozen PNP is acceptable 1
• Perform mixing without preincubation, within 30 minutes 1
• LA cannot be conclusively determined if thrombin time (TT) is significantly prolonged 1

Interpretation methods:

• Calculate Index of Circulating Anticoagulant (ICA): [(patient mix time - normal plasma time) / patient time] × 100 1
• Establish cut-off at 99th percentile of healthy donor distribution 1
• Results suggest LA when clotting times or ICA exceed local cut-off 1

Critical caveat: The mixing step is particularly important for patients on vitamin K antagonists to avoid false-positive results, as discrepancies between two-step and three-step procedures are most pronounced in this population 3, 4

Step 3: Confirmatory Testing

Confirmation principle: Demonstrate phospholipid-dependence by showing correction with increased phospholipid concentration 1

Confirmatory procedure:

• Perform the same screening test(s) with increased phospholipid concentration 1
• Use bilayer or hexagonal (II) phase phospholipids 1
• Calculate percentage correction: [(screen time - confirm time) / screen time] × 100 1
• Establish cut-off as the mean of individual % corrections from healthy donors 1
• Results are confirmatory of LA if % correction exceeds the local cut-off 1

Alternative integrated approach: Some commercial assays combine screening and confirmation in a single procedure by testing at low and high phospholipid concentrations simultaneously, calculating either percentage correction or LA ratio (screen/confirm) 1

Managing Anticoagulant Interference

Patients on Vitamin K Antagonists (VKA)

Preferred approach: Discontinue VKA for 1-2 weeks or until INR <1.5 before testing 1

• Bridge with LMWH, administering last dose >12 hours before blood draw 1

Alternative if discontinuation impossible: If INR is 1.5 to <3.0, use 1:1 dilution of patient plasma with PNP 1

• Major limitation: Interpretation remains difficult and LA titer will be diluted 2-fold 1

Patients on Direct Oral Anticoagulants (DOACs) or Heparin

These medications interfere with LA testing and produce unreliable results 5

• Testing should be performed after adequate washout period 5
• Unfractionated heparin requires particular caution during acute thromboembolic events 1

Testing During Acute Thrombosis

Exercise caution: Acute-phase reactants (especially Factor VIII) may be elevated during acute events 1

• Antibody levels may decrease due to deposition at the thrombotic site, potentially rising again after the acute phase 5
• Consider retesting after resolution of the acute event if clinical suspicion remains high 5

Testing During Pregnancy

Factor VIII increases during pregnancy can mask LA by shortening aPTT 5

• Approximately 25% of pregnant patients who were LA-positive earlier become negative in second or third trimester 5
• Retest postpartum if clinical suspicion persists 5

Integrated Testing and Clinical Context

Complete antiphospholipid antibody profile required: LA results must be interpreted alongside anticardiolipin (aCL) and anti-β2-glycoprotein I (aβ2GPI) antibodies by ELISA 1, 5

• Triple positivity (LA + aCL + aβ2GPI of same isotype, usually IgG) identifies patients at highest risk for thrombosis 1, 5
• Isolated LA positivity is more frequent in subjects without clinical events and may represent false-positive results, especially if mild in potency, found in elderly patients, or diagnosed for the first time 1

Confirmation timing: Positive tests must be confirmed after at least 12 weeks to distinguish persistent from transient antibody positivity 5

• This 12-week rule applies only to positive results, not negative results 5
• Two positive LA tests spaced at least 12 weeks apart are required for APS diagnosis 5

Result reporting: Provide a report with clear explanation of results, expressing all values as patient-to-PNP ratios for screening, mixing, and confirmatory procedures 1

Common Pitfalls to Avoid

• Inadequate platelet removal: Single centrifugation leaves residual platelets that cause false-positive results; double centrifugation is mandatory 1
• Testing on anticoagulation: VKA, DOACs, and heparin all interfere with LA detection; discontinue before testing when possible 1, 5
• Skipping the mixing step: This leads to false-positives, particularly in VKA-treated patients with factor deficiencies 3, 4
• Using manufacturer cut-offs without validation: Local cut-offs based on 99th percentile of your laboratory's healthy donor population are essential 1, 6
• Single test approach: Using only one screening test (either dRVVT or aPTT alone) misses LA-positive patients; both tests are required 1, 2