WO2015040441A1 - Procedure and measuring system for determining blood coagulation characteristics - Google Patents
Procedure and measuring system for determining blood coagulation characteristics Download PDFInfo
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- WO2015040441A1 WO2015040441A1 PCT/HU2014/000084 HU2014000084W WO2015040441A1 WO 2015040441 A1 WO2015040441 A1 WO 2015040441A1 HU 2014000084 W HU2014000084 W HU 2014000084W WO 2015040441 A1 WO2015040441 A1 WO 2015040441A1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/49—Blood
- G01N33/4905—Determining clotting time of blood
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
Definitions
- the subject of the invention relates to a procedure and measuring system for determining blood coagulation characteristics, during which real-time liquid phase tests are performed in a capillary whole blood sample using the same principle applied during traditional laboratory liquid phase measurements giving results comparable with the results of traditional laboratory liquid phase measurements and a user-friendly, easy-to-use measuring system consisting of a measuring device and reagent set is set up to perform the tests.
- the frequency of the doctor-patient meeting may be favourably formed in a personalised way.
- a possibility for in vitro POC testing of haemostasis disorders accompanying perioperative or emergency conditions is, for example, the characterisation of the viscoelastic changes of blood using sonoclot, thromboelastometry and thromboelastography [summarised by Ganter M.T., Hofer C.K. (2008): Anesth.Analg. 106: 1366-1375.; Enriquez L.J.,Shore-Lesserson L.(2009): Br.J.Anaesth. 103: il4-i22.].
- the viscoelastic measurements are not performed in the blood flow but instead under static conditions.
- sample collection, sample preparation When performing traditional laboratory liquid phase blood coagulation tests first of all plasma is created from the one millilitre volume of anticoagulated vein blood sample, therefore the traditional laboratory liquid phase analysis is preceded by a pre-analytic period of varying duration ("sample collection, sample preparation").
- sample preparation After sample preparation the characteristics of the biochemical process of blood coagulation are measured by monitoring prothrombin-thrombin activation, fibrinogen-fibrin transformation catalysed by the formed thrombin and the fibrin polymerisation induced by various inducing processes, like, for example, determining Prothrombin Time, in which case the prothrombin-thrombin activation is initialised by thromboplastin.
- the monitoring of the fibrin polymerisation may be realised by detecting optical change (for example light dispersion change, nephelometry or turbidity change, automatic laboratory coagulometers detecting based on the principle of turbidimetry), or by measuring the changes of viscoelastic characteristics (for example, automatic laboratory coagulometers detecting using a mechanical principle) [Gogstad G.O. et al. (1986): Clin.Chem. 32: 1857-1862.; Hoffmann J.J.M.L.,Verhappen M.A.L.(1988): Clin.Chem. 34: 2135-2140.].
- optical change for example light dispersion change, nephelometry or turbidity change, automatic laboratory coagulometers detecting based on the principle of turbidimetry
- viscoelastic characteristics for example, automatic laboratory coagulometers detecting using a mechanical principle
- the parameters characteristic of the sample may be determined with a POC measuring device, then they may be converted using the software of the measuring device and then forwarded to a central data store for diagnostic, comparative and risk-analysis purposes.
- the advantage of the POC test is that the analysis of the blood sample takes place at the currently selected location, usually using a few tens of microlitres of capillary blood mostly originating from the fingertip [Perry D.J. et al.(2010): Br.J.Haematol. 150: 501-514.].
- the POC tests used to date do not usually follow the entire process due to the complex composition of the sample (enzymatically active blood plasma and active cellular elements), instead they usually examine thrombin activity. Therefore the comparison of the results obtained in traditional laboratory liquid phase measurements with blood plasma testing and the result obtained in POC tests with whole blood testing usually requires special assessment, as in the case of blood plasma testing the effect of the active cellular elements on thrombin activity and fibrin polymer structure cannot be assessed, furthermore, during the time between sample taking and traditional laboratory processing various changes have to be taken into account in the blood sample components, primarily in the coagulation factors (deactivation- activation).
- Patent numbers WO 94/16095 and WO 95/30770 concern dry chemistry POC tests set up to determine blood coagulation characteristics.
- recombinant thromboplastin reagent is applied to a solid absorbent carrier, for example onto plastic polymer strips, onto a reaction zone with a diameter of approximately 0.5 cm by drying-lyophilisation, then the liquid phase component containing the test sample is reacted with this zone.
- the progress of the enzyme-substrate reaction is measured with sensing electrodes formed on the carrier material, with the change in electrical resistance of the reaction zone while connected to a POC measuring device, the measured value is then displayed on a monitor in a measurable way.
- test reagents are immobilised in the detection zone of a non-porous carrier card serving for carrying out POC Prothrombin Time (PT), Activated Coagulation Time (ACT), Activated Partial Thromboplastin Time (APPT), platelet aggregation and fibrinogen tests. Following this the detection zone is reacted with a liquid component containing the test sample.
- PT Prothrombin Time
- ACT Activated Coagulation Time
- APPT Activated Partial Thromboplastin Time
- platelet aggregation platelet aggregation and fibrinogen tests.
- the other disadvantage here also is that it is not the blood coagulation process that is characterised, instead one of its physical consequences is detected, and for this redox particles ensuring ion migration are dissolved into the liquid component of the reaction, the presence of which may influence the blood coagulation processes that require space and surface area.
- the third disadvantage is apparent in the limited ability to assess blood flow-free viscosity measurement.
- test sample is transported using capillarity to the detection zone of a non-porous carrier card prepared with a coagulation reagent.
- the elegant solution's advantage is its disadvantage: these in vitro reactions cannot be compared with enzyme substrate reactions taking place in in vivo aqueous reaction spaces.
- the reactions of whole blood samples or blood samples treated with anticoagulant and reagents in a cuvette are monitored so that the haematocrit value is determined by optical measurement of the reaction and the Prothrombin Time INR value is determined with rheological measurement.
- the advantage of the solution is that the blood coagulation reaction takes place in a cuvette, which provides space and surface area for the process similarly to blood coagulation reactions taking place in in vivo aqueous reaction spaces.
- the disadvantage of the solution is that it is complex, and also that rheological measurement does not appear in traditional laboratory liquid phase measurement solutions, due to which the comparability of the results is questionable.
- the surface of the depression created on the test card is covered with a blood coagulation initiator, for example with thromboplastin.
- the key participant in the test arrangement is a blade wheel moving element with which the blood sample is taken into the reaction chamber, the reaction mixture is mixed and the coagulate created is removed, and the blood coagulation in the plasma sample or whole blood sample is detected by the completion of the electrical circuit performed by the coagulate.
- the advantage of this solution is the blade wheel moving element. Its advantage is also its disadvantage: the solution is complex.
- the objective of with our invention was to overcome the disadvantages of the aforementioned solutions and to elaborate a simple, fast, specific and reproducible procedure in which real time, liquid phase tests are performed in a capillary whole blood sample for determining blood coagulation equivalent to the principle used in traditional laboratory liquid phase measurements and with results comparable to the results of the measurements of traditional laboratory liquid phase measurement, and in order to perform the tests a user friendly, easy-to-handle measuring system of a measuring device and reagent set is set up. Furthermore, our objective with our invention was to determine blood coagulation characteristics by monitoring the optical changes over time accompanying the entire blood coagulation process, both its cellular and non-cellular reactions, which is solved by detecting optical turbidity as a function of time.
- test reaction space is created in an easy-to-handle, small volume reaction vessel, measuring cuvette.
- PT Prothrombin Time
- PT-INR Prothrombin International Normalised Ratio
- ACT Activated Coagulation Time
- the active perishable component of the reagent set is stored in the measuring cuvette forming a part of the reagent set in a vacuum-dried/lyophilised state until used.
- the measuring cuvette is fitted into the heat- regulated measuring position of the measuring device of the measuring system and the dried content in the measuring cuvette is dissolved with the liquid stable component of the reagent set. With this step the measuring system is now suitable for receiving the test sample.
