FIBRINOGEN TEST

Abstract

The present invention is related to a novel and direct method for measuring the fibrinogen level in a sample, which is particularly useful in emergency situations. The novel method is independent of thrombin formation and is not interfered by the presence of oral anti-coagulation drugs or other chemicals contrary to the commonly used clotting assays.

Claims

1. A method for measuring the fibrinogen level in a sample wherein the blood coagulation cascade is inhibited, preferably inhibition of both intrinsic and extrinsic pathway.

2. A method according to claim 1 comprising an enzymatic cleavage reaction, wherein the enzymatic cleavage of fibrinogen competes with enzymatic cleavage of a detection substrate present in the sample.

3. A method according to claim 1 or comprising the use of a serine endopeptidase for enzymatic cleavage of fibrinogen.

4. A method according to claim 2, wherein the speed of enzymatic cleavage depends on the fibrinogen level in the sample.

5. A method according to claim 2 comprising measuring the proteolytic activity of a serine endopeptidase which is inversely proportional to the fibrinogen level in said sample.

6. A method according to claim 1, said method being performed in the absence of CaCl2 and/or in the absence of thrombin activity.

7. A method according to claim 1, said method comprising the presence of protease inhibitors, preferably inhibitors of fibrin polymerization, more preferably thrombin inhibitors.

8. A method according to claim 1 which does not include the generation of a calibration curve and/or the generation/presence of a fibrinogen standard.

9. A method according to claim 1, wherein the sample is selected from blood or plasma.

10. A method according to claim 1 which is used in centralized haematological or clinical laboratories, emergency rooms, emergency situations occurring even outside hospitals, medical practices, private home, paddocks, barns, or point-of-care testing (POCT) environment.

11. A diagnostic kit used for performance of a method according to claim 1.

12. A serine endopeptidase [EC 3.4.21], preferably snake venom serine endopeptidase, more preferably venombin A [EC 3.4.21.74] used in a method according to claim 1.

13. A detection substrate, preferably artificial detection substrate, particularly in combination with the serine endopeptidase according to claim 12.

14. A detectable moiety, preferably in combination with the serine endopeptidase according to claim 12.

Description

FIGURES

[0070] FIG. 1. The relationship between the fibrinogen levels and their signals (here as an e.g. absorption at 405 nm indicated on the y-axis) is shown in dependence of the time in sec (x-axis). The plain line indicates high concentration of fibrinogen, the dotted line indicates low concentration of fibrinogen and the dashed line indicates zero fibrinogen in the sample. For more explanation see text.

[0071] FIG. 2. The relationship between the fibrinogen level (given in g/L on the x-axis) is inversely proportional to the electrical signal generated by methoxydiphenylamine (y-axis) when using the i-STAT system.

[0072] FIG. 3. The relationship between the fibrinogen level (given in g/L on the x-axis) is inversely proportional to the electrical signal generated by phenylenediamine (y-axis) when using the CoaguChek system.

[0073] FIG. 4. Modeling the enzymatic kinetics of human plasma fibrinogen in either 0, 20% or 40% commercially available human plasma with PefachromTH as artificial substrate and batroxobin as enzyme. The K.sub.m of PefachromTH-batroxobin was increased about 2-fold and more than 5-fold in the presence of fibrinogen at 0.67 and 1.35 g/L, respectively, while holding the V.sub.max at similar speed. The substrate concentration is given on the x-axis, the enzyme activity is given on the y-axis. For more explanation, see text.

[0074] FIG. 5. Modeling the enzymatic kinetics of human plasma fibrinogen in either 0, 30% or 60% commercially available human plasma with PefachromTH as artificial substrate and batroxobin as enzyme. The K.sub.m of PefachromTH-batroxobin was increased about 2.5-fold and more than 5-fold in the presence of fibrinogen at 0.8 and 1.56 g/L, respectively, while holding the V.sub.max at similar speed. The substrate concentration is given on the x-axis, the enzyme activity is given on the y-axis. For more explanation, see text.

