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The Nijmegen Hemostasis Assay: Simultaneous Fluorogenic Measurement of Thrombin and Plasmin Generation in a Single Well
Published on: February 27, 2026
Pre-analytical storage and reagent-dependent sensitivity as sources of variability in prothrombin time and activated
A D van Dam1, M M Bioch2, E H Jeninga3
1Radboud Laboratory of Diagnostics, Department of Laboratory Medicine, Radboudumc, Nijmegen, the Netherlands; Clinical Chemical Hematology Laboratory, Elisabeth-Tweesteden Hospital, Tilburg, the Netherlands; Laboratory of Clinical Chemistry and Hematology, Jeroen Bosch Hospital, 's-Hertogenbosch, the Netherlands.
Background:
Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are widely used coagulation assays whose results can vary due to pre-analytical and analytical factors. Understanding these sources of variability is essential for reliable laboratory practice and external quality assessment (EQA).
Objectives:
This study evaluates the impact of pre-analytical storage and method-dependent sensitivity on PT and aPTT measurements and provides guidance for EQA programmes and clinical interpretation.
Methods:
Plasma samples from 42 healthy volunteers were analysed within 4 h of collection or after storage at -20 °C, -80 °C, or snap-frozen in liquid nitrogen (LN₂) and subsequent storage at -80 °C. Additionally, pooled plasma was spiked with direct oral anticoagulants, heparins, and lupus anticoagulant antibodies to assess reagent-specific sensitivity. PT and aPTT were measured on multiple routine platforms using commonly used reagents from 4 different manufacturers.
Results:
Storage at -80 °C or LN₂ preserved PT and aPTT values, whereas storage at -20 °C caused variable prolongation. In healthy volunteers, PT and aPTT showed marked inter-method variability. Sensitivity to anticoagulants and lupus anticoagulant also differed considerably between reagents, with the largest variation observed for edoxaban. This study provides an overview of inter-method variability, which may affect clinical decision-making and inter-laboratory comparability.
Conclusions:
PT and aPTT are screening tests whose accurate interpretation depends on optimal sample handling and awareness of reagent- and method-specific sensitivities. These differences can affect clinical decision-making in anticoagulated patients. EQA programmes could increase their value by using frozen plasma, including clinically relevant anticoagulants, and implementing case-based scenarios rather than reporting only clotting times.