- the active, perishable component(s) of the reagent set is (are), among others, a blood coagulation component(s) sensitive to oxidative processes, temperature fluctuations, pH and ion shifts like, for example thromboplastin and fibrinogen.
- a very preferable solution that can be well compared with traditional laboratory liquid phase measurements is if the real time reactions are detected as a function of time not indirectly but directly by optically monitoring the change in turbidity accompanying the entire process of the blood coagulation process (thrombin activation enzyme cascade, thrombin activity, fibrinogen-fibrin transformation, fibrin polymerisation, and possibly the enzymatic cross-linking of the fibrin network), the sum total of the cellular and non-cellular reactions, preferably with the help of nephelometry and turbidimetry.
- the subject of the invention then is a procedure for the real-time determination of the blood coagulation characteristics in blood samples, for the optical detection of reactions taking place in in vivo aqueous reaction spaces in in vitro reaction spaces, preferably in a small- volume reaction vessel, in a measuring cuvette by using a reagent set.
- the essence of the procedure is that real time, liquid phase tests are performed on blood samples equal to the principle used in traditional laboratory liquid phase measurements and with results comparable to the results of the measurements of traditional laboratory liquid phase measurement in such a way that the active, perishable component(s) and the stable component(s) of the reagent set are handled in a separate way, in separate phases.
- a coagulation curve is produced (figure 2a), i.e. the optical changes occurring due to the fibrin network, fibrin fibres created during coagulation in the entire volume of the measuring cuvette are monitored as a function of time, from which the blood coagulation characteristics may be determined and then preferably displayed.
- the rotation of the mixing element creates optical noise in the path of the light, which noise may also be detected in the optically opaque reaction mixture (e.g. determination of APTT, ACT with whole blood), and which noise increases during the creation and thickening of the fibrin fibres.
- the subject of the invention then relates to a further procedure for the real-time determination of blood coagulation characteristics in blood samples, to the optical detecting of reactions taking place in in vivo aqueous reaction spaces in in vitro reaction spaces, preferably in a reaction vessel with a small volume, with the use of a reagent set.
- a mechanical mixing element is placed in the measuring cuvette, preferably balls rotated on the base of the measuring cuvette for the entire time of the examined coagulation process, as a result of which fibrin polymerisation is initiated on the bordering interface of the mechanical mixing element and the reaction mixture. Then the mechanical mixing is continued until the breaking off of the polymerised fibrin from the bordering interface of the mechanical mixing element and the reaction mixture is achieved, at which breaking-off time the simultaneous, sudden and abrupt change in the optical transparency and optical noise level is detected, and then the breaking off time is assessed as being the coagulation time.
- the change in blood coagulation characteristic is determined using the principle of turbidimetry.
- the blood coagulation characteristic is Prothrombin Time (PT), and/or Prothrombin International Normalised Ratio (PT-INR), and/or Activated Coagulation Time (ACT), and/or D-Dimer.
- the determination of Prothrombin Time is realised through the optical monitoring of fibrin polymerisation.
- the blood coagulation characteristic of Prothrombin International Normalised Ratio is derived by calculation using the reagent-specific calibration data, from the determination of the Prothrombin Time (PT).
- the opto-active biochemical component in the active, perishable component of the reagent set is blood coagulation initiating recombinant thromboplastin.
- the blood coagulation initiating component is silicate particles.
- the subject of the invention furthermore, also relates to a measuring system for the realtime determination of blood coagulation characteristics in blood samples, which has a reagent set and a measuring device with a central unit performing the optical detecting of the blood samples.
- the measuring system is set up so that the measuring device contains a measuring unit supplied with an optical measuring cell suitable for accommodating the measuring cuvette forming a part of the reagent set.
- the active, perishable component required for the detecting of the blood coagulation characteristics, to which an opto-active biochemical component is also added, is stored in the measuring cuvette in a lyophilised state.
- the liquid, stable component of the reagent set is placed in the measuring cuvette placed in the optical measuring cell.
- the optical measuring cell is supplied with a mixing element and heating unit connected to the central unit via a mixing controller and a heating controller.
- the measuring cuvette connected to the heating controller and placed in the optical measuring cell has a temperature sensor that measures the temperature, furthermore, the reagent set also contains a reagent dispenser and a blood sample dispenser.
- the mixing element is preferably moved at the optical measuring cell with the help of a rotating magnetic field.
- a preferable embodiment of the measuring system is when a reagent-identifying radio frequency RPID device is built into the measuring device that is set up to input calibration and control data and perform data registration.
- FIG. 1 - figures la and lb show the previously presented two-step and single-step coagulation curves
- - figure 2a depicts the coagulation curve obtained by turbidimetry detecting
- figure 2b depicts the coagulation curve obtained with the application of the further procedure according to the invention
- - and figure 3 depicts the block diagram of the measuring system.
- the curve marked A is the result of a measuring arrangement in which the active, perishable component of the reagent set is stored in the measuring cuvette forming a part of the reagent set in a lyophilised state, and following placing the measuring cuvette at the optical measuring cell, the liquid, stable component of the reagent set was added for the purpose of dissolving and incubation.
- the curve marked B is the result of a measuring arrangement in which the active, perishable component of the reagent set was stored in the measuring cuvette forming a part of the reagent set in the liquid state preceding lyophilising, and following placing the measuring cuvette at the optical measuring cell, the liquid, stable component of the reagent set was added for the purpose of incubation.
- the increased optical sensitivity established with the addition of fibrinogen and the lyophilised arrangement is striking (figure lb, curve A).
- FIG 2a the optical change caused by the fibrin network, fibrin fibres built up during the coagulation is shown as a function of time with the traditional coagulation curve.
- the measuring device M As soon as the measuring device M is ready for performing measurements (the block temperature is at 37 °C), it reads in the reagent set R calibration and expiry data with the help of the RFID device 8 from the unique RFID label of the reagent set R. If it finds that the expiry date of the reagent set R is appropriate, after inputting the sample/patient identifier it notifies the user, for example, via the display 7 connected to the central unit 1 , to position the measuring cuvette forming a part of the reagent set R into the measuring unit 10. Every measuring cuvette contains the active, perishable component Rl in a lyophilised, dry state.
- D-Dimer is determined via the optical monitoring of latex agglutination.
- Blood sample dispenser R4 for accommodating at the most, preferably 20 ⁇ of blood sample
- the following table 1 serves to support that stated above, in which the results of Prothrombin International Normalised Ratio (PT-INR) tests are compared with the measurement results of Prothrombin International Normalised Ratio (PT-INR) tests performed on the same blood sample series by another POC system,
- Measuring system A The measuring system according to the invention
- Laboratory device B laboratory device detecting using the principle of nephelometry
Abstract
Procedure for real-time determination of blood coagulation characteristics in blood samples, for optical detection of in vivo aqueous reactions carried out in reaction spaces in vitro, preferably in a small-volume reaction vessel, in a measuring cuvette by using a reagent set, in which real time, liquid phase tests of whole blood samples based on identical principles used in traditional laboratory liquid phase measurements are performed and with results comparable to the results of the measurements of traditional laboratory liquid phase measurement. In the tests the active, perishable component(s) and the stable component(s) of the reagent set are handled in separate phases.
Description
PROCEDURE AND MEASURING SYSTEM FOR DETERMINING BLOOD
COAGULATION CHARACTERISTICS
The subject of the invention relates to a procedure and measuring system for determining blood coagulation characteristics, during which real-time liquid phase tests are performed in a capillary whole blood sample using the same principle applied during traditional laboratory liquid phase measurements giving results comparable with the results of traditional laboratory liquid phase measurements and a user-friendly, easy-to-use measuring system consisting of a measuring device and reagent set is set up to perform the tests.