[0075] FIG. 6. Determination of fibrinogen levels in defined samples. FIG. 6A shows the pNA-release curves at different plasma Citrol-1 (PL) concentrations. PL was reconstituted and diluted to the indicated concentrations of 1.6-150%, with theoretical fibrinogen (Fg) concentrations of 0.04-3.75 g/L in the reaction carried out at room temperature in the presence of batroxobin, Pefachrom TH and Pefabloc FG. The recorded OD 405 values by a plate reader (Clariostar, BMG Labtech) at each minute were normalized against the initial background OD 405 values. The averages of the OD 405 normalized values are plotted at Y-axis, with error bars of standard deviation from 3 samples, while X-axis shows the recording time of up to 10 minutes. Each curve representing different fibrinogen concentration (represented by different shape and shade, see figure legend for details) is plotted and linked with a straight line between each recording. FIG. 6B shows the typical standard curves depicting the relationship between fibrinogen concentration and OD 405 normalized at different recording time. The X-axis is the calculated fibrinogen concentrations in the reaction, while Y-axis the OD 405 normalized from 3 replicates. Each curve represents the fibrinogen concentration-signal relationship at different recording time, e.g. 4, 6 7, 10 and 15 minutes (legend). The regression lines (solid lines) and their 95% confidence areas (contained within the dotted lines between the solid lines) at different recording time were generated by GraphPad Prism 7. FIG. 6C shows the pNA-release curves at different plasma PL concentrations and 2 other commercially available control plasmas, Control plasma P and Low abnormal control assayed plasma (Low PL). All plasmas were reconstituted according the instructions to 100% plasmas. PL was serially diluted to create standard curve spanning fibrinogen concentrations of 0.08-1.25 g/L in the reaction, for clarity, only 3.1% and 50% dilutions are plotted. Two other plasmas, Control plasma P (Siemens) and Low abnormal control assayed plasma (IL), were included in the same experimental run in which the reaction was carried out at room temperature in the presence of batroxobin, Pefachrom TH and Pefabloc FG. The recorded OD 405 values at each minute were normalized against the initial background OD 405 values. The averages of the OD 405 normalized values are plotted at Y-axis, with error bars of standard deviation from 3 samples, while X-axis shows the recording time of up to 10 minutes. Each curve representing different fibrinogen concentration (represented by different shape and shade, see figure legend for details) is plotted and linked with a straight line between each recording. FIG. 6D shows the standard curve depicting the relationship between fibrinogen concentration and OD 405 normalized at the recording time at the 10th minute. The X-axis is the calculated fibrinogen concentrations in the reaction using PL, while Y-axis the OD 405 normalized from 3 replicates. The solid regression line was generated by GraphPad Prism 7. To estimate the fibrinogen concentrations of the Control plasma P and Low PL, the OD 405 normalized values of the 2 plasmas were interpolated (dotted lines with arrows), hence giving the conversion of OD signals to fibrinogen concentrations when the plasmas were at 50% concentration in the reaction. FIG. 6E shows the estimated fibrinogen concentrations of Control plasma P (Siemens) and Low abnormal control assayed plasma (HemosIL) at different time points. Y-axis denotes the fibrinogen concentrations of these 2 plasmas, Control plasma P (filled circle) and Low abnormal control assayed plasma (open circle) when they are undiluted, with error bar representing the standard deviation. The X-axis is the recording time of up to 10 minutes of the reaction explained in FIGS. 6c and 6d. The shaded areas within the dotted lines represent the 95% confidence intervals of these 2 plasmas, Control plasma P shaded by dots and Low abnormal control assayed plasma shaded by hatching lines. For more explanation, see text.

