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.

Conflicts of Interest

The authors declare no conflicts of interest.

Open Research

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Supporting Information

References

1H. Bussey and J. L. Francis, “Heparin Overview and Issues,” Pharmacotherapy 24, no. 8 Pt 2 (2004): 103S–107S.
10.1592/phco.24.12.103S.36109
CAS PubMed Web of Science® Google Scholar
2C. Kearon, E. A. Akl, A. J. Comerota, et al., “Antithrombotic Therapy for VTE Disease: Antithrombotic Therapy and Prevention of Thrombosis, 9th Ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines,” Chest 141, no. 2 Suppl (2012): e419S–e496S, https://doi.org/10.1378/chest.11-2301.
10.1378/chest.11-2301
CAS PubMed Web of Science® Google Scholar
3M. Hoffman, “Heparins: Clinical Use and Laboratory Monitoring,” Laboratoriums Medizin 41, no. 10 (2010): 621–626.
Google Scholar
4A. Derbalah, S. Duffull, F. Newall, K. Moynihan, and H. Al-Sallami, “Revisiting the Pharmacology of Unfractionated Heparin,” Clinical Pharmacokinetics 58, no. 8 (2019): 1015–1028.
PubMed Web of Science® Google Scholar
5X. Delavenne, E. Ollier, S. Chollet, et al., “Pharmacokinetic/Pharmacodynamic Model for Unfractionated Heparin Dosing During Cardiopulmonary Bypass,” British Journal of Anaesthesia 118, no. 5 (2017): 705–712.
10.1093/bja/aex044
CAS PubMed Web of Science® Google Scholar
6R. A. Marlar, B. Clement, and J. Gausman, “Activated Partial Thromboplastin Time Monitoring of Unfractionated Heparin Therapy: Issues and Recommendations,” Seminars in Thrombosis and Hemostasis 43, no. 3 (2017): 253–260.
PubMed Web of Science® Google Scholar
7C. M. Takemoto, M. B. Streiff, K. M. Shermock, et al., “Activated Partial Thromboplastin Time and Anti-Xa Measurements in Heparin Monitoring: Biochemical Basis for Discordance,” American Journal of Clinical Pathology 139, no. 4 (2013): 450–456.
10.1309/AJCPS6OW6DYNOGNH
CAS PubMed Web of Science® Google Scholar
8S. Samuel, T. A. Allison, S. Sharaf, et al., “Antifactor Xa Levels vs. Activated Partial Thromboplastin Time for Monitoring Unfractionated Heparin. A Pilot Study,” Journal of Clinical Pharmacy and Therapeutics 41, no. 5 (2016): 499–502.
10.1111/jcpt.12415
CAS PubMed Web of Science® Google Scholar
9R. A. Wahking, R. H. Hargreaves, S. M. Lockwood, S. K. Haskell, and K. W. Davis, “Comparing Anti-Factor Xa and Activated Partial Thromboplastin Levels for Monitoring Unfractionated Heparin,” Annals of Pharmacotherapy 53, no. 8 (2019): 801–805.
10.1177/1060028019835202
CAS PubMed Web of Science® Google Scholar
10S. Kitchen and F. E. Preston, “The Therapeutic Range for Heparin Therapy: Relationship Between Six Activated Partial Thromboplastin Time Reagents and Two Heparin Assays,” Thrombosis and Haemostasis 75 (1996): 734–739.
10.1055/s-0038-1650358
CAS PubMed Web of Science® Google Scholar
11K. McLaughlin, J. Rimsans, K. W. Sylvester, et al., “Evaluation of Antifactor-Xa Heparin Assay and Activated Partial Thromboplastin Time Values in Patients on Therapeutic Continuous Infusion Unfractionated Heparin Therapy,” Clinical and Applied Thrombosis/Hemostasis 25 (2019): 1076029619876030.
10.1177/1076029619876030
Web of Science® Google Scholar
12P. Toulon, M. Smahi, and N. Pooter, “APTT Therapeutic Range for Monitoring Unfractionated Heparin Therapy. Significant Impact of the Anti-Xa Reagent Used for Correlation,” Journal of Thrombosis and Haemostasis 19, no. 8 (2021): 2002–2006.
10.1111/jth.15264
