ISTH Guidelines for Lupus Anticoagulant Testing

Pre‑Analytical Requirements

Timing of blood draw – Collect blood before any anticoagulant therapy or after an adequate wash‑out period to avoid false‑positive results (ISTH) 1.
Anticoagulant for collection – Use 0.109 M sodium citrate in a 9:1 blood‑to‑anticoagulant ratio (ISTH) 1.
Double centrifugation – First spin at 2000 g for 15 min at room temperature; transfer plasma to a non‑activating tube and re‑centrifuge at >2500 g for 10 min to obtain platelet‑poor plasma (ISTH) 1.
Plasma filtration – Not recommended because it yields variable results, reduces von Willebrand factor, and adds cost (ISTH) 1.
Delayed testing – Freeze plasma promptly at ≤ ‑70 °C to preserve coagulation factors (ISTH) 1.
Thawing – Thaw frozen plasma in a 37 °C water bath for 5 min, then mix thoroughly before analysis (ISTH) 1.

First screening assay – Perform dilute Russell viper venom time (dRVVT) as the initial test (ISTH) 1.
Second screening assay – Perform a phospholipid‑sensitive activated partial thromboplastin time (aPTT) with low phospholipid and silica activator as the second test (ISTH) 1.
Positive definition – Lupus anticoagulant (LA) is considered present if either the dRVVT or the sensitive aPTT yields a positive result (ISTH) 1.
Local cut‑off establishment – Determine assay cut‑offs using plasma from healthy donors and set the threshold at the 99th percentile of the normal distribution (ISTH) 1.
Result expression – Report all screening results as a patient‑to‑pooled normal plasma (PNP) ratio (ISTH) 1.

Indication – Perform mixing studies for every positive screening test to differentiate an inhibitor from a factor deficiency (ISTH) 1.
Mixing protocol – Mix patient plasma with PNP in a 1:1 ratio; PNP may be prepared in‑house or obtained commercially (ISTH) 1.
Timing – Conduct the mixing without pre‑incubation and complete the assay within 30 minutes of mixing (ISTH) 1.
Thrombin time limitation – LA cannot be conclusively interpreted if the thrombin time is markedly prolonged (ISTH) 1.
Index of Circulating Anticoagulant (ICA) – Calculate ICA = [(mixing time – normal plasma time) / patient time] × 100; establish the ICA cut‑off at the 99th percentile of healthy donors (ISTH) 2.
Interpretation – LA is suggested when either the mixing clotting time or the ICA exceeds the locally defined cut‑off (ISTH) 2.

Principle – Demonstrate phospholipid dependence by showing correction of clotting time when phospholipid concentration is increased (ISTH) 1.
Procedure – Repeat the screening assay(s) with a high‑phospholipid reagent, using bilayer or hexagonal II phase phospholipids (ISTH) 1.
Percentage correction – Calculate % correction = [(screening time – confirmatory time) / screening time] × 100; set the cut‑off as the mean % correction obtained from healthy donors (ISTH) 5.
Positive confirmation – LA is confirmed when the % correction exceeds the locally established cut‑off (ISTH) 5.
Integrated commercial assays – Some platforms combine low‑ and high‑phospholipid testing in a single run, reporting either % correction or an LA ratio (screen/confirm) (ISTH) 6.

Vitamin K antagonists (VKA) – Discontinue VKA for 1–2 weeks or until the INR is < 1.5 before LA testing; if bridging is required, use low‑molecular‑weight heparin with the last dose > 12 h before phlebotomy (ISTH) 6.
If VKA cannot be stopped – When INR is 1.5–< 3.0, dilute patient plasma 1:1 with PNP before testing; note that this dilutes the LA titer approximately two‑fold and interpretation remains challenging (ISTH) 6.
Unfractionated heparin – Exercise special caution when testing during acute thromboembolic events, as heparin can interfere with LA assays (ISTH) 6.
Acute‑phase reactants – Elevated factor VIII during acute thrombosis may shorten aPTT and affect LA results; consider retesting after the acute phase resolves (ISTH) 6.

Comprehensive antiphospholipid profile – LA results must be interpreted together with anticardiolipin (aCL) and anti‑β2‑glycoprotein I (aβ2GPI) antibody testing (ISTH) 6.
Risk stratification – Triple positivity (LA + aCL + aβ2GPI of the same isotype, usually IgG) identifies patients at the highest risk for thrombosis (ISTH) 6.
Isolated LA positivity – More common in individuals without clinical events and may represent a false‑positive, especially when potency is low, in elderly patients, or at first diagnosis (ISTH) 6.
Result reporting – Provide a clear report that includes patient‑to‑PNP ratios for screening, mixing, and confirmatory steps (ISTH) 1.

Inadequate platelet removal – Single centrifugation leaves residual platelets that cause false‑positive LA; double centrifugation is mandatory (ISTH) 1.
Testing while on anticoagulants – VKA, DOACs, and heparin interfere with LA detection; discontinue or appropriately manage anticoagulation before testing (ISTH) 6.
Skipping the mixing step – Omitting mixing leads to false‑positives, particularly in VKA‑treated patients (ISTH) – note: this recommendation is supported by non‑ignored references.
Using manufacturer cut‑offs without local validation – Laboratories must establish their own 99th‑percentile cut‑offs based on healthy donor data (ISTH) 1.
Relying on a single screening test – Employing only dRVVT or only aPTT misses LA‑positive cases; both assays are required (ISTH) 1.