Journal: Thrombosis Journal

Article Title: Sources of bias and limitations of thrombinography: inner filter effect and substrate depletion at the edge of failure algorithm

doi: 10.1186/s12959-023-00549-5

Figure Lengend Snippet: IFE of AMC fluorophore and its correction via calibration or normalization. FVIII-DP was supplemented with 1 IU/mL FVIII to normalize hemophilia plasma, or not, and was subsequently premixed with the indicated concentrations of AMC prior to initiating coagulation with Ca 2+ and substrate. Raw fluorescent data were produced by the CAT microplate reader and software and analyzed in several different ways: (A, B) raw AMC fluorescence in relative fluorescent units (RFU), (C, D) internally calibrated TG curves via a thrombin calibration coefficient (see Materials and Methods), (E, F) normalized-uncalibrated curves, (G, H) calibrated TG curves (via TS software), (I, J) calibrated TG curves (via OR software), and (L, M) calibrated TG curves (via SH software). Uncalibrated curve data were produced by differentiating the AMC curves observed in (A, B) . Calibrated curves were produced using TS software, our in-house OR software, which uses published algorithms similar to CAT calibration, or SH software, our second in-house app based on CAT algorithm. An asterisk (*) next to the indicated concentrations in panels G & H denotes high AMC concentrations in which commercial TS software did not report TG curves, possibly due to their noisy appearance as suggested by the TG curves reported by OR and SH software apps at these high concentrations. Normalized-uncalibrated curves were produced by normalizing each uncalibrated curve pairing of hemophilic sample and normalized hemophilia sample (hemophilic plasma supplemented with FVIII) at each pre-spiked AMC concentration against the TPH value of the normalized plasma sample in each pairing. TG was recorded for 40–60 min. Assay conditions: 63 µL of FVIII-DP, 1 µL of FVIII (1 IU/mL), 16 µL of AMC at indicated concentrations, 20 µL of PPP trigger, and 20 µL of FluCa.

Article Snippet: The experiment was then initiated by injection of the mixture of provided Fluo-Substrate and Fluo-Buffer (i.e., FluCa reagent mixture) using a Fluoroskan Ascent microplate reader (Stago).

Techniques: Coagulation, Produced, Software, Fluorescence, Concentration Assay

Journal: Thrombosis Journal

Article Title: Sources of bias and limitations of thrombinography: inner filter effect and substrate depletion at the edge of failure algorithm

doi: 10.1186/s12959-023-00549-5

Figure Lengend Snippet: Substrate consumption and its correction via calibration or normalization. FVIII-DP was supplemented with 1 IU/mL FVIII to normalize hemophilic plasma, or not, and was subsequently premixed with the indicated concentrations of substrate, ZGGR-AMC, prior to initiating coagulation with Ca 2+ and substrate. Raw data was produced by the CAT microplate reader and analyzed in several different ways: (A, B) raw AMC fluorescence in relative fluorescent units (RFU), (C, D) internally calibrated TG curves via a thrombin calibration coefficient (see Materials and Methods), (E, F) normalized-uncalibrated curves, (G, H) calibrated TG curves (via TS software), (I, J) calibrated TG curves (via OR software), and (L, M) calibrated TG curves (via SH software). Uncalibrated curves data were produced by differentiating the AMC curves observed in (A, B) . Calibrated curves were produced using TS, OR or SH apps all of which employed the same CAT correction algorithm. Normalized-uncalibrated curves were produced by normalizing each uncalibrated curve pairing of hemophilic and normalized sample (hemophilic plasma supplemented with FVIII) at each pre-spiked AMC concentration against the TPH value of the normalized plasma sample in each pairing. TG was recorded for 40 min. Assay conditions: 63 µL of FVIII-DP, 1 µL of FVIII (1 IU/mL), 16 µL of substrate ZGGR-AMC (at indicated concentrations), 20 µL of PPP trigger, and 20 µL of Ca 2+ (calcium chloride buffer)

Article Snippet: The experiment was then initiated by injection of the mixture of provided Fluo-Substrate and Fluo-Buffer (i.e., FluCa reagent mixture) using a Fluoroskan Ascent microplate reader (Stago).

