Fluorescence artifact correction in the thrombin generation assay: Necessity for correction algorithms in procoagulant samples

William C Chang; Joseph W Jackson; Kellie R Machlus; Alisa S Wolberg; Mikhail V Ovanesov

doi:10.1002/rth2.12499

Fluorescence artifact correction in the thrombin generation assay: Necessity for correction algorithms in procoagulant samples

Res Pract Thromb Haemost. 2021 Mar 26;5(3):447-455. doi: 10.1002/rth2.12499. eCollection 2021 Mar.

Authors

William C Chang¹, Joseph W Jackson¹, Kellie R Machlus^{2

3

4}, Alisa S Wolberg², Mikhail V Ovanesov¹

Affiliations

¹ Office of Tissues and Advanced Therapies Center for Biologics Evaluation and Research US Food and Drug Administration Silver Spring MD USA.
² Department of Pathology and Laboratory Medicine and UNC Blood Research Center University of North Carolina at Chapel Hill Chapel Hill North Carolina USA.
³ Vascular Biology Program, Department of Surgery Boston Children's Hospital and Harvard Medical School Boston MA USA.
⁴ Present address: Brigham and Women's Hospital Harvard Medical School Boston MA USA.

Abstract

Introduction: The thrombin generation (TG) test is a global hemostasis assay sensitive to procoagulant conditions. However, some TG assays may underestimate elevated TG when the thrombin fluorogenic substrate is depleted or fluorescence is attenuated by the inner filter effect (IFE).

Objectives: We sought to elucidate the extent to which procoagulant conditions require correcting for fluorogenic substrate depletion and/or IFE.

Methods: We analyzed corrections for substrate depletion and IFE and their effect on TG parameters in plasma samples with elevated blood coagulation factors in the presence or absence of thrombomodulin via commercial calibrated automated thrombogram (CAT) platform and in-house software capable of internal thrombin calibration with or without CAT-like artifact correction.

Results: Elevated thrombin peak height (TPH) and endogenous thrombin potential (ETP) were detected with 2× and 4× increases in blood coagulation factors I, V, VIII, IX, X, and XI, or prothrombin in the presence or absence of artifact correction. The effect of the CAT algorithm was evident in TG curves from both low procoagulant (thrombomodulin-supplemented) and procoagulant (factor-supplemented) plasma samples. However, in all samples, with the exception of elevated prothrombin, CAT's correction was small (<10%) and did not affect detection of procoagulant samples versus normal plasma. For elevated prothrombin samples, uncorrected TPH or ETP values were underestimated, and CAT correction produced drastically elevated TG curves.

Conclusions: Our data suggest that correction for substrate consumption and IFE, as offered by the CAT algorithm, is critical for detecting a subset of extremely procoagulant samples, such as elevated prothrombin, but is not necessary for all other conditions, including elevated factors XI and VIII.

Keywords: blood coagulation factors; calibration; hemostasis; plasma; thrombin.