[0076] FIG. 7. Interference with anti-coagulation drugs in PT and aPTT were tested. FIG. 7A shows the Michaelis-Menten enzyme kinetics plot of batroxobin-Pefachrome TH reaction, in the presence of different concentrations of cOmplete protease inhibitor cocktail from Roche. The X-axis represents the increasing concentration of Pefachrome TH, while Y-axis represents the enzyme activity of batroxobin in room temperature. The curves represent the relationships of the enzyme activities in the absence (control) and 0.03x-2x of recommended usage concentrations of cOmplete protease inhibitor cocktail from Roche. Increasing usage of the protease inhibitor cocktail suppressed the enzyme activity of batroxobin. The suppression was of mixture of types of inhibitions, where Vmax was reduced and Km was increased. FIG. 7B a 7C show the Michaelis-Menten constance (Km) (FIG. 7B) and Vmax (FIG. 7C) of batroxobin-Pefachrome TH substrate, in the presence of different concentrations of cOmplete protease inhibitor cocktail from Roche, as shown in FIG. 7a. The parameters were estimated by GraphPad Prism 7. The Km was increased when the concentration of the inhibitor cocktail was increased in the batroxobin-Pefachrome TH substrate reaction, while the reverse was true for Vmax. Error bar is representing the 95% confidence interval around the average value of the Km or Vmax, while the error bars of those values obtained from the reaction performed in higher inhibitor concentrations were omitted due to the extremely large confidence interval. The batroxobin-Pefachrome TH substrate reaction was affected by a cocktail of general protease inhibitors, but not by the typical therapeutic and non-therapeutic inhibitors in blood coagulation. For more explanation, see text.

[0077] FIG. 8. Interference studies with different DOACs using the inventive batroxobin-Pefachrome TH enzymatic reaction. FIG. 8A shows the Michaelis-Menten enzyme kinetics plot of batroxobin-Pefachrom TH reaction in the presence of different concentrations of Dabigatran. The X-axis represents the increasing concentration of Pefachrome TH, while Y-axis represents the enzyme activity of batroxobin in room temperature. The curves represent the relationships of the enzyme activities in the presence of 0 ng/mL (as negative control) and 31-500 ng/mL of Dabigatran. Increasing usage of the Dabigatran did not significantly suppress the enzyme activity of batroxobin. FIG. 8B shows the Michaelis-Menten enzyme kinetics plot of batroxobin-Pefachrom TH reaction in the presence of different concentrations of 0.13-2.0 g/mL Argatroban. Testing was performed as in FIG. 8A. FIG. 8C shows the Michaelis-Menten enzyme kinetics plot of batroxobin-Pefachrom TH reaction in the presence of different concentrations of 38-600 ng/mL Rivaroxaban. Testing was performed as in FIG. 8A. FIG. 8C shows the Michaelis-Menten enzyme kinetics plot of batroxobin-Pefachrom TH reaction in the presence of different concentrations of Dabigatran, Argatroban and Rivaroxaban. The X-axis represents the increasing concentration of Pefachrome TH, while Y-axis represents the enzyme activity of batroxobin in room temperature. For more detail plot of each drug treatment, please refer to the individual graph (8a: Dabigatran, 8b: Argatroban, 8c: Rivaroxaban). Increasing usage of the drugs did not significantly suppress the enzyme activity of batroxobin. FIG. 8D shows Michaelis-Menten constant (Km), FIG. 8E shows Vmax of batroxobin-Pefachrome TH substrate, in the presence of different concentrations of Dabigatran, Argatroban or Rivaroxaban, as shown in FIG. 8a-8d. The Km was not significantly affected by all concentrations of all inhibitors. Since Km of batroxobin-Pefachrome TH substrate reaction was much more influenced by the presence of fibrinogen, this fibrinogen test principle should be well resistant to the presence of DM and DXaIs. The presence of very high doses of drug could reduce slightly Vmax, but the effect on the fibrinogen assay can be considered negligible. Error bar is representing the 95% confidence interval around the average value of the Km or Vmax. For more explanation, see text.

[0078] FIG. 9. Interference with chemicals is shown. FIG. 9A shows the Michaelis-Menten enzyme kinetics plot of batroxobin-Pefachrom TH reaction in the presence of chemicals known to inhibit coagulation and fibrinolysis pathways. The X-axis represents the increasing concentration of Pefachrome TH, while Y-axis represents the enzyme activity of batroxobin in room temperature. The curves represent the relationships of the enzyme activities in the presence of 6 U/mL of Fragmin (a low molecular weight heparin available from Pfizer), as well as combined 10 TIU/mL aprotinin and 0.1 M 6-aminocaproic acid. Comparing to the untreated reaction (control), these agents did not significantly influence the activity. FIG. 9B shows Michaelis-Menten constant (Km), FIG. 9C shows Vmax of batroxobin-Pefachrome TH substrate in the presence of chemicals known to inhibit coagulation and fibrinolysis pathways. The Km was not significantly affected by all concentrations of all inhibitors. Since Km of batroxobin-Pefachrome TH substrate reaction was much more influenced by the presence of fibrinogen, this fibrinogen test principle should be well resistant to the presence of these inhibitors. Error bar is representing the 95% confidence interval around the average value of the Km or Vmax. For more explanation, see text.

