Analytical Validation of the High Concentrated Thrombin Time-To-Reptilase Time Ratio: A Proposed Assay for Monitoring Unfractionated Heparin in Patients With Low Fibrinogen Levels.

Abstract

Introduction: This study aimed to evaluate the analytical performance of the high-concentrated thrombin time-to-reptilase time (hcTT/RT) ratio as a novel assay to neutralize fibrinogen effects and improve accuracy in unfractionated heparin (UFH) monitoring and to validate its use in clinical samples with low fibrinogen levels.

Methods: A total of 240 heparin-spiked plasma samples, prepared from 30 normal plasma samples with varying UFH concentrations, were analyzed. The hcTT/RT ratio's correlation with anti-FXa activity and its sensitivity and specificity were compared with the hcTT assay. Additionally, 89 clinical samples from UFH-treated patients with low fibrinogen levels were analyzed to validate the assay in clinical settings.

Results: Both hcTT and the hcTT/RT ratio demonstrated strong correlations with anti-FXa activity (R² = 0.76 and 0.75, respectively). The hcTT/RT ratio outperformed hcTT in detecting subtherapeutic UFH levels, achieving higher diagnostic accuracy (AUC: 0.99 vs. 0.98, p < 0.001), greater sensitivity (89.2% vs. 86.7%), and perfect specificity (100.0% vs. 98.3%), with comparable performance for supratherapeutic UFH levels. Notably, the hcTT/RT ratio remained unaffected by low fibrinogen concentrations. In the validation study, the hcTT/RT ratio showed a stronger correlation with anti-FXa activity than activated partial thromboplastin time and hcTT alone (R² = 0.72 vs. 0.63 and 0.72 vs. 0.67, respectively) and had no significant correlation with fibrinogen levels (Spearman's r = -0.01).

Conclusions: The hcTT/RT ratio is a reliable assay for monitoring UFH, especially in patients with low fibrinogen levels. Further large-scale clinical studies are needed to evaluate its practical application in clinical settings.