In the world of in vitro diagnostics the fast spreading of Point-Of-Care, in short POC, tests [Webster S. (2006): Eur.Clin.Lab. 25: 10-1 1.; Huckle D. (2008): Expert Rev.Mol.Diagn. 8: 679-688.] was promoted by their characteristic that, as opposed to the traditional laboratory analysis of test samples collected from patients, they provide test results at the location where the sample was taken, in real time, with fast availability. In the control and monitoring of blood coagulation, haemostasis disorders associated with congenital or chronic diseases, possibly requiring medicinal therapy, this real-time measurement is time after time a key and life-saving factor both from the point of view of the practicing doctor and the patient. With the application of sensitive, specific, reproducible and user-friendly blood coagulation tests (for example Prothrombin Time - PT, Activated Coagulation Time - ACT, Activated Partial Thromboplastin Time - APTT) the frequency of the doctor-patient meeting may be favourably formed in a personalised way.
In oral anticoagulation therapy affecting an increasingly greater proportion of the population, the demand for the individual, personalised monitoring of the blood coagulation pathways in the medicine and dose-finding phase, then during the following continuous medicine therapy and in various perioperative states clearly favours the application of POC tests as opposed to traditional laboratory procedures [Jackson CM. et al.(2005): Clin.Chem. 51 : 483-485.; Perry D.J. et al.(2010): Br.J.Haematol. 150: 501-514.; Wu H.M. et al.(2010): Per.Med. 7: 65-73.]. All this gives extra significance to the creation of POC tests the measurement results of which are comparable to the results received with the use of traditional laboratory procedures.
A possibility for in vitro POC testing of haemostasis disorders accompanying perioperative or emergency conditions is, for example, the characterisation of the viscoelastic changes of blood using sonoclot, thromboelastometry and thromboelastography [summarised by Ganter M.T., Hofer C.K. (2008): Anesth.Analg. 106: 1366-1375.; Enriquez L.J.,Shore-Lesserson L.(2009): Br.J.Anaesth. 103: il4-i22.]. When considering the measurement results a significant aspect is that the viscoelastic measurements are not performed in the blood flow but instead under static conditions.
When performing traditional laboratory liquid phase blood coagulation tests first of all plasma is created from the one millilitre volume of anticoagulated vein blood sample, therefore the traditional laboratory liquid phase analysis is preceded by a pre-analytic period of varying duration ("sample collection, sample preparation"). After sample preparation the characteristics of the biochemical process of blood coagulation are measured by monitoring prothrombin-thrombin activation, fibrinogen-fibrin transformation catalysed by the formed thrombin and the fibrin polymerisation induced by various inducing processes, like, for example, determining Prothrombin Time, in which case the prothrombin-thrombin activation is initialised by thromboplastin. The monitoring of the fibrin polymerisation may be realised by detecting optical change (for example light dispersion change, nephelometry or turbidity change, automatic laboratory coagulometers detecting based on the principle of turbidimetry), or by measuring the changes of viscoelastic characteristics (for example, automatic laboratory coagulometers detecting using a mechanical principle) [Gogstad G.O. et al. (1986): Clin.Chem. 32: 1857-1862.; Hoffmann J.J.M.L.,Verhappen M.A.L.(1988): Clin.Chem. 34: 2135-2140.]. The common advantage of these methods is that they provide information about the entire blood coagulation process taking place in the blood plasma, and about all its biochemical part-processes (thrombin-activated enzyme cascade, thrombin activation, fibrinogen-fibrin transformation, fibrin polymerisation, and possibly the enzymatic cross-linking of the fibrin network), their disadvantage is that due to the nature of the sample they are unsuitable for characterising the activity of the cellular components of blood that influences blood coagulation.
In POC test solutions after the test blood sample is taken in the unit performing the desired in vitro reaction the parameters characteristic of the sample, like, for example, the optical signals accompanying the course of the reaction, may be determined with a POC measuring
device, then they may be converted using the software of the measuring device and then forwarded to a central data store for diagnostic, comparative and risk-analysis purposes. The advantage of the POC test is that the analysis of the blood sample takes place at the currently selected location, usually using a few tens of microlitres of capillary blood mostly originating from the fingertip [Perry D.J. et al.(2010): Br.J.Haematol. 150: 501-514.]. In traditional laboratory liquid phase blood coagulation diagnostics tests, the in vitro determination of in vivo reactions taking place in aqueous reaction spaces takes place in reaction-specific buffer systems. When setting up POC tests it is precisely because of this that the adaptation of blood coagulation reactions taking place in in vivo aqueous reaction spaces to in vitro conditions is a critical factor.
During the determination of the Prothrombin Time blood coagulation characteristic the POC tests used to date do not usually follow the entire process due to the complex composition of the sample (enzymatically active blood plasma and active cellular elements), instead they usually examine thrombin activity. Therefore the comparison of the results obtained in traditional laboratory liquid phase measurements with blood plasma testing and the result obtained in POC tests with whole blood testing usually requires special assessment, as in the case of blood plasma testing the effect of the active cellular elements on thrombin activity and fibrin polymer structure cannot be assessed, furthermore, during the time between sample taking and traditional laboratory processing various changes have to be taken into account in the blood sample components, primarily in the coagulation factors (deactivation- activation).
Patent numbers WO 94/16095 and WO 95/30770 concern dry chemistry POC tests set up to determine blood coagulation characteristics. In one possible embodiment of these tests recombinant thromboplastin reagent is applied to a solid absorbent carrier, for example onto plastic polymer strips, onto a reaction zone with a diameter of approximately 0.5 cm by drying-lyophilisation, then the liquid phase component containing the test sample is reacted with this zone. The progress of the enzyme-substrate reaction is measured with sensing electrodes formed on the carrier material, with the change in electrical resistance of the reaction zone while connected to a POC measuring device, the measured value is then displayed on a monitor in a measurable way. With this solution it became possible to perform certain blood coagulation tests in a simple and user-friendly way. However, the
ability to compare results obtained in this way and the results of traditional laboratory liquid phase measurement results is questionable, as in vitro reactions performed with reagents dried onto a solid carrier are not equivalent to enzyme substrate reactions taking place in in vivo aqueous reaction spaces. The comparability of the mentioned traditional and POC results is also made difficult by that in the latter, user-friendly solution it is not the blood coagulation process itself that is characterised, instead the final result (thrombin activity) of the biochemical reaction series that forms a part of it is detected. The basis of the detecting here is not fibrinogen, the natural cleaved substrate of thrombin, instead it takes place in an indirect way, the electrochemical signal change is created with the enzymatic digestion of the low molecular weight substrate.
In patent numbers US 6060323, US 6338821, and US6673622B1 test reagents are immobilised in the detection zone of a non-porous carrier card serving for carrying out POC Prothrombin Time (PT), Activated Coagulation Time (ACT), Activated Partial Thromboplastin Time (APPT), platelet aggregation and fibrinogen tests. Following this the detection zone is reacted with a liquid component containing the test sample. During the blood coagulation process, as a result of the change in the viscosity of the blood the modified migration of the redox particles (for example, ferricyanide, ferrocyanide, cadmium chloride) dissolved with the liquid component results in a change in current strength and impedance measurable in the detection zone. With the formation of measuring electrodes the viscosity of the blood may be measured. One of the disadvantages of this user-friendly solution is similar to those mentioned in the case of the previous patent specification, i.e. in vitro reactions performed with a reagent immobilised on a solid carrier are not equivalent to enzyme substrate reactions taking place in in vivo aqueous reaction spaces. The other disadvantage here also is that it is not the blood coagulation process that is characterised, instead one of its physical consequences is detected, and for this redox particles ensuring ion migration are dissolved into the liquid component of the reaction, the presence of which may influence the blood coagulation processes that require space and surface area. The third disadvantage is apparent in the limited ability to assess blood flow-free viscosity measurement.