[0079] FIG. 10. The pNA-release curves at 2 different PL concentrations to demonstrate the adaptability of the batroxobin-Pefachrome TH reaction. PL was diluted to 4% and 24% to create reactions with fibrinogen concentrations of roughly 0.1 and 0.7 g/L, respectively, in the reaction run at 37 C. in the appropriate amount of batroxobin and Pefachrome TH (refer to the legend for details). The recorded OD 405 values at each minute (X-axis) were normalized against the initial background OD 405 values, hence the OD 405 normalized as Y-axis. The averages of the OD 405 normalized values are plotted at Y-axis, with error bars of standard deviation from 3 samples, while X-axis shows the recording time of up to 10 minutes. Each curve represented by straight line linking the averages of the OD 405 normalized values depicts the reaction speed at different conditions (refer to the legend for details).

EXAMPLES

Example 1: Whole Blood Fibrinogen Level Measurement on PoC Device with PT-INR and ACT Functionalities

[0080] Fibrinogen level measurement in a sample using the i-STAT point of care system (Axonlab/Abbott) is described herein, which should enable the user of the device to determine the fibrinogen level from the test object (patient) within a very short time.

[0081] The test should work very similar to the existing prothrombin time (PT) offered by i-STAT, except giving INR information triggered by tissue factor. The new fibrinogen utilizes the snake venom protein, batroxobin, to convert fibrinogen into fibrin. In the presence of an artificial detection-substrate including PPAAM or PefachromeTH for thrombin-like serine protease, and batroxobin, said artificial substrate is competing with the fibrinogen. With fixed amounts of batroxobin and PPAAM in the test, the relationship of fibrinogen concentration and of the electrochemical signal generated by the amount of the detection-substrate PPAAM can be determined. The fibrinogen is competing with PPAAM for batroxobin, resulting in a relationship between fibrinogen levels and electrochemical signals which are inversely proportional (FIG. 2).

Example 2: Fibrinogen Assay with CoaguChek from Roche Diagnostics

[0082] Fibrinogen level measurement in a sample using the CoaguChek XS point of care device from Roche Diagnostics GmbH is described herein, which should enable the user of the device to determine the fibrinogen level in the whole blood sample from the test object (patient) within a very short time.

[0083] The test should work very similar to the existing prothrombin time (PT) test offered by CoaguChek XS. The new fibrinogen test utilizes the snake venom protein, batroxobin, to convert sample fibrinogen into fibrin. In the presence of an artificial detection substrate including Electrozyme TH or PefachromeTH, i.e. substrates for thrombin-like serine protease, and batroxobin said artificial substrate is competing with the fibrinogen. With fixed amounts of batroxobin and the detection-substrate in the test, the relationship of fibrinogen concentration and electrochemical signal generated by the amount of active detection-substrate can be determined. Since the fibrinogen is competing with the detection-substrate for batroxobin, the relationship between fibrinogen levels and electrochemical signals are inversely proportional to each other (FIG. 3).

Example 3: Fibrinogen Assay with a Standard Spectrophotometer

[0084] Measurement of the fibrinogen level using PefachromTH containing the detectable moiety para-nitroaniline (pNA) and batroxobin together with various human plasma concentrations was used with CLARIOstar (BMG Labtech). Again, competition between fibrinogen as natural substrate and the artificial substrate PefachromeTH for cleavage by the enzyme batroxobin (E) was measured.

