Abstract:
The system includes: an assortment ( 10 ) of sensors ( 10   a,    10   b,    10   c ), wherein the working electrode ( 14   a ) of each sensor is covered with a specific reagent of a given proteolytic enzyme, including a substrate capable of releasing leaving groups (LG) via the action of the enzyme; a measuring apparatus ( 20 ) having an electronic circuit imposing a current, whose intensity or voltage may or may not be variable, between the electrodes, and for receiving in return a signal representative of the release of the LG; and an electronic apparatus ( 30 ) for processing the transmitted signal and displaying, on a display screen ( 32 ), an indication representative of the release of the LG as a function of time. The system may be used to determine, in a plasma or whole blood sample, factors responsible for a coagulation anomaly.

Description:
This application claims priority from European Patent Application No. 05014751.1, filed Jul. 7, 2005, the entire disclosure of which is incorporated herein by reference. 
     FIELD OF THE INVENTION 
     The invention concerns a system for implementing a test for the differential determination in real time of the evolution of a proteolytic enzyme level in a small bodily fluid sample, and as a function of this dynamic differential analysis, for anticipating the tendency of a patient to develop a given pathology. 
     The invention will be more particularly illustrated by the endogenous thrombin potential test (ETP), wherein the continuous measurement of certain plasmatic coagulation factors allows abnormal levels to be detected and forestall, via an appropriate treatment, a risk of haemophilia, or conversely, thrombosis. 
     The invention also concerns a laboratory system, which could be adapted to take the measurements at the patient&#39;s bedside. 
     BACKGROUND TO THE INVENTION 
     Finding out the blood coagulation time, designated as prothrombin time (PT), i.e. the aptitude of different proteolytic enzymes, also known as “factors”, to contribute to the formation of a clot, or conversely, to prevent it, forms part of routine examinations, or even daily examinations in numerous acquired, traumatic, pre or post-operative pathological situations. It is, for example, necessary, during anticoagulant treatment for heart disease, to be able to adjust the dosage of an anti-thrombotic medicine, for example warfarin or heparin, in order to prevent any risk of haemorrhagy in the event of an overdose, or conversely, the risk of thrombosis if the anticoagulant dose is insufficient. 
     This determination of prothrombin time (PT) or partially activated thromboplastin time (APPT) has long been carried out in a laboratory by direct visual observation of the time necessary for a clot to form, then with the help of more or less complex and cumbersome apparatus usually relying upon optical detection, such as those disclosed for example in U.S. Pat. Nos. 5,302,348 and 5,154,082. 
     According to most recent methods, the principle consists in using a chemical substrate incorporating at least one chemical reactant, an end link of which can be cut by a specific enzyme to release a group (LG) whose presence can be detected in the measuring medium by a signal representative of the enzyme activity. 
     This method of detection corresponds for example to that disclosed in EP Patent No. 0 679 193. In the method disclosed, a sensor includes a chemical substrate, an end link of which can be cut by the enzyme being analysed to release a group (LG) whose concentration representative of activity of the enzyme in the medium can be measured by optical means based on alterations in colorimetry, luminescence or fluorescence. When the bodily fluid being analysed is whole blood, the red blood cells have to be removed, either by prior centrifugation of the sample, or by providing a membrane forming a barrier to the red blood cells on the sensor. This method thus has the drawback of requiring a relatively long, even expensive analysis time, to remove the red blood cells. 
     The aforementioned drawback can be greatly reduced, even removed, with the method proposed in EP Patent No. 1 031 830 and in U.S. Pat. No. 6,352,630 B1, both of which are incorporated in this Application by reference. The method, which concerns the blood coagulation measurement time, also relies on the indirect determination of the activity of a proteolytic enzyme by means of a chemical substrate able to release, via the action of the enzyme, leaving groups which will alter the electric properties of the medium, the resulting signal being in this case analysed by amperometry and correlated with a PT or APTT value representative of the coagulation time. With this non-colorimetric method, the prior preparation to obtain clear plasma is omitted, and determination can be carried out more quickly on whole blood. 
     All of the methods that have just briefly been recalled only allow an overall determination to be carried out and do not identify, among all the enzymes involved in the coagulation phenomenon, the enzyme responsible for a coagulation anomaly, whether this be haemophilia or thrombosis. 