CAS PubMed Web of Science® Google Scholar
13D. R. J. Arachchillage, F. Kamani, S. Deplano, W. Banya, and M. Laffan, “Should We Abandon the APTT for Monitoring Unfractionated Heparin?,” Thrombosis Research 157 (2017): 157–161.
10.1016/j.thromres.2017.07.006
CAS PubMed Web of Science® Google Scholar
14R. C. Gosselin and R. A. Marlar, “Preanalytical Variables in Coagulation Testing: Setting the Stage for Accurate Results,” Seminars in Thrombosis and Hemostasis 45, no. 5 (2019): 433–448.
10.1055/s-0039-1692700
PubMed Web of Science® Google Scholar
15T. Helin, T. Tirri, H. Korkala, K. Lappalainen, and L. Joutsi-Korhonen, “Laboratory Assessment of Unfractionated Heparin (UFH) With Activated Clotting Time (ACT) and Anti-Xa Activity During Peripheral Arterial Angiographic Procedure,” Diagnostics 13, no. 8 (2023): 1489.
10.3390/diagnostics13081489
CAS PubMed Web of Science® Google Scholar
16J. E. Wehner, M. Boehne, S. David, K. Brand, A. Tiede, and R. Bikker, “Activated Clotting Time (ACT) for Monitoring of Low-Dose Heparin: Performance Characteristics in Healthy Adults and Critically Ill Patients,” Clinical and Applied Thrombosis/Hemostasis 26 (2020): 1076029620975494.
10.1177/1076029620975494
Web of Science® Google Scholar
17M. Smahi, N. Pooter, M. J. Hollestelle, and P. Toulon, “Monitoring Unfractionated Heparin Therapy: Lack of Standardization of Anti-Xa Activity Reagents,” Journal of Thrombosis and Haemostasis 18, no. 10 (2020): 2613–2621.
10.1111/jth.14969
CAS PubMed Web of Science® Google Scholar
18S. Burki, B. Brand, R. Escher, W. A. Wuillemin, and M. Nagler, “Accuracy, Reproducibility and Costs of Different Laboratory Assays for the Monitoring of Unfractionated Heparin in Clinical Practice: A Prospective Evaluation Study and Survey Among Swiss Institutions,” BMJ Open 8, no. 6 (2018): e022943.
10.1136/bmjopen-2018-022943
PubMed Web of Science® Google Scholar
19F. N. Croles, M. V. Lukens, R. Mulder, M. P. M. Maat, A. B. Mulder, and K. Meijer, “Monitoring of Heparins in Antithrombin-Deficient Patients,” Thrombosis Research 175 (2019): 8–12.
10.1016/j.thromres.2019.01.007
CAS PubMed Web of Science® Google Scholar
20M. J. Ray, E. J. Perrin, I. R. Smith, and G. A. Hawson, “A Proposed Model to Monitor Heparin Therapy Using the Concentrated Thrombin Time Which Allows Standardisation of Reagents and Improved Estimation of Heparin Concentrations,” Blood Coagulation & Fibrinolysis 5 (1996): 515–521.
10.1097/00001721-199607000-00002
Google Scholar
21J. S. Wang, C. Y. Lin, and R. B. Karp, “Comparison of High-Dose Thrombin Time With Activated Clotting Time for Monitoring of Anticoagulant Effects of Heparin in Cardiac Surgical Patients,” Anesthesia & Analgesia 79, no. 1 (1994): 9–13.
10.1213/00000539-199407000-00003
CAS PubMed Web of Science® Google Scholar
22L. Shore-Lesserson, H. E. Manspeizer, M. Bolastig, D. Harrington, F. Vela-Cantos, and M. DePerio, “Anticoagulation for Cardiac Surgery in Patients Receiving Preoperative Heparin: Use of the High-Dose Thrombin Time,” Anesthesia & Analgesia 90, no. 4 (2000): 813–818.
10.1213/00000539-200004000-00008
CAS PubMed Web of Science® Google Scholar
23D. Apipongrat, P. Police, R. Lamool, P. Butthep, and W. Chantkran, “Validation of High Concentrated Thrombin Time Assay for Unfractionated Heparin Monitoring,” Journal of Clinical Laboratory Analysis 36, no. 10 (2022): e24695.
10.1002/jcla.24695
CAS PubMed Web of Science® Google Scholar