[0085] Commercially available human plasma in different dilutions, 30% and 60%, was used as source of fibrinogen. Samples of different fibrinogen levels, 0.8 and 1.56 g/L, respectively, were prepared. The enzymatic activity was calculated based on the amount of pNA released per minute. The amount of pNA is directly proportional to the absorption at 405 nm. Different concentrations of PefachromTH and fibrinogen were tested in the presence of (E) in terms of the velocity of pNA release, the results and analysis are summarized in the FIG. 4 and Table 2.

[0086] Based on the Michaelis-Menten enzyme kinetic modeling, the presence of fibrinogen was altering the Michaelis-Menten constants (K.sub.m) of the enzyme-substrate reactions, while the maximum enzymatic reaction speed (V.sub.max) of the reactions were not significantly altered. The increased K.sub.m and similar V.sub.max consistently demonstrated the non-inhibitory competition between the fibrinogen and PefachromTH.

[0087] Based on the Michaelis-Menten equation, wherein K.sub.cat, which is almost constant in this case, denotes the maximum number of substrate molecules per active site per second, and the concentrations of both (S) and (E) are the same too in the reactions, the increased in K.sub.m significantly affects the enzymatic reaction speed (v). The change of the enzymatic reaction speed, due to the presence of fibrinogen, can easily be measured and provides the estimation of fibrinogen concentration. Using PefachromTH as the artificial substrate (S), we monitored the v of the pNA generation by batroxobin as enzyme (E) in the presence of different levels of human plasma derived fibrinogen, as shown in Table 2. The changes in v were due to the presence of fibrinogen, and the decrease in v was directly proportional to the increase in fibrinogen concentration, which was due to the increase in K.sub.m based on the Michaelis-Menten enzyme kinetics.

TABLE-US-00002 TABLE 2 The enzymatic reaction of PefachromTH via cleavage by batroxobin in the presence of different concentrations of human plasma-derived fibrinogen. The enzymatic reaction time after 7 or 10 min was measured at 405 nm. The data is based on 2 independent measurements. For more details see text. Plasma Fibrinogen OD405 OD405 conc. [%] [g/l] (7 min) (10 min) 0 0 0.1175 0.158 20 0.674 0.095 0.132 40 1.348 0.063 0.085

[0088] Continuing with PefachromTH as the artificial substrate (S) here, we monitored the v of the pNA generation by batroxobin in the presence of different levels of human plasma derived fibrinogen (Table 2). The changes in v were due to the presence of fibrinogen, and the decrease in v was inversely proportional to the increase in fibrinogen concentration, which was due to the increase in K.sub.m based on the Michaelis-Menten enzyme kinetics.

Example 4: Fibrinogen Assay with a Standard Spectrophotometer

[0089] Measurement of the fibrinogen level using PefachromTH containing the detectable moiety para-nitroaniline (pNA) and batroxobin together with various human plasma concentrations was used with CLARIOstar (BMG Labtech). Again, competition between fibrinogen as natural substrate and the artificial substrate PefachromeTH for cleavage by the enzyme batroxobin (E) was measured.

[0090] Commercially available human plasma in different dilutions, 30% and 60%, was used as source of fibrinogen. Samples of different fibrinogen levels, 0.8 and 1.56 g/L, respectively, were prepared. The enzymatic activity was calculated based on the amount of pNA released per minute. The amount of pNA is directly proportional to the absorption at 405 nm. Different concentrations of PefachromTH and fibrinogen were tested in the presence of (E) in terms of the velocity of pNA release, the results and analysis are summarized in the FIG. 5 and Table 3.

[0091] Based on the Michaelis-Menten enzyme kinetic modeling, the presence of fibrinogen was altering the Michaelis-Menten constants (K.sub.m) of the enzyme-substrate reactions, while the maximum enzymatic reaction speed (V.sub.max) of the reactions were not significantly altered. The increased K.sub.m and similar V.sub.max consistently demonstrated the non-inhibitory competition between the fibrinogen and PefachromTH.