     Until recent times, in order to obtain this kind of information, the method consisted in separating a blood sample into several samples and causing reactions with various anti-bodies to identify which enzyme was defective. This method required a relatively large blood sample, necessitated a lot of time and could only be carried out in a laboratory. 
     More recently, International Patent Application No. WO 03/093831 discloses a method for determining in real time the evolution of thrombin activity in a blood sample, but preferably in a plasma sample, relying upon fluorometric determination, compared to a calibration curve. This method has the same drawbacks as those previously cited for overall prothrombin time determination, concerning in particular the relatively large volume of the sample (approximately 160 μl, 80 μl of which is for the calibration solution), and the rather long measuring time (approximately 45 minutes). 
     SUMMARY OF THE INVENTION 
     It is thus an object of the present invention to overcome the drawbacks of the aforecited prior art by providing a differential determination test of the evolution of a proteolytic enzyme over time and particularly to perform a kind of screening of the activity of the enzymes involved in the coagulation phenomenon in a sample of whole blood or a small volume of plasma, and in a relatively short time. 
     The invention therefore concerns a system for electrochemically determining the evolution of the concentration or activity of at least one proteolytic enzyme to detect any deficiency thereof or abnormal activity in a small sample of bodily fluid, such as plasma or whole blood. 
     The system includes an assortment of electrochemical sensors, a measuring apparatus and an electric signal processing apparatus. 
     Each sensor has the shape of a tongue of small dimensions carrying at least one reference electrode and one working electrode on which a specific reactant for a given proteolytic enzyme is immobilised, whose composition incorporates at least one chemical substrate, an end link of which can be cut by the enzyme to release leaving groups (LG). 
     The measuring apparatus includes at least one connection slot for receiving a sensor, and an electronic circuit powered by an energy source for imposing an electric current between the electrodes of the sensor whose intensity or voltage may or may not be variable, and receiving in return an electric signal representative of the release of the leaving groups (LG). 
     In a preferred embodiment, the measuring apparatus allows chrono-amperometric determination to be carried out. 
     The electronic apparatus includes software for processing the signal emitted by the measuring apparatus and displaying an indication representative of the release of leaving groups (LG) over time on a display screen. This data can be given on the screen in alphanumerical form, or in the form of curves displayed sequentially or in a mosaic. 
     Thus, in accordance with a first embodiment of the present invention, a system for the electrochemical determination of the evolution of the concentration or activity of at least one proteolytic enzyme for detecting a deficiency or abnormal activity thereof in a small sample of bodily fluid is provided, wherein the system includes: (a) an assortment of electrochemical sensors, each having the shape of a tongue of small dimensions carrying at least one reference electrode and one working electrode on which a specific reagent for a given proteolytic enzyme is immobilised, and the composition of which includes at least one chemical substrate, an end link of which can be cut by the enzyme to release leaving groups; (b) a measuring apparatus including at least one connecting slot for receiving a sensor and whose electronic circuit, powered by an energy source, imposes, between the electrodes of the sensor, an electric current whose intensity or voltage may or may not be variable, and for receiving in return an electric signal representative of the release of the leaving groups, and (c) an electronic apparatus including software for processing the signal transmitted by the measuring apparatus to display on a display screen an indication representative of the release of the leaving groups as a function of time. In accordance with a second embodiment of the present invention, the first embodiment is modified so that the electronic circuit of the measuring apparatus is arranged for carrying out a chrono-amperometric determination. In accordance with a third embodiment of the present invention, the third embodiment is modified so that each assortment of sensors is made up of several sensors, each having a specific reagent for a given proteolytic enzyme, wherein the measuring apparatus can include as many connecting slots as there are sensors in an assortment and in that the electronic apparatus software can differentiate the sensors to display curves sequentially or in a mosaic. In accordance with a fourth embodiment of the present invention, the first embodiment is modified so that each sensor of an assortment includes a material mark corresponding to the determination of a specific enzyme, wherein the mark on the sensor is complementary to a material mark on the connection with the measuring apparatus. In accordance with a fifth embodiment of the present invention, the first embodiment is modified so that the assortment of electrochemical sensors also includes a calibration sensor. 