In patent number WO2010/122158A1 the test sample is transported using capillarity to the detection zone of a non-porous carrier card prepared with a coagulation reagent. The elegant
solution's advantage is its disadvantage: these in vitro reactions cannot be compared with enzyme substrate reactions taking place in in vivo aqueous reaction spaces.
In patent US 5522255 and, as its continuation, US 6189370B1, and US 6575017 the whole blood sample or one treated with an anticoagulant is applied to a solid carrier strip, or is placed in a cuvette of a POC test measuring device monitored by its LED light sources and photodiode sensors, in a determined volume. In the case of the latter the blood coagulation process is characterised by the deviating migration of particles added to the sample or the reagent that sense changes to the surrounding magnetic field. The advantages of the cuvette volume in the enzyme substrate reaction are hindered by the disadvantage of the solution that in this POC test it is not the blood coagulation process that is characterised, instead one of its physical consequences is detected.
In one of the embodiments of the POC tests according to patent application numbers WO 2004/111656A1 and WO 2005/054847A1 the reactions of whole blood samples or blood samples treated with anticoagulant and reagents in a cuvette are monitored so that the haematocrit value is determined by optical measurement of the reaction and the Prothrombin Time INR value is determined with rheological measurement. The advantage of the solution is that the blood coagulation reaction takes place in a cuvette, which provides space and surface area for the process similarly to blood coagulation reactions taking place in in vivo aqueous reaction spaces. The disadvantage of the solution is that it is complex, and also that rheological measurement does not appear in traditional laboratory liquid phase measurement solutions, due to which the comparability of the results is questionable.
In patent application number US2003/0180824A1 the surface of the depression created on the test card, the reaction chamber is covered with a blood coagulation initiator, for example with thromboplastin. The key participant in the test arrangement is a blade wheel moving element with which the blood sample is taken into the reaction chamber, the reaction mixture is mixed and the coagulate created is removed, and the blood coagulation in the plasma sample or whole blood sample is detected by the completion of the electrical circuit performed by the coagulate. As compared to the other card arrangements, the advantage of this solution is the blade wheel moving element. Its advantage is also its disadvantage: the solution is complex.
The objective of with our invention was to overcome the disadvantages of the aforementioned solutions and to elaborate a simple, fast, specific and reproducible procedure in which real time, liquid phase tests are performed in a capillary whole blood sample for determining blood coagulation equivalent to the principle used in traditional laboratory liquid phase measurements and with results comparable to the results of the measurements of traditional laboratory liquid phase measurement, and in order to perform the tests a user friendly, easy-to-handle measuring system of a measuring device and reagent set is set up. Furthermore, our objective with our invention was to determine blood coagulation characteristics by monitoring the optical changes over time accompanying the entire blood coagulation process, both its cellular and non-cellular reactions, which is solved by detecting optical turbidity as a function of time.
From earlier experiences we understood and recognised that in the in vitro adaptation of enzyme substrate-like, enzyme activation reactions/series of reactions taking place in in vivo aqueous reaction spaces, as opposed to the various absorbent surfaces it is preferable if a test reaction space is created in an easy-to-handle, small volume reaction vessel, measuring cuvette. With this solution more reliable information is obtained in vitro about space and surface-demanding blood coagulation processes taking place in in vivo aqueous reaction spaces. Besides this a result is obtained that is comparable with traditional laboratory liquid phase measurements.
It was also deemed preferable to create a user friendly, easy-to-use measuring system of a measuring device and set of reagents to quickly determine blood coagulation characteristics in a personalised way like, for example, Prothrombin Time (PT), or Prothrombin International Normalised Ratio (PT-INR), or Activated Coagulation Time (ACT), or D- Dimer tests. A measuring cuvette is set up in the measuring system forming a part of the reagent set fitted into a heat-regulated measuring position.
It was observed that from the point of view of the user it is simple and preferable if when setting up the real time measurement test that its active perishable component(s) and its stable, solvent and buffer component(s) are handled in a separate way, in a different phase. Therefore, the active perishable component of the reagent set is stored in the measuring cuvette forming a part of the reagent set in a vacuum-dried/lyophilised state until used.
Immediately before measurement is performed the measuring cuvette is fitted into the heat- regulated measuring position of the measuring device of the measuring system and the dried content in the measuring cuvette is dissolved with the liquid stable component of the reagent set. With this step the measuring system is now suitable for receiving the test sample. The active, perishable component(s) of the reagent set is (are), among others, a blood coagulation component(s) sensitive to oxidative processes, temperature fluctuations, pH and ion shifts like, for example thromboplastin and fibrinogen.
The aforementioned separate-phase formulation of the perishable component(s) of the reagent set and of the liquid, stable component(s) of the reagent set, the maintenance of the blood coagulation reaction in the liquid phase, is especially preferable in such situations, countries where the water required for the performance of the liquid phase reaction is unavailable, or is difficult to obtain or expensive. When determining blood coagulation characteristics in real time a very preferable solution that can be well compared with traditional laboratory liquid phase measurements is if the real time reactions are detected as a function of time not indirectly but directly by optically monitoring the change in turbidity accompanying the entire process of the blood coagulation process (thrombin activation enzyme cascade, thrombin activity, fibrinogen-fibrin transformation, fibrin polymerisation, and possibly the enzymatic cross-linking of the fibrin network), the sum total of the cellular and non-cellular reactions, preferably with the help of nephelometry and turbidimetry.
This train of thought was assisted by the unexpected observation (figures la and lb) that when determining Prothrombin Time (PT) in a capillary whole blood sample, on the effect of fibrinogen added to the active, perishable component of the reagent set for the purpose of improving the optical characteristics of the reaction, instead of obtaining the two-step coagulation reaction, and the two-step coagulation curve (figure la, with the arrows indicating the two steps) illustrating this, when performing the earlier, fibrinogen-free tests we obtained a single-step, large-amplitude curve (figure lb).
It was also an unexpected observation that on the effect of the recombinant thromboplastin reagent initialising the blood coagulation and added to the active, perishable component of
the reagent set, the optical characteristics of the reaction were significantly improved. It was possible to elicit the described phenomenon with our own recombinant thromboplastin reagent produced with our own standard fermentation procedure and optimised lipidation technology [see patent submittal number WO 2011/148207]. Contrary to this, we were unable to repeat that described above with natural thromboplastin reagents of varying quality and activity obtained from various natural sources.
The subject of the invention then is a procedure for the real-time determination of the blood coagulation characteristics in blood samples, for the optical detection of reactions taking place in in vivo aqueous reaction spaces in in vitro reaction spaces, preferably in a small- volume reaction vessel, in a measuring cuvette by using a reagent set. The essence of the procedure is that real time, liquid phase tests are performed on blood samples equal to the principle used in traditional laboratory liquid phase measurements and with results comparable to the results of the measurements of traditional laboratory liquid phase measurement in such a way that the active, perishable component(s) and the stable component(s) of the reagent set are handled in a separate way, in separate phases. For the purpose of jointly detecting the blood coagulation cellular and non-cellular processes together opto-active biochemical components are added to the active, perishable component of the reagent set, preferably fibrinogen or recombinant thromboplastin, and the active, perishable component supplemented in this way is stored in a lyophilised state in the measuring cuvette forming a part of the reagent set. Immediately before measurement the liquid, stable component of the reagent set is placed in the measuring cuvette, then the content of the measuring cuvette is homogenised with the help of a mixing element. The content of the measuring cuvette prepared in this way is incubated at the temperature suitable for the test reaction. The test blood sample, preferably capillary whole blood is placed in the measuring cuvette. The real-time determination of the blood coagulation characteristics is performed by monitoring the optical changes as a function of time elicited by the biochemical reaction causing changes in the blood coagulation characteristics through the joint detection of the cellular and non-cellular processes of blood coagulation in the measuring cuvette.
During a further preferable realisation of the procedure according to the invention while monitoring the optical changes as a function of time a coagulation curve is produced (figure
2a), i.e. the optical changes occurring due to the fibrin network, fibrin fibres created during coagulation in the entire volume of the measuring cuvette are monitored as a function of time, from which the blood coagulation characteristics may be determined and then preferably displayed.