[0092] Based on the Michaelis-Menten equation, wherein K.sub.cat, which is almost constant in this case, denotes the maximum number of substrate molecules per active site per second, and the concentrations of both (S) and (E) are the same too in the reactions, the increased in K.sub.m significantly affects the enzymatic reaction speed (v):

[00001]$v=\frac{k_{\mathrm{cat}}\left[E\right]\left[S\right]}{K_m+\left[S\right]}$

[0093] The change of the enzymatic reaction speed, due to the presence of fibrinogen, can easily be measured and provides the estimation of fibrinogen concentration. Using PefachromTH as the artificial substrate (S), we monitored the v of the pNA generation by batroxobin as enzyme (E) in the presence of different levels of human plasma derived fibrinogen, as shown in Table 3. The changes in v were due to the presence of fibrinogen, and the decrease in v was inversely proportional to the increase in fibrinogen concentration, which was due to the increase in K.sub.m based on the Michaelis-Menten enzyme kinetics.

TABLE-US-00003 TABLE 3 The enzymatic reaction of PefachromTH via cleavage by batroxobin in the presence of different concentrations of human plasma-derived fibrinogen. The enzymatic reaction time after 7, 11 or 16.5 min was measured at 405 nm. The data is based on 2 independent measurements. For more details see text. Fibrinogen OD405 OD405 OD405 [g/l] (7 min) (10 min) (16.5 min) 0 0.159 0.262 0.394 0.8 0.131 0.209 0.310 1.56 0.062 0.105 0.161 3.1 0.031 0.050 0.080

[0094] Continuing with PefachromTH as the artificial substrate (S) here, we monitored the v of the pNA generation by batroxobin in the presence of different levels of human plasma derived fibrinogen (Table 3). The changes in v were due to the presence of fibrinogen, and the decrease in v was directly proportional to the increase in fibrinogen concentration, which was due to the increase in K.sub.m based on the Michaelis-Menten enzyme kinetics.

Example 5: Plasma Fibrinogen Concentrations Determined by Batroxobin Enzyme Kinetics

[0095] Measurement of the fibrinogen level was performed using well characterized plasmas, which were commercially available, to challenge the feasibility of this innovative principle of fibrinogen measurement in blood sample.

[0096] The current well accepted fibrinogen assay is clot-based Clauss test. The control plasmas, available from Siemens and Instrumentation Laboratory (IL), are used in the standard Clauss test as controls in fibrinogen measurement, and the fibrinogen concentrations were well characterized (Table 4). To test the feasibility of this chromogenic fibrinogen assay in plasma fibrinogen determination, briefly, the calibration curves was obtained from serially diluted Citrol-1, a control plasma from Siemens (FIG. 6a,6b). The other 2 plasmas with different fibrinogen levels (Table 4), Control plasma P (Siemens) and Low abnormal control assayed plasma (IL), were assayed to estimate their fibrinogen concentrations by intrapolating from the calibration curve created using Citrol-1 (see FIGS. 6c, 6d and 6e for details).

TABLE-US-00004 TABLE 4 Plasma samples and their fibrinogen concentrations. Control plasma P (from Siemens), low abnormal control assayed plasma (from IL) and control plasma Citrol-1 (from Siemens) as stated in the product inserts were extracted and summarized in this table. Each fibrinogen concentration is displayed in average value and confidence interval (in bracket) determined by different instrument/analyzer and reagent. For more details see text. Fibrinogen concentration [g/l] Analyzer reagent Control plasma P HaemosiL Citrol-1 Siemens CA Multifibren U 1.0 (0.6-1.4) 2.5 (2.2-2.8 Cl) systems Dade Thrombin 0.8 (0.4-1.2) 2.5 (2.2-2.8 Cl) Reagent BCS XP Multifibren U 1.0 (0.6-1.4) 2.6 (2.3-2.9 Cl) PT-Fibrinogen 1.5 (1.1-1.9) ACL classic PT-fibrinogen 1.8 (1.4-2.2) HS PLUS Fibrinogen-C 1.9 (1.5-2.3) PT-Fibrinogen 1.4 (1.0-1.8) ACL TOP PT-fibrinogen 1.8 (1.4-2.2) HS PLUS Fibrinogen-C 1.9 (1.4-2.4)

[0097] Based on the current conditions described in FIG. 6, the assay was able to have good differentiation or separation between fibrinogen levels of 0.05 and 0.3 g/L (FIG. 6a & 6b). The inversely proportional relationship between the fibrinogen concentration and the measurable signal as OD at 405 nm was clearly demonstrated in this clinically characterized control plasma (FIG. 6a & 6b). A calibration curved was produced and the fibrinogen concentrations of the 2 plasmas were estimated (FIG. 6c & 6d). The estimations of these 2 plasmas were matching very well with the values given by the plasma suppliers (FIG. 6e/Table 4). Hence, the clinically relevant plasma samples of abnormally low fibrinogen levels were correctly estimated using the inventive method, based on batroxobin enzyme kinetics.

