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

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...

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Autores principales: Chang, William C., Jackson, Joseph W., Machlus, Kellie R., Wolberg, Alisa S., Ovanesov, Mikhail V.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035796/
https://www.ncbi.nlm.nih.gov/pubmed/33870030
http://dx.doi.org/10.1002/rth2.12499
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author Chang, William C.
Jackson, Joseph W.
Machlus, Kellie R.
Wolberg, Alisa S.
Ovanesov, Mikhail V.
author_facet Chang, William C.
Jackson, Joseph W.
Machlus, Kellie R.
Wolberg, Alisa S.
Ovanesov, Mikhail V.
author_sort Chang, William C.
collection PubMed
description 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.
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spelling pubmed-80357962021-04-15 Fluorescence artifact correction in the thrombin generation assay: Necessity for correction algorithms in procoagulant samples Chang, William C. Jackson, Joseph W. Machlus, Kellie R. Wolberg, Alisa S. Ovanesov, Mikhail V. Res Pract Thromb Haemost Original Articles ‐ Thrombosis 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. John Wiley and Sons Inc. 2021-03-26 /pmc/articles/PMC8035796/ /pubmed/33870030 http://dx.doi.org/10.1002/rth2.12499 Text en © 2021 International Society on Thrombosis and Haemostasis (ISTH). This article is a U.S. Government work and is in the public domain in the USA. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ (https://creativecommons.org/licenses/by-nc-nd/4.0/) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.
spellingShingle Original Articles ‐ Thrombosis
Chang, William C.
Jackson, Joseph W.
Machlus, Kellie R.
Wolberg, Alisa S.
Ovanesov, Mikhail V.
Fluorescence artifact correction in the thrombin generation assay: Necessity for correction algorithms in procoagulant samples
title Fluorescence artifact correction in the thrombin generation assay: Necessity for correction algorithms in procoagulant samples
title_full Fluorescence artifact correction in the thrombin generation assay: Necessity for correction algorithms in procoagulant samples
title_fullStr Fluorescence artifact correction in the thrombin generation assay: Necessity for correction algorithms in procoagulant samples
title_full_unstemmed Fluorescence artifact correction in the thrombin generation assay: Necessity for correction algorithms in procoagulant samples
title_short Fluorescence artifact correction in the thrombin generation assay: Necessity for correction algorithms in procoagulant samples
title_sort fluorescence artifact correction in the thrombin generation assay: necessity for correction algorithms in procoagulant samples
topic Original Articles ‐ Thrombosis
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035796/
https://www.ncbi.nlm.nih.gov/pubmed/33870030
http://dx.doi.org/10.1002/rth2.12499
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