In the case of a preferably solution of the procedure, the change in blood coagulation characteristic is determined using the principle of nephelometry.
In the case of a further preferable embodiment of the procedure the change in blood coagulation characteristic is determined using the principle of turbidimetry.
According to our experiences in the case of those tests where the optical characteristics of the reaction mixture in the measuring cuvette make it difficult to produce the coagulation curve it is preferable to realise a further procedure according to the invention.
When realising the further procedure according to the invention during the entire duration of the examined coagulation process a mechanical mixing (e.g. balls) element is rotated at the base of the measuring cuvette. The fibrin created during the coagulation is polymerised in the form of fibres at the bordering interface between the mechanical mixing element and the reaction mixture. Via this a dual optical effect is achieved, which involves the following:
1. The rotation of the mixing element creates optical noise in the path of the light, which noise may also be detected in the optically opaque reaction mixture (e.g. determination of APTT, ACT with whole blood), and which noise increases during the creation and thickening of the fibrin fibres.
2. The development-thickening of the fibrin fibres uses-extracts the fibrinogen in the reaction mixture, which reduces the optical transparency, due to which the optical transparency measured in the measuring cuvette increases.
The further procedure according to the invention led to an unexpected recognition according to which a state is reached during the thickening of the fibrin fibres when the fibrin fibres break off the interface bordering the mixing element and the reaction mixture. At such a
time the optical transparency in the measuring cuvette changes suddenly and abruptly, and simultaneously with this the optical noise level also shows a sudden and abrupt difference. All this may be seen on the coagulation curve created as a function of time (figure 2b), with the help of which curve the coagulation time is given by projecting the breaking off of the fibrin fibres to the time axis (figure 2b).
The subject of the invention then relates to a further procedure for the real-time determination of blood coagulation characteristics in blood samples, to the optical detecting of reactions taking place in in vivo aqueous reaction spaces in in vitro reaction spaces, preferably in a reaction vessel with a small volume, with the use of a reagent set. Real time, liquid phase tests are performed on blood samples equal to the principle used in traditional laboratory liquid phase measurements and with results comparable to the results of the measurements of traditional laboratory liquid phase measurement in such a way that when setting up the reaction mixture according to the test the active, perishable component(s) and the stable component(s) of the reagent set are handled in a separate way, in separate phases, and for the purpose of jointly detecting the blood coagulation cellular and non-cellular processes together opto-active biochemical components are added to the active, perishable component of the reagent set. The active, perishable component of the reagent set is stored in a lyophilised state in the measuring cuvette forming a part of the reagent set, immediately before measurement the liquid, stable component of the reagent set is placed in the measuring cuvette, then the content of the measuring cuvette is homogenised with the help of a mixing element. The content of the measuring cuvette prepared in this way is incubated at the temperature suitable for the test reaction, the test blood sample, preferably capillary whole blood is placed in the measuring cuvette and the real-time determination of the blood coagulation characteristics is performed by monitoring the optical changes as a function of time in the total volume of the measuring cuvette elicited by the biochemical reaction(s) causing changes in the blood coagulation characteristics through the joint detection of the cellular and non-cellular processes of blood coagulation in the measuring cuvette. When this is performed a mechanical mixing element is placed in the measuring cuvette, preferably balls rotated on the base of the measuring cuvette for the entire time of the examined coagulation process, as a result of which fibrin polymerisation is initiated on the bordering interface of the mechanical mixing element and the reaction mixture. Then the mechanical mixing is continued until the breaking off of the polymerised fibrin from the bordering
interface of the mechanical mixing element and the reaction mixture is achieved, at which breaking-off time the simultaneous, sudden and abrupt change in the optical transparency and optical noise level is detected, and then the breaking off time is assessed as being the coagulation time.
In the case of a preferable embodiment of the further procedure according to the invention the change in blood coagulation characteristic is determined using the principle of turbidimetry. Preferably, during testing, the blood coagulation characteristic is Prothrombin Time (PT), and/or Prothrombin International Normalised Ratio (PT-INR), and/or Activated Coagulation Time (ACT), and/or D-Dimer.
During the procedure it may be preferable if the determination of Prothrombin Time (PT) is realised through the optical monitoring of fibrin polymerisation. Then the blood coagulation characteristic of Prothrombin International Normalised Ratio (PT-INR) is derived by calculation using the reagent-specific calibration data, from the determination of the Prothrombin Time (PT). In the case of determining the Prothrombin Time (PT), or the Prothrombin International Normalised Ratio (PT-INR) it is very preferable if the opto-active biochemical component in the active, perishable component of the reagent set is blood coagulation initiating recombinant thromboplastin. In the case of a further, very preferable embodiment of the procedure when determining Activated Coagulation Time the blood coagulation initiating component is silicate particles.
In the case of a further possible solution of the procedure according to the invention it is very preferable if the determination of the Activated Coagulation Time is realised by the optical monitoring of fibrin polymerisation, and the determination of D-Dimer is realised by the optical monitoring of latex agglutination.
The subject of the invention, furthermore, also relates to a measuring system for the realtime determination of blood coagulation characteristics in blood samples, which has a reagent set and a measuring device with a central unit performing the optical detecting of the blood samples. The measuring system is set up so that the measuring device contains a measuring unit supplied with an optical measuring cell suitable for accommodating the measuring cuvette forming a part of the reagent set. The active, perishable component required for the detecting of the blood coagulation characteristics, to which an opto-active biochemical component is also added, is stored in the measuring cuvette in a lyophilised state. The liquid, stable component of the reagent set is placed in the measuring cuvette placed in the optical measuring cell. Also, the optical measuring cell is supplied with a mixing element and heating unit connected to the central unit via a mixing controller and a heating controller. The measuring cuvette connected to the heating controller and placed in the optical measuring cell has a temperature sensor that measures the temperature, furthermore, the reagent set also contains a reagent dispenser and a blood sample dispenser.
The mixing element is preferably moved at the optical measuring cell with the help of a rotating magnetic field.
A preferable embodiment of the measuring system is when a reagent-identifying radio frequency RPID device is built into the measuring device that is set up to input calibration and control data and perform data registration.
A possible, example solution of the measuring system according to the invention is presented in detail on the basis of the appended figures, without limiting the sphere of protection to this example only, where
- figures la and lb show the previously presented two-step and single-step coagulation curves, - figure 2a depicts the coagulation curve obtained by turbidimetry detecting, figure 2b depicts the coagulation curve obtained with the application of the further procedure according to the invention,
- and figure 3 depicts the block diagram of the measuring system.
The Prothrombin Time (PT) coagulation curves INR=1 according to figures la and lb were drawn by detecting the absorbance at the optimum wavelength of 700 nm (y axis, absorbance) in fingertip blood, i.e. capillary whole blood, taken from healthy individuals as a function of time - seconds - (x axis, seconds). Due to the effect of the fibrinogen added to the active, perishable component of the reagent set required for the reaction for the purpose of improving the optical characteristics of the reaction, instead of the two-step coagulation reaction and the two-step coagulation curve presenting it (figure la, two arrows indicating the steps) obtained in the case of fibrinogen-free tests, large amplitude curves were obtained (figure lb). In figure lb the curve marked A is the result of a measuring arrangement in which the active, perishable component of the reagent set is stored in the measuring cuvette forming a part of the reagent set in a lyophilised state, and following placing the measuring cuvette at the optical measuring cell, the liquid, stable component of the reagent set was added for the purpose of dissolving and incubation. In figure lb the curve marked B is the result of a measuring arrangement in which the active, perishable component of the reagent set was stored in the measuring cuvette forming a part of the reagent set in the liquid state preceding lyophilising, and following placing the measuring cuvette at the optical measuring cell, the liquid, stable component of the reagent set was added for the purpose of incubation. The increased optical sensitivity established with the addition of fibrinogen and the lyophilised arrangement is striking (figure lb, curve A).
