Functional Assessment of Platelet‐Derived Phospholipid Activity in Pooled Normal Plasmas by Low‐Phospholipid Thrombin Generation Assay and Its Impact on Lupus Anticoagulant Mixing Tests

BACKGROUND: Pooled normal plasma (PNP) used for lupus anticoagulant (LA) mixing tests may carry platelet-derived phospholipid (PL) activity introduced during freezing/lyophilization, potentially attenuating LA-mediated prolongation of clotting times. OBJECTIVES: To determine whether thrombin generation assay (TGA) under low-PL conditions functionally captures endogenous, platelet-derived PL activity in commercial PNP and relates to LA mixing outcomes. METHODS: Four commercial PNPs (CRYOcheck PNP, Coagtrol N, SHP, Ci-Trol 1) were tested by TGA at PL 4, 1, 0.1, and 0 μM with 1 pM tissue factor and 1.6 μM corn trypsin inhibitor. LA weak-positive and LA positive controls were mixed 1:1 with each PNP and assessed by five APTT reagents to compute the index of circulating anticoagulant (ICA). Platelet factor 4 (PF4) levels were measured by ELISA. RESULTS: SHP and Ci-Trol 1 showed consistently higher TGA peak heights than CRYOcheck PNP and Coagtrol N across all PL concentrations; thrombin generation persisted at 0 μM PL, consistent with activity in the absence of added PL. The other TGA parameters exhibited the same trend. Mean ICA values were inversely correlated with TGA peak height at 4 and 1 μM PL (r ≤ -0.985), with direction preserved at 0.1 and 0 μM. PF4 levels were markedly higher in SHP and Ci-Trol 1 and paralleled higher TGA peak height and lower ICA. CONCLUSIONS: Endogenous, platelet-derived PL activity in commercial PNP is a key determinant of the LA mixing test performance. TGA under low-PL conditions, complemented by PF4 measurement, offers a practical approach to qualify PNP lots and reduce the under recognition of LA.