Example 6: Interference Study of Direct Thrombin (DTI) and Direct FXa Inhibitor (DXaI) in the Fibrinogen Measurement Based on Batroxobin Enzyme Kinetics

[0098] In this example the advantageous property of the inventive method has been tested against interference from direct thrombin or FXa inhibitors.

[0099] In the emergency situation when a patient needed a fibrinogen level estimation, the fibrinogen test has to be free of as many interfering factors as possible. The use of direct oral anti-coagulants (DOACs), including DM and DXaIs, are getting more common to prevent thrombosis in many diseases. In this example, we tested 3 protease inhibitors Dabigatran (a DTI), Argatroban (a DTI) and Rivaroxaban (aDXaI) in our new method to assess the interference of these representative drugs of this class in our fibrinogen measurement method. To evaluate the inhibitory effect of these pharmaceutical agents, we looked into the effects of these agents in the enzyme kinetics between batroxobin and its substrate Pefachrome TH. The batroxobin-Pefachrome TH enzymatic reactions were tested in the presence of a cocktail of protease inhibitors obtained from Roche, cOmplete protease inhibitor cocktail (see FIG. 7a). The enzyme kinetic parameters like Vmax and Km were estimated based on Michaelis-Menten enzyme kinetic model, and the inhibitory effects of the cocktails on the enzymatic reaction was clearly visible (FIG. 7a), and the Michaelis-Menten parameters, Vmax and Km, indicate mixtures of inhibitions (FIG. 7b & 7c).

[0100] With the establishment of enzyme kinetic study, the interference of the DM and DXaI was started by testing the potency of these drugs in the two common blood coagulation tests, namely Prothrombin Time (PT) and activated Partial Thromboplastin Time (aPTT). In the presence of these DTI and DXaI, these two tests will display delays in clotting time. Based on this principle, we assessed the potency of these drugs by applying the reported peak and trough plasma concentrations in PT and aPTT. The results are shown in Table 5: the direct thrombin and FXa inhibitors, denoted as DTI and DXaI respectively, were able to delay blood clotting time based on Prothrombin Time (PT) and activated Partial Thromboplastin Time (aPTT). The reported maximum and trough concentrations of the Dabigatran was between 447-10 ng/mL, while Rivaroxaban was 535-6 ng/mL. The control plasma was spiked individually with different amounts and kinds of inhibitors, and the clotting times of PT and aPTT were recorded by BCS-XP (Siemens).

TABLE-US-00005 TABLE 5 Summary of the inhibitory effects of DTIs and DXaI within the clinical range of concentrations which were also tested on the influence of batroxobin-mediated fibrinogen assay. They are denoted by the name of the inhibitor along with the final concentration in the plasma. The negative control (denoted as neg. control) was the control plasma without any spiking of inhibitor. Another negative control (denoted as ISTH) was the recommended control plasma by International Society on Thrombosis and Hemostasis (ISTH), also without any spiking of inhibitor. PT-test aPTT-test CT Mean CT Mean Drug (sec) (sec) delay (sec) (sec) delay Dabigatran 19.81 19.90 97% 91.09 90.98 183% 500 ng/ml 19.98 90.87 Dabigatran 10.58 10.64 6% 41.3 41.28 28% 31 ng/ml 10.69 41.2 Rivaroxaban 19.42 19.37 92% 80.73 80.40 150% 600 ng/ml 19.32 80.07 Rivaroxaban 10.74 10.68 6% 39.46 39.45 23% 38 ng/ml 10.61 39.44 Argatroban 12.26 12.25 22% 55.77 55.62 73% 2.0 g/ml 12.24 55.46 Argatroban 10.18 10.20 1% 34.62 34.62 8% 0.13 g/ml 10.21 34.62 ISTH 9.65 9.68 28.77 28.71 9.70 28.64 Neg. control 10.10 10.08 32.16 32.14 10.05 32.12

[0101] A spectrum of potencies of strong to weak based on the modes of action and concentrations was detected, and the results were consistent with literatures.

