Interpretation of Prothrombin Time and Activated Partial Thromboplastin Time Mixing Studies Using Predefined Algorithms

Background: Mixing test plasma with prolonged Prothrombin Time (PT) and activated partial thromboplastin time (aPTT) with normal plasma in a 1:1 ratio, helps distinguish between clotting factor deficiency and inhibitors. In our clinical laboratory, the upper reference interval (URI) is routinely used to determine correction (or no correction) in mixing studies. Aims and objectives: We aimed to use the International Council for Standardization in Haematology. (ICSH) 2024 recommendations for interpreting PT/aPTT mixing studies following validation of the defined cutoffs. Materials and Methods: From December 2022 to December 2024, plasma samples with prolonged PT/aPTT were tested by mixing them with normal pooled plasma at a 1:1 ratio. A cut-off of ≥70% correction or ≤15% Rosner Index was used to identify clotting factor deficiency per ICSH, and this was compared with URI. Results: After validating the cutoffs, the analysis of mixing results and factor assays was performed on 243 patients with prolonged PT (n=54), aPTT (n=56), and PT/aPTT (n=133). All patients exhibited either single or multiple clotting factor deficiencies. Inhibitor detection varied depending on the interpretive criteria: the upper reference interval method identified 12/243 cases (4.9%), the 70% correction criterion identified 20/243 cases (8.2%) overall, and the Rosner Index identified 7/189 cases (3.7%) among samples with prolonged aPTT, highlighting method-dependent discordance. Agreement between the various methods was 90.0% or higher. Conclusion: The ICSH recommendations accurately identified clotting factor deficiencies after local validation of the cut-offs. Clinical correlation and appropriate laboratory testing are essential for detecting concurrent inhibitors in clotting factor deficiency.