24Z. Meng, Y. Zhao, and Y. He, “Fibrinogen Level Predicts Outcomes in Critically Ill Patients With Acute Exacerbation of Chronic Heart Failure,” Disease Markers 2021 (2021): 6639393.
10.1155/2021/6639393
PubMed Web of Science® Google Scholar
25G. Zhang, L. Zhang, S. Si, et al., “Fibrinogen and Antithrombin III Are Associated With In-Hospital Mortality Among Critically Ill Patients With Acute Kidney Injury,” Renal Failure 44, no. 1 (2022): 1938–1947, https://doi.org/10.1080/0886022X.2022.2142138.
10.1080/0886022X.2022.2142138
PubMed Web of Science® Google Scholar
26C. Yao, G. Zhang, N. Zhang, et al., “Fibrinogen Is Associated With Prognosis of Critically Ill Patients With Sepsis: A Study Based on Cox Regression and Propensity Score Matching,” Mediators of Inflammation 2023 (2023): 1–13.
10.1155/2023/7312822
Web of Science® Google Scholar
27N. Omidkhoda, F. Abedi, V. Ghavami, et al., “The Effect of Heparin and Its Preparations on Disseminated Intravascular Coagulation Mortality and Hospitalization: A Systematic Review,” International Journal of Clinical Practice 2022 (2022): 2226761.
10.1155/2022/2226761
PubMed Web of Science® Google Scholar
28H. Karapetian, “Reptilase Time (RT),” Methods in Molecular Biology 992 (2013): 273–277.
10.1007/978-1-62703-339-8_20
CAS PubMed Google Scholar
29D. M. Adcock, D. M. Hoefner, K. Kottke-Marchant, R. A. Marlar, D. I. Szarmozi, and D. J. Wuranek, “Collection, Transport, and Processing of Blood Specimen for Testing Plasma-Based Coagulation Assays and Molecular Hemostasis Assays; Approved Guideline-Fifth Edition,” CLSI Document H21–A5 28, no. 5 (2008): 1–33.
Google Scholar
30 CLSI, ed., “ One-Stage Prothrombin Time (PT) Test and Activated Partial Thromboplastin Time (APTT) Test; Approved Guideline,” in CLSI Document H47–A2, 2nd ed. (Clinical and Laboratory Standards Institute, 2008).
Google Scholar
31J. A. Hanley and B. J. McNeil, “A Method of Comparing the Areas Under Receiver Operating Characteristic Curves Derived From the Same Cases,” Radiology 148, no. 3 (1983): 839–843.
10.1148/radiology.148.3.6878708
CAS PubMed Web of Science® Google Scholar
32T. Wada, A. Shiraishi, S. Gando, et al., “Association of Antithrombin With Development of Trauma-Induced Disseminated Intravascular Coagulation and Outcomes,” Frontiers in Immunology 13 (2022): 1026163.
10.3389/fimmu.2022.1026163
CAS PubMed Web of Science® Google Scholar
33J. S. Larry, “Laboratory Diagnosis of the Lupus Anticoagulant,” Clinical Laboratory Science 30, no. 1 (2017): 7–14.
10.29074/ascls.30.1.7
Google Scholar
34I. Mackie, A. Casini, M. Pieters, R. Pruthi, C. Reilly-Stitt, and A. Suzuki, “International Council for Standardisation in Haematology Recommendations on Fibrinogen Assays, Thrombin Clotting Time and Related Tests in the Investigation of Bleeding Disorders,” International Journal of Laboratory Hematology 46, no. 1 (2024): 20–32.
10.1111/ijlh.14201
PubMed Web of Science® Google Scholar

Volume47, Issue4

August 2025

Pages 738-747

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

Methods

Results

Conclusions

Conflicts of Interest

Open Research

Data Availability Statement

Supporting Information

References

References

Information

About Wiley Online Library

Help & Support

Opportunities

Connect with Wiley

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

Methods

Results

Conclusions

Conflicts of Interest

Open Research

Data Availability Statement

Supporting Information

References

References

Related

Information