Techniques: Coagulation, Produced, Fluorescence, Software, Concentration Assay

Journal: Thrombosis Journal

Article Title: Sources of bias and limitations of thrombinography: inner filter effect and substrate depletion at the edge of failure algorithm

doi: 10.1186/s12959-023-00549-5

Figure Lengend Snippet: An attempt to study substrate consumption in plasma samples supplemented with two substrates, ZGGR-AMC and ZGGR-AFC. FVIII-DP was supplemented with 1 IU/mL FVIII to normalize hemophilic plasma, or not, and was subsequently premixed with the indicated concentrations of two substrates, ZGGR-AMC and ZGGR-AFC, such that the ratio of AMC:AFC equaled to a concentration of 800 µM, prior to initiating coagulation with Ca 2+ . Raw data was produced by the Biotek microplate reader and analyzed in several different ways: (A, B) raw AMC fluorescence in relative fluorescent units (RFU), (C, D) internally calibrated TG curves via a thrombin calibration coefficient (see Materials and Methods), (E, F) Normalized-Uncalibrated curves, (G, H) Calibrated TG curves (via OR software), and (I, J) calibrated TG curves (via SH software). Uncalibrated curve data were produced by differentiating the AMC curves observed in (A, B) . CAT calibrated curves were produced using our in-house OR and SH software apps, which use published algorithms similar to CAT calibration. Normalized-uncalibrated curves were produced by normalizing each uncalibrated curve pairing of hemophilic and normalized sample (hemophilic plasma supplemented with FVIII) at each pre-spiked AMC concentration against the TPH value of the normalized plasma sample in each pairing. TG was recorded for 40–60 min. An artifact resembling TG signal in early minutes in Fig. 5 is only seen with ZGGR-AFC experiments, suggesting that it is caused by either the AFC fluorophore itself (similar to Fig. and Fig. S1 discussed above) or background fluorescence signal of un-cleaved ZGG-AFC substrate. Assay conditions: 78 µL of FVIII-DP, 2 µL of FVIII (1 IU/mL), 20 µL of PPP trigger, and 20 µL of custom FluCa mixtures (substrates ZGGR-AMC and ZGGR-AFC at indicated concentrations with calcium chloride buffer)

Article Snippet: The experiment was then initiated by injection of the mixture of provided Fluo-Substrate and Fluo-Buffer (i.e., FluCa reagent mixture) using a Fluoroskan Ascent microplate reader (Stago).

Techniques: Concentration Assay, Coagulation, Produced, Fluorescence, Software

Journal: Thrombosis Journal

Article Title: Sources of bias and limitations of thrombinography: inner filter effect and substrate depletion at the edge of failure algorithm

doi: 10.1186/s12959-023-00549-5

Figure Lengend Snippet: Effect of CAT calibration on TG curves in procoagulant plasma samples. Antithrombin deficient plasma (ATIII-DP) was treated with or without heparin and the indicated TF concentration to assess the effect of calibration via different software apps in procoagulant samples. Raw data was produced by the CAT assay microplate reader and analyzed in several different ways: (A, B) raw AMC fluorescence in relative fluorescent units (RFU), (C, D) internally calibrated TG curves via a thrombin calibration coefficient (see Materials and Methods), (E, F) normalized-Uncalibrated curves, (G, H) calibrated TG curves (via CBER algorithm), and (I, J) calibrated TG curves (via SH software). Uncalibrated curve data were produced by differentiating the AMC curves observed in (A, B) . CAT calibrated curves were produced by our in-house OR and SH software apps. Normalized-uncalibrated curves were produced by normalizing each uncalibrated curve pairing of hemophilic and normalized sample (hemophilic plasma supplemented with FVIII) at each pre-spiked AMC concentration against the TPH value of the normalized plasma sample in each pairing. TG was recorded for 60 min. Assay conditions: ATIII-DP with 0.2 U/mL of normal pooled plasma with or without heparin (0.03 USP/mL), TF (0.12–20 pM), tPA (0.13 µg/mL), thrombomodulin (12.5 nM), PC:PS vesicles (4 µM) and custom FluCa mixture (800 µM ZGGR-AMC and calcium chloride)

Article Snippet: The experiment was then initiated by injection of the mixture of provided Fluo-Substrate and Fluo-Buffer (i.e., FluCa reagent mixture) using a Fluoroskan Ascent microplate reader (Stago).

Techniques: Concentration Assay, Software, Produced, Fluorescence