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Updated: May 25, 2026
The Nijmegen Hemostasis Assay: Simultaneous Fluorogenic Measurement of Thrombin and Plasmin Generation in a Single Well
Published on: February 27, 2026
Development and application of an automated chromogenic thrombin generation assay that is sensitive to defects in the
Laura Green1, Omid Safa, Samuel J Machin
1Haemostasis Research Unit, Department of Haematology, University College London, London, UK.
Background:
Activated protein C resistance (APCR) within a thrombin generation test (TGT) system is associated with an increased risk of venous thromboembolism (VTE). However, application of the TGT is restricted by the analytical platforms used to monitor thrombin generation. Using a routine coagulation analyser we have developed an automated chromogenic TGT that is sensitive to defects in the protein C pathway.
Method:
The TGT was performed on a TOP500 analyser, in the presence and absence of Protac. The reaction was monitored using a substrate with slow kinetics for thrombin (S-2444). Results were expressed as the area under the curve normalised ratio (AUCnr). Assay results were compared with Coatest APCR (expressed as APC-ratio [CoAPCr]).
Patients:
Samples were obtained from 35 healthy subjects and 91 patients with previous history of VTE. Of these patients, 19, 17, and 9 had heterozygous factor V Leiden (FVL), antiphospholipid syndrome (APS), and protein C/protein S deficiencies (PC/PS) respectively.
Results:
Inter-assay imprecision in the presence and absence of Protac were 20% and <5% respectively. There was a significant difference between the AUCnr of normals (median [IQR]: 2.8 [2.4-4.7]) compared to: FVL (1.0 [0.7-1.2]); PC/PS (1.1 [0.9-1.2]); and APS (1.1 [0.8-1.4]); p<0.001 for each comparison. No significant difference was seen between the AUCnr of normals and other VTE patients. The detection rate of AUCnr and CoAPCr were: 100% and 56% for FVL; 88% and 44% for PC/PS; and 64% and 45% for APS respectively.
Conclusion:
The automated TGT exhibited good sensitivity to defects in the protein C pathway.