     In accordance with a sixth embodiment of the present invention, the first embodiment is modified so that the measuring apparatus further includes a closing device for insulating the sensors inserted into the apparatus from the external medium, and a thermoregulation device for keeping the sensors at a determined constant temperature during the entire measurement. In accordance with a seventh embodiment of the present invention, the first embodiment is modified so that the measuring apparatus further includes a thermal probe for measuring the ambient temperature and in that the electronic apparatus software enables the reference curve to be selected as a function of the ambient temperature. In accordance with an eighth embodiment of the present invention, the first embodiment is modified so that the measuring apparatus further includes a secondary display screen for displaying an overall or instantaneous parameter of the measurement that is carried out. In accordance with a ninth embodiment of the present invention, the eighth embodiment is further modified so that the displayed parameter is the prothrombin time or the activated partial thromboplastin time when the bodily fluid is plasma or whole blood. 
     In accordance with a tenth embodiment of the present invention, the first embodiment is modified so that the measuring apparatus and the electronic apparatus are united in a single operating unit. In accordance with an eleventh embodiment of the present invention, the first embodiment is modified so that the volume of the sample of bodily fluid taken is less than 10 μl. In accordance with a twelfth embodiment of the present invention, the first embodiment is modified so that it displays an indication of the measurement carried out on the display screen in graphic or other form. In accordance with a thirteenth embodiment of the present invention, the first embodiment is modified so that the biological fluid is blood, particularly whole blood, in which the evolution of the concentration of coagulation factors or conversely coagulation inhibitors is determined to detect any deficiency, or conversely an excess, or abnormal activity. In accordance with a fourteenth embodiment of the present invention, the twelfth embodiment is further modified so that the composition of the specific reagent includes at least one oligopeptide substrate, an end link of which can be separated by a coagulation factor to give a leaving group, a thromboplastin and a buffer medium. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Other features and advantages of the present invention will appear more clearly upon reading the following description of an example embodiment, given by way of non-limiting illustration, with reference to the annexed drawings, in which: 
         FIG. 1  shows in perspective a measuring system according to the invention; 
         FIG. 1A  shows a sensor variant; 
         FIG. 2  is a diagram of the process leading to a signal that can be exploited by the measuring apparatus, then by the software of the electronic apparatus; 
         FIG. 3  includes a curve representative of a factor II deficiency; 
         FIG. 4  includes a curve representative of abnormal activity of factor V Leiden; 
         FIG. 5  includes a curve representative of a factor VII deficiency, and 
         FIG. 6  includes a curve representative of a protein S deficiency. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Referring first of all to  FIG. 1 , a system according to the invention is shown by way of example for screening a patient&#39;s blood, possibly at his bedside. The system includes an assortment  10  of electrochemical sensors, a measuring apparatus  20  and an electronic apparatus  30 , these three elements not being shown on the same scale. 
     In the example shown, assortment  10  includes three sensors  10   a ,  10   b  and  10   c  shown for convenience on a larger scale. Each sensor has the shape of a tongue approximately 40 mm long and 8 mm wide. 
     Referring more particularly to sensor  10   a , which is, for example, for detecting a prothrombin deficiency (also called factor II), it can be seen that it includes a thin plastic support  11 , made for example of PET, carrying two current collectors  14 ,  15 , over its entire length, separated by a small space  13  which insulates them electrically. 
     Support  11  and collectors  14 ,  15  are covered with an insulating coating  12  in which two apertures  16 ,  17  are cut, for example by stamping, close to each end and making visible portions of collectors  14 ,  15 . A first aperture  16  electrically connects sensor  10   a  to measuring apparatus  20 . The second aperture  17  forms the measuring zone, the visible portions of the collectors respectively forming the working electrode  14   a  and the reference electrode  15   a.    
     Working electrode  14   a  is made for example by laminating a thin strip of platinum and reference electrode  15   a  is made by laminating a thin strip of silver that is previously or subsequently chlorinated. It is also possible to provide a counter-electrode in the measuring zone. Working electrode  14   a  is coated with a specific reagent  34  described in detail hereinafter. 
     In the sensor model shown, it can be seen that measuring aperture  17  is covered with a transparent cap  18 , forming a transverse capillary channel  18   a  for bringing the blood sample to be analysed into contact with electrodes  14   a  and  15   a.    