In figure 2a the optical change caused by the fibrin network, fibrin fibres built up during the coagulation is shown as a function of time with the traditional coagulation curve. The sensing of the change in light transmittance experienced in the total volume of the measuring cuvette as a function of time (in the graph optical signal change from the minimum level to the maximum level) took place with turbidimetric detecting. The determination of the Coagulation Time is performed from the traditional coagulation curve by projecting the curve inflexion point (indicated with IP in the figure) to the time axis (sec = seconds, min = minimum, max = maximum, Δ = optical signal level difference).
In figure 2b the coagulation curve is presented created with the further procedure according to the invention with the indication of the turbidimetric detection results as a function of
time. During the further procedure according to the invention a mechanical mixing element is rotated (balls in this concrete case) on the base of the measuring cuvette, which elicits optical noise in the detecting light path. The fibrin created during the coagulation polymerises in the form of fibres at the border interface between the mechanical mixing element and the reaction mixture, which increases the optical noise elicited by the mechanical mixing element. When due to its thickness the fibrin fibres break off the border interface between the mechanical mixing element and the reaction mixture, the transparency in the measuring cuvette suddenly and abruptly changes (in figure 2b Δ difference), and at the same time as this the optical noise level also displays a sudden and abrupt difference (in figure 2b indications Z and ZZ). The Coagulation Time is given from the coagulation curve drawn with the further procedure according to the invention by projecting the breaking off of the fibrin fibres to the time axis (sec = seconds, min = minimum, max = maximum, Δ = optical signal level difference). Figure 3 displays the measuring system according to the invention, preferably for the realisation of the procedure(s) presented previously, which has a reagent set R, and a measuring device M, with a central unit 1 , which performs the optical detecting of the blood coagulation characteristics of blood samples. The measuring device M contains a measuring unit 10, supplied with an optical measuring cell 9, suitable for accommodating the measuring cuvette forming a part of the reagent set R. The active, perishable components Rl supplemented with an opto-active biochemical component required for the detecting the blood coagulation characteristics are stored in the measuring cuvette in a lyophilised state. The liquid, stable component R2 of the reagent set R is placed in the measuring cuvette placed in the optical measuring cell 9. The optical measuring cell 9 is supplied with a mixing element 4 and a heating unit 5 connected to the central unit 1 via a mixing controller 2 and a heating controller 3. The optical measuring cell 9 has a temperature sensor 6, which is connected to the heating controlled 3. The central unit 1 is connected to a display 7. Furthermore, the reagent set R also contains a reagent dispenser R3 and a blood sample dispenser R4.
Preferably the mixing element 4 is moved at the optical measuring cell 9 with the help of a rotating magnetic field.
A preferred embodiment of the measuring system is when a radio frequency RFID device 8 for identifying the reagents and for inputting and registration of calibration and control data is built into the measuring device M. The operation of the measuring system according to the invention for the real-time determination of blood coagulation characteristics in blood samples is presented in detail in the following.
As soon as the measuring device M is ready for performing measurements (the block temperature is at 37 °C), it reads in the reagent set R calibration and expiry data with the help of the RFID device 8 from the unique RFID label of the reagent set R. If it finds that the expiry date of the reagent set R is appropriate, after inputting the sample/patient identifier it notifies the user, for example, via the display 7 connected to the central unit 1 , to position the measuring cuvette forming a part of the reagent set R into the measuring unit 10. Every measuring cuvette contains the active, perishable component Rl in a lyophilised, dry state. Every measuring cuvette contains the mixing element 4, preferably mixing balls, serving to mix the reagents between layers of film for insulating-sealing, which must be placed in the measuring cuvette before removing the film with the push of a finger. After sensing the positioning of the measuring cuvette in the optical measuring cell 9 the measuring device M instructs the dispensing of the liquid, stable component R2 of the reagent set R (using a reagent dispenser R3 of the appropriate volume, preferably 200 μΐ). After sensing the dispensing of the liquid, stable component R2, the dissolving of the lyophilised active, perishable component Rl with the liquid, stable component R2 starts, which is performed with the help of the mixing element 4, preferably mixing balls, moving in a rotating magnetic field. The duration of the dissolving, which the measuring device M indicates for information purposes, is also the incubation of the reaction mixture to the working temperature (preferably 37 °C). On completion of the dissolving/incubation the measuring device M instructs that the blood sample be dispensed. Tried and tested procedures and equipment serve to take the capillary blood (puncture, placing the appropriate volume of blood sample R4 into the dispenser), through which the appropriate volume of blood sample, preferably 22 μΐ is placed by the user into the measuring cuvette. The measuring device M senses the input of the blood sample and initiates a short (preferably 10-30 seconds) mixing operation, following the completion of which the
measuring starts. At the same time when the mixing starts the measuring device instructs the user to close the slide-cover of the optical measuring cell 9 in order to ensure that the measuring process is undisturbed. The measuring device M displays information on the progress of the measuring. During measurement the measuring unit 10 of the measuring device M monitors the change in cloudiness caused by the coagulation reaction, when the coagulation is completed it stops the measurement. The measuring device M determined the Prothrombin Time (PT), then from this, with the help of reagent-specific calibration data, the central unit 1 derives by calculation the blood coagulation characteristic of Prothrombin International Normalised Ratio (PT-INR).
In the solution according to the invention the Activated Coagulation Time in blood samples is determined via the optical monitoring of fibrin polymerisation.
In the case of a further, preferable embodiment of the invention D-Dimer is determined via the optical monitoring of latex agglutination.
Example of the preferable composition of the reagent set R:
A possible, exemplary composition of the active, perishable component Rl in wet state (before lyophilisation):
■ 30-40 ml of an aqueous solution of human fibrinogen, preferably at a concentration of 15-25 g/1
■ 2-3 ml Na-citrate, preferably at a concentration of 3.8 g/1
■ 3-4 ml of an aqueous emulsion of human recombinant thromboplastin, the PT value of which measured during a coagulation normal control using a traditional coagulometer is between 7-9 seconds
■ 2-3 g Bovine Serum Albumin
- 1.5-2.5 g PEG 4000
■ 0.0- 1.0 ml Distilled water
The preferable composition of the liquid, stable component R2:
■ 2-2.5 g/1 Tricine
■ 3-4 g/1 Glycine
■ 2-2.5 g/1 CaCl2 2H20
1-1.2 g/l Na-azide
1-1.5 mg/1 Polybrene
Distilled water Reagent dispenser R3 : for accommodating at least, preferably 200 μΐ of reagent
Blood sample dispenser R4: for accommodating at the most, preferably 20 μΐ of blood sample
As a result of the invention, then, a solution has been created for determining blood coagulation characteristics during which real-time liquid phase tests are performed in a capillary whole blood sample using the same principle applied during traditional laboratory liquid phase measurements giving results comparable with the results of traditional laboratory liquid phase measurements. A very favourably usable measuring device M and reagent set R also tried out in practice, as well as a user-friendly, easy-to-use measuring system of these - designated by us as the CoagS or CoagV measuring system - was successfully created for the determination of blood coagulation characteristics.
For example, the following table 1 serves to support that stated above, in which the results of Prothrombin International Normalised Ratio (PT-INR) tests are compared with the measurement results of Prothrombin International Normalised Ratio (PT-INR) tests performed on the same blood sample series by another POC system,
a traditional laboratory device detecting using a mechanical principle,
- a traditional laboratory device detecting using the principle of nephelometry.
Four measurement series are presented in table 1 below. In every measurement series one of the above devices is the reference device, with which the results are compared. The results obtained with non-selected blood sample series associated with two indicated INR ranges conforming to clinical practice well illustrate that the Prothrombin International Normalised Ratio (PT-INR) test performed with the measuring system according to the invention harmonise excellently with the measurement results of the other POC system, and even
surpass them, and expressly better approach the measurement results of traditional laboratory devices detecting using the mechanical principle and the nephelometry principle.