[0102] Having demonstrated the potency of the drugs in inhibiting thrombin and FXa, the interfering effects of Dabigatran, Argatroban and Rivaroxaban were studied in the batroxobin-Pefachrome TH enzymatic reaction. In the enzyme kinetic study, we included from 0-500 ng/mL of Dabigatran into the batroxobin-Pefachrome TH reaction (see FIG. 8a). The similar study was carried for the drug Argatroban between the range of 0-2 g/mL (see FIG. 8b). Additionally, Rivaroxaban of 0-600 ng/mL was applied to this enzyme kinetic study (see FIG. 8c). In this study, we did not observe the strong inhibitory effects with DTIs and DXaI as with the cocktail of protease inhibitors from Roche (comparing FIGS. 8a to 8c). There might be very slight reduction in Vmax at the highest dose of each drug, but the Km stayed very constant throughout the different drugs and concentrations (FIG. 8e & 8f). Since Km is the main parameter being shifted according to the presence of fibrinogen, i.e. the higher the fibrinogen concentration, the larger Km is increased (refer to Example 3 & 4 for details), we can safely rule out the assay will be interfered by the typical DTIs and DXaIs or common DOACs.

Example 7: Interference Study of Chemicals Known to Affect Clot-Based Assay

[0103] In this example the advantageous property of the inventive method was tested against chemical interference known to affect clot-based assays.

[0104] Similar to the test performed in the previous example, enzyme kinetics between batroxobin and its substrate Pefachrome TH were evaluated. Potential interfering substances, which have been demonstrated to interfere in clot-based assays, are heparins (including unfractionated and low molecular weight heparins, UFH and LMWH), hirudin, EDTA and fibrinogen degradation products (FDPs). Heparins and hirudin are therapeutic substances in the treatment of thrombosis. Increased FDPs presence in plasma is due to conditions that increase fibrinolysis and fibrinogen lysis. The normal FDP level is around 5-8 g/mL. Higher FDP concentration is known to inhibit clot formation. Pharmaceutical substances to inhibit fibrinolysis in the treatment of hemorrhages like aprotinin and 6-aminocaproic acid were also included in this study. Additionally, a colloid hydroxyethyl starch (HES), used in the plasma expander solution, was subjected to interfering activity study.

[0105] First, very high concentrations of low molecular weight heparin (Fragmin), aprotinin and 6-aminocaproic acid were tested for their interference in the batroxobin-Pefachrome TH enzyme reaction (see FIG. 9a). The predicted Vmax and Km were not significantly different from the untreated control (FIG. 9b & 9c). Hence, it is very safe to conclude that the inventive method for fibrinogen measurement will not be interfered by these substances.

[0106] We furthermore assessed the interference of unfractionated heparin (Liquemin: till 4 U/mL), calcium-chelator EDTA (till 8 mM), hirudin (till 4 U/mL), HES (till 5 mg/mL), FDP (till 57 g/mL) using the same methodology. We failed to see significant interference coming from all these substances, indicating again the independence or non-interference of the inventive method against these substances (data not shown).

Example 8: Adaptable Enzymatic Conditions

[0107] Since the typical PoC devices, i.e. iSTAT and CoaguCheck, warm up their blood samples to body temperature during testing, we studied this principle when operated in body temperature. We adjusted a few parameters so that we could increase the signal output and allow good differentiation at the low fibrinogen concentrations.

[0108] The adaptation of the inventive fibrinogen detection method based on batroxobin enzyme kinetics to body temperature was successfully performed. Parameters like the concentrations of the enzyme and substrates were adjusted to produce desirable performance at low fibrinogen concentrations in plasma (see FIG. 10).