     It can be seen that the end of the sensor includes a specific marking  19  of a sensor type from the assortment, enabling measuring apparatus  20  to “recognise” it. For sensor  10   a , this marking is formed by a raised portion  19   a  located along the axis of the sensor. For sensor  10   b , this raised portion  19   b  is offset to the right and for sensor  10   c , shown in place in measuring apparatus  20 , the raised portion  19   c  (not shown) is offset to the left. It is also possible to envisage other types of marking, for example a small extension  19   d  of the end of the sensor, as shown in  FIG. 1A , or conversely, a small notch (not shown). The advantage of these marking means will appear more clearly with the description of measuring apparatus  20 . 
     It will also be observed that assortment  10  can include a larger number of sensors, advantageously including a calibration sensor. 
     Measuring apparatus  20  includes a case  21  constructed by assembling two moulded plastic shells  21   a ,  21   b , the bottom shell  21   b  extending slightly beyond top shell  21   a . These two shells delimit a housing for an energy source and for an electronic circuit (not shown) for processing signals transmitted by the leaving groups (LG). This electronic circuit is an adaptation of the circuits used for dosing glucose, for example by amperometry as disclosed in U.S. Pat. No. 5,378,628. It differs only in the different setting of the electric signal representative of the release of groups LG by the thrombin, or by other proteolytic enzymes. 
     Measuring apparatus  20  also includes as many connecting slots  24   a ,  24   b ,  24   c  as there are sensors  10   a ,  10   b ,  10   c  in the measuring system assortment. These connecting slots are made between and in shells  21   a  and  21   b  forming case  21 . In the embodiment shown, the top shell has a notch and the bottom shell a hollow groove for inserting and removing the disposable sensor after use. 
     According to the embodiment shown, measuring apparatus  20  further includes a cap  23  that can be folded back and which insulates the sensors  10   a ,  10   b ,  10   c  introduced into the apparatus, which then includes a thermostat (not shown) for keeping the measuring zone at a constant temperature (for example 37° C.). As will be seen hereinafter, temperature has a very great influence on the generation of thrombin. Alternatively, it would be possible to omit a thermostated chamber by providing a probe (not shown) for measuring the ambient temperature and selecting a calibration curve from a group of curves, stored in the memory of the measuring apparatus, or even better in the electronic apparatus, as a function of various temperatures. 
     It will also be observed that each connecting slot  24   a ,  24   b , and  24   c  includes a notch  29   a ,  29   b  and  29   c  (not visible) complementary to each raised mark  19   a ,  19   b  and  19   c , i.e. preventing any inversion of the sensor and clearly designating, without any error, the curve or the data which will be displayed on the display screen. 
     With the type of marking shown in  FIG. 1A , “recognition” of a determined sensor can be carried out by electronic means by measuring apparatus  20 , such that any connecting slot can receive any sensor. This type of marking also increases the number of different sensors that an assortment  10  can include. For example, with a number of extensions  19   d  comprised between zero and 3 able to occupy three different positions, eight sensors can be differentiated. 
     It will be observed finally that measuring apparatus  20  could include a secondary display  25 , able to serve as a screen for checking proper operation, for example by displaying ON or OFF depending upon whether control button  17  has been pressed or not, or by providing visual end of measurement data, enabling the sensors to be removed in complete security. The secondary display can also display, by way of complementary data, an overall measurement value, such as PT or APTT. 
     The measuring apparatus  20  that has just been described includes three connecting slots  24   a ,  24   b  and  24   c , but it is clear that it could include a larger number, to enable a larger number of simultaneous measurements to be carried out. 
     Measuring apparatus  20  can be connected via a cord  31  to an electronic apparatus  30  including a display screen  32 . In the example shown, the electronic apparatus is a portable computer, in which software for processing the signals received from measuring apparatus  20  has been installed, for displaying curves or data regarding the measurement being carried out on screen  32 . The computer also enables the practitioner to store data useful to him for interpreting the curves, and/or enabling him to follow the pathology of a given patient, and to carry out the ordinary tasks possible with a computer. 
     In the example of  FIG. 1 , measuring apparatus  20  and electronic apparatus  30  are shown as separate elements, but it is entirely conceivable to unite them in a single operating unit. It is even possible to design the assembly in the form of a briefcase including a housing for storing sensor assortments  10 . 