The designations in the following table 1 are as follows:
Measuring system A = The measuring system according to the invention
Measuring system B = Other POC system
Laboratory device A - Laboratory device detecting using a mechanical principle
Laboratory device B = laboratory device detecting using the principle of nephelometry
Table 1
PT-INR Measuring Measuring Reference Laboratory device determination system A system B A
Test sample Fingertip blood Fingertip Anticoagulated blood plasma blood
INR < 2 1.25 1.22 1.27
Deviation from 1% 4%
reference
2 < INR < 4,5 2.69 2.33 2.61
Deviation from 3% 10%
reference
PT-INR Measuring Measuring Reference Measuring system A determination system A system B
Test sample Fingertip blood Anti- Fingertip blood
coagulated
blood plasma
INR < 2 1.35 1.35 1.26
Deviation from 7% 7%
reference
2 < INR < 4,5 2.77 2.76 2.46
Deviation from 13% 12%
reference
PT-INR
determination Measuring Measuring Reference Laboratory device system A system B B
Test sample Fingertip blood Fingertip Anti-coagulated blood plasma blood
INR < 2 1.60 1.52 1.50
Deviation from 7% 1%
reference
2 < INR < 4,5 2.87 2.63 2.82
Deviation from 2% 7%
reference
PT-INR Measuring Laboratory Reference Measuring system B determination system A device B
Test sample Fingertip blood Anti- Fingertip blood
coagulated
blood plasma
INR < 2 1.62 1.60 1.55
Deviation from 4% 3%
reference
2 < INR < 4,5 3.04 2.93 2.75
Deviation from 10% 6%
reference
Claims
Procedure for real-time determination of blood coagulation characteristics in blood samples, for optical detection of in vivo aqueous reactions carried out in reaction spaces in vitro, preferably in a small-volume reaction vessel, in a measuring cuvette by using a reagent set, c h ar a c te r i s e d b y that real time, liquid phase tests are performed on blood samples equal to the principle used in traditional laboratory liquid phase measurements and with results comparable to the results of the measurements of traditional laboratory liquid phase measurement in such a way that when setting up the reaction mixture according to the test the active, perishable component(s) and the stable component(s) of the reagent set are handled in a separate way, in separate phases, for the purpose of jointly detecting the blood coagulation cellular and non-cellular processes together an opto-active biochemical component is added to the active, perishable component of the reagent set and the active, perishable component is stored in a lyophilised state in the measuring cuvette forming a part of the reagent set, immediately before measurement the liquid, stable component of the reagent set is placed in the measuring cuvette, then the content of the measuring cuvette is homogenised with the help of a mixing element, and the content of the measuring cuvette prepared in this way is incubated at the temperature suitable for the test reaction, the test blood sample, preferably capillary whole blood, is placed in the measuring cuvette, and the real-time determination of the blood coagulation characteristics is performed by monitoring the optical changes as a function of time elicited by the biochemical reaction(s) causing changes in the blood coagulation characteristics through the joint detection of the cellular and non-cellular processes of blood coagulation in the entire volume of the measuring cuvette, during the monitoring of the optical changes occurring as a function of time a coagulation curve is drawn from which the blood coagulation characteristics are determined and then preferably displayed.
Procedure for real-time determination of blood coagulation characteristics in blood samples, for optical detection of in vivo aqueous reactions carried out in reaction spaces in vitro, preferably in a small-volume reaction vessel, in a measuring cuvette by using a reagent set, c h ar act e ri s e d b y that real time, liquid phase tests are performed on blood samples equal to the principle used in traditional laboratory liquid phase measurements and with results comparable to the results of the measurements of
traditional laboratory liquid phase measurement in such a way that when setting up the reaction mixture according to the test the active, perishable component(s) and the stable component(s) of the reagent set are handled in a separate way, in separate phases, for the purpose of jointly detecting the blood coagulation cellular and non-cellular processes together an opto-active biochemical component is added to the active, perishable component of the reagent set and the active, perishable component is stored in a lyophilised state in the measuring cuvette forming a part of the reagent set, immediately before measurement the liquid, stable component of the reagent set is placed in the measuring cuvette, then the content of the measuring cuvette is homogenised with the help of a mixing element, and the content of the measuring cuvette prepared in this way is incubated at the temperature suitable for the test reaction, the test blood sample, preferably capillary whole blood, is placed in the measuring cuvette, and the real-time determination of the blood coagulation characteristics is performed by monitoring the optical changes as a function of time elicited by the biochemical reaction(s) causing changes in the blood coagulation characteristics through the joint detection of the cellular and non-cellular processes of blood coagulation in the entire volume of the measuring cuvette, during the realisation of which the measuring cuvette is supplied with a mechanical mixing elemen, preferably balls continuously rotating at the base of the measuring cuvette during the entire duration of the examined coagulation process, as a result of which a thickening polymerised fibrin fiber generating between the mechanical mixing element and the surface of the reaction mixture, then the mechanical mixing is continued until the breaking off of fibrin fibre polymerised between the mechanical mixing element and the surface of the reaction mixture achieved at which breaking-off time the simultaneous, sudden and abrupt change in the optical transparency and optical noise level is detected, and is assessed as being the coagulation time.
Procedure according to claim 1, c har ac t e ri s e d b y that the change in blood coagulation characteristic is determined using the principle of nephelometry.
Procedure according to claim 1 or 2, c har a c t e ri s e d b y that the change in blood coagulation characteristic is determined using the principle of turbidimetry.
5. Procedure according to any of claims l-4,characterised by that during testing the blood coagulation characteristic is Prothrombin Time (PT), and/or Prothrombin International Normalised Ratio (PT-INR), and/or Activated Coagulation Time (ACT), and/or D-Dimer
6. Procedure according to claim 5, characterised by that the determination of Prothrombin Time (PT) is realised through the optical monitoring of fibrin polymerisation.
7. Procedure according to claim 6, characterised by that the blood coagulation characteristic of Prothrombin International Normalised Ratio (PT-INR) is derived by calculation using reagent-specific calibration data, from the determination of the Prothrombin Time (PT).
8. Procedure according to any of claims 5-7, characterised by that in the case of determining the Prothrombin Ido (PT), or the Prothrombin International Normalised Ratio (PT-INR) the opto-active biochemical component in the perishable component of the reagent set is blood coagulation initiating recombinant thromboplastin.
9. Procedure according to claim 5, characterised by that when determining Activated Coagulation Time the blood coagulation initiating component is silicate particles
10. Procedure according to claim 5 or 9, characterised by that the determination of the Activated Coagulation Time is realised by the optical monitoring of fibrin polymerisation.
11. Procedure according to claim 5, characterised by that the determination of D- Dimer is realised by the optical monitoring of latex agglutination.
12. Measuring system for the real-time determination of blood coagulation characteristics in blood samples, preferably for the realisation of any of the procedures according to any of claims 1-11, which has a reagent set and a measuring device with a central unit
performing the optical detecting of the blood samples, c h ar act e r i s e d b y that the measuring device (M) contains a measuring unit (10) supplied with an optical measuring cell (9) suitable for accommodating the measuring cuvette forming a part of the reagent set ( ), the active, perishable component (Rl) required for the detecting of the blood coagulation characteristics, to which an opto-active biochemical component is also added, is stored in the measuring cuvette in a lyophilised state, the liquid, stable component (R2) of the reagent set (R) is placed in the measuring cuvette placed in the optical measuring cell (9), also, the optical measuring cell (9) is supplied with a mixing element (4) and heating unit (5) connected to the central unit (1) via a mixing controller (2) and a heating controller (3), the measuring cuvette connected to the heating controller (3) and placed in the optical measuring cell (9) has a temperature sensor (6) that measures the temperature, furthermore, the reagent set also contains a reagent dispenser (R3) and a blood sample dispenser (R4).
