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.