       FIG. 2  is a schematic diagram of the reaction that generates a current between electrodes  14   a  and  15   a , which are connected via an electronic detection circuit that is not shown. The substrate is represented schematically by the formula R 1 -AA 2 -AA 1 -Arg-LG in which AA 1  and AA 2  represent amino-acids such as those described in U.S. Pat. No. 4,303,853 or 6,352,853, but it is entirely possible to use other peptides. Group R 1  represents a group connecting with working electrode  14   a  for orienting the oligopeptide and LG represents a leaving group, such as one of the groups described in the aforecited U.S. Pat. No. 6,352,853. In the left part of the diagram, it can be seen that the thrombin enzyme selectively cuts the connection between the arginine and the leaving group LG. In the right part of the diagram, it can be seen that the released leaving group can migrate towards electrode  15   a  and generate a current that will be proportional to the number of released leaving groups LG and thus to the quantity of thrombin formed in the medium per unit of time. In other words, determination of the activity of a given proteolytic enzyme relies on a chrono-amperometric measurement for tracing a curve representing the variation in intensity in μA/cm 2  over time expressed in seconds as shown in the graphs of  FIGS. 3 to 6 . This chrono-amperometric determination also allows, by means of an appropriate calculation algorithm, the measuring results to be displayed, for example in ETP value (endogenous thrombin potential). 
     In the method concerning an overall determination (PT or APPT), the retained value is for example that of the inflexion point, measured approximately in the 15 seconds following the start of the reaction and this value only represents around 10% of the total thrombin. With the test according to the invention, the reaction time is considerably longer, able to reach up to 45 min, preferably between 2 and 30 min and particularly between 3 and 10 min. This takes account of important parameters for the practitioner concerning the thrombin generating “dynamics” as explained in more detail with reference to  FIG. 3 . 
       FIG. 3  shows a graph showing a factor II deficiency, namely prothrombin, in the plasma. It was achieved with a sensor whose working electrode surface is 0.054 cm 2  using the oligopeptide TOS-Gly-Pro-Arg-3chloro-4-hydroxyanilide, 2HCL as the substrate. 
     It is evidently possible to make other choices, both as regards the nature of the substrate and the surface of the working electrode. 
     The recording was carried out with normal plasma at a constant temperature of 23.5° C. for reference curve R 1 , and for measuring curve FII. In each case the quantity of sample deposited, or a reference solution, is 10 μl. The measurement was carried out over a period of 10 minutes. 
       FIG. 3  also shows a second reference curve R 2 , with normal plasma, at a temperature of 24.5° C., which clearly shows that a difference of only 1° C. causes a significant movement in the curve, and thus in the parameters usually taken into account, in particular: 
     PH (peak high): maximum signal value 
     TTP (time to peak): time to reach the peak; 
     LT (lag time): reaction time 
     ETP (endogenous thrombin potential) or AUC (area under curve) 
     Any shift in one of these parameters in relation to the reference value can be interpreted by the practitioner to detect an anomaly in the coagulation phenomenon. 
     Thus, when curves R 1  and FII are compared, measured under in the same conditions and at the same temperature, it can be seen that the PH value is greatly reduced and that the TTP value is considerably increased for FII. This can be interpreted as a prothrombin deficiency or a triggering factor. 
       FIG. 4  shows a graph shown on a different scale, showing the result of a measurement of a plasma sample with a factor V Leiden deficiency. As can be seen in  FIG. 4 , the PH value is much greater than the reference PH value. A greater TTP value than that of the reference value (curve R 1 ) is observed to reach a slightly greater PH value, which means attenuated factor VII activity. 
       FIG. 5  shows, on the same scale as  FIG. 3 , a graph showing a factor VII deficiency, namely proconvertin, whose presence also contributes to increasing the conversion of prothrombin into thrombin. A larger TTP value than the reference value (curve R 1 ) will be observed to reach an only slightly greater PH value, which means attenuated factor VII activity. 
       FIG. 6  shows, on the same scale as  FIG. 4 , a graph showing a protein S deficiency in the coagulation phenomenon. As can be seen, the TTP value is hardly changed compared to that of the reference value (curve R 2 ) and the corresponding PH value presents a significant increase that can be interpreted as a protein S deficiency. 
     By using other appropriate specific reagents, it is possible to determine which other factors might be responsible for a coagulation phenomenon anomaly, such as factor VIII or factor IX, a deficiency of which corresponds to a tendency towards haemophilia, protein C, antithrombin III, or lupus anticoagulants. 
     Likewise, without departing from the scope of the invention, the system could be applied to other bodily fluids by choosing appropriate substrates.