13. The measuring system according to claim 12, c h arac te ri s e d b y that the mixing element (4) is moved at the optical measuring cell (9) with the help of a rotating magnetic field.
14. The measuring system according to claim 12 or 13, characterised by that a reagent- identifying radio frequency RFID device (8) is built into the measuring device (M) that is set up to input calibration and control data and perform data registration.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| HUP1300542 | 2013-09-19 | ||
| HU1300542A HUP1300542A2 (en) | 2013-09-19 | 2013-09-19 | Method and measuring system for determination of parameters of coagulation of blood |
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| WO2015040441A1 true WO2015040441A1 (en) | 2015-03-26 |
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|---|---|---|---|
| PCT/HU2014/000084 WO2015040441A1 (en) | 2013-09-19 | 2014-09-19 | Procedure and measuring system for determining blood coagulation characteristics |
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| WO (1) | WO2015040441A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017123266A1 (en) * | 2016-01-16 | 2017-07-20 | Hewlett-Packard Development Company, L.P. | Blood characteristic measurement |
| JP2019507345A (en) * | 2016-02-18 | 2019-03-14 | ダイアグノスティカ スターゴ | Method for assaying specific D-dimers for venous thromboembolism and use thereof for diagnosing pulmonary embolism and deep vein thrombosis |
| CN111551701A (en) * | 2020-04-03 | 2020-08-18 | 深圳大学 | Flexible micro-column ring array for detecting clot contractility and preparation method and application thereof |
| CN112394183A (en) * | 2021-01-19 | 2021-02-23 | 潍坊库恩曼动力机械有限公司 | Intelligent blood detection system based on cross matching |
Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1013192A (en) * | 1962-11-13 | 1965-12-15 | Warner Lambert Pharmaceutical | Diagnostic reagent composition for use in determining blood coagulation factors |
| US3228841A (en) * | 1965-06-10 | 1966-01-11 | Warner Lambert Pharmaceutical | Diagnostic reagent composition for determining blood coagulation factors and method of use |
| US4458015A (en) * | 1981-04-02 | 1984-07-03 | Boehringer Mannheim Gmbh | Reagent for the optical determination of the blood coagulation |
| US4784944A (en) * | 1984-04-09 | 1988-11-15 | Behringwerke Aktiengesellschaft | Prothrombin time determining reagent and methods of using and preparing the same |
| WO1994016095A1 (en) | 1993-01-13 | 1994-07-21 | Avocet Medical, Inc. | Method, test article, and system for performing assays |
| US5443960A (en) * | 1991-11-13 | 1995-08-22 | Dahlbaeck; Bjoern | Method for the diagnosis of blood coagulation disorders |
| WO1995030770A1 (en) | 1994-05-06 | 1995-11-16 | Avocet Medical, Inc. | Dry prothrombin time assay |
| US5522255A (en) | 1993-08-31 | 1996-06-04 | Boehringer Mannheim Corporation | Fluid dose, flow and coagulation sensor for medical instrument |
| US6060323A (en) | 1997-06-27 | 2000-05-09 | Hemosense, Inc. | Method and device for measuring blood coagulation or lysis by viscosity changes |
| US20030180824A1 (en) | 2002-03-25 | 2003-09-25 | Mpock Emmanuel C. | System for performing blood coagulation assays and measuring blood clotting times |
| WO2004111656A1 (en) | 2003-06-15 | 2004-12-23 | Zafena Ab | Coagulation tests at ambient temperature |
| WO2005054847A1 (en) | 2003-12-02 | 2005-06-16 | Zafena Aktiebolag | Hematocrit and analyte concentration determination |
| WO2010122158A1 (en) | 2009-04-23 | 2010-10-28 | Dublin City University | A lateral flow assay device for coagulation monitoring and method thereof |
| WO2011148207A1 (en) | 2010-05-28 | 2011-12-01 | Diagon Kft. | Procedure for biphasic preparation of liposomes and application thereof in manufacturing diagnostic reagents |
| WO2013072710A1 (en) * | 2011-11-16 | 2013-05-23 | Diagon Kft. | Procedure and automatic apparatus for in vitro blood coagulation diagnostic tests |
-
2013
- 2013-09-19 HU HU1300542A patent/HUP1300542A2/en unknown
-
2014
- 2014-09-19 WO PCT/HU2014/000084 patent/WO2015040441A1/en active Application Filing
Patent Citations (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1013192A (en) * | 1962-11-13 | 1965-12-15 | Warner Lambert Pharmaceutical | Diagnostic reagent composition for use in determining blood coagulation factors |
| US3228841A (en) * | 1965-06-10 | 1966-01-11 | Warner Lambert Pharmaceutical | Diagnostic reagent composition for determining blood coagulation factors and method of use |
| US4458015A (en) * | 1981-04-02 | 1984-07-03 | Boehringer Mannheim Gmbh | Reagent for the optical determination of the blood coagulation |
| US4784944A (en) * | 1984-04-09 | 1988-11-15 | Behringwerke Aktiengesellschaft | Prothrombin time determining reagent and methods of using and preparing the same |
| US5443960B1 (en) * | 1991-11-13 | 1999-01-12 | T A C Thrombosis And Coagulati | Method for the diagnosis of blood coagulation disorders |
| US5443960A (en) * | 1991-11-13 | 1995-08-22 | Dahlbaeck; Bjoern | Method for the diagnosis of blood coagulation disorders |
| WO1994016095A1 (en) | 1993-01-13 | 1994-07-21 | Avocet Medical, Inc. | Method, test article, and system for performing assays |
| US6189370B1 (en) | 1993-08-31 | 2001-02-20 | Roche Diagnostics Corporation | Fluid dose, flow and coagulation sensor for medical instrument |
| US5522255A (en) | 1993-08-31 | 1996-06-04 | Boehringer Mannheim Corporation | Fluid dose, flow and coagulation sensor for medical instrument |
| US6575017B1 (en) | 1993-08-31 | 2003-06-10 | Roche Diagnostics Corporation, Inc. | Fluid dose, flow and coagulation sensor for medical instrument |
| WO1995030770A1 (en) | 1994-05-06 | 1995-11-16 | Avocet Medical, Inc. | Dry prothrombin time assay |
| US6060323A (en) | 1997-06-27 | 2000-05-09 | Hemosense, Inc. | Method and device for measuring blood coagulation or lysis by viscosity changes |
| US6338821B1 (en) | 1997-06-27 | 2002-01-15 | Arvind N. Jina | Method and device for measuring blood coagulation or lysis by viscosity changes |
| US6673622B1 (en) | 1997-06-27 | 2004-01-06 | Hemosense, Inc. | Coagulation or lysis assays by measuring impedance |
| US20030180824A1 (en) | 2002-03-25 | 2003-09-25 | Mpock Emmanuel C. | System for performing blood coagulation assays and measuring blood clotting times |
| WO2004111656A1 (en) | 2003-06-15 | 2004-12-23 | Zafena Ab | Coagulation tests at ambient temperature |
| WO2005054847A1 (en) | 2003-12-02 | 2005-06-16 | Zafena Aktiebolag | Hematocrit and analyte concentration determination |
| WO2010122158A1 (en) | 2009-04-23 | 2010-10-28 | Dublin City University | A lateral flow assay device for coagulation monitoring and method thereof |
| WO2011148207A1 (en) | 2010-05-28 | 2011-12-01 | Diagon Kft. | Procedure for biphasic preparation of liposomes and application thereof in manufacturing diagnostic reagents |
| WO2013072710A1 (en) * | 2011-11-16 | 2013-05-23 | Diagon Kft. | Procedure and automatic apparatus for in vitro blood coagulation diagnostic tests |
Non-Patent Citations (10)
| Title |
|---|
| ANTON VAN DEN BESSELAAR ET AL: "Preparation of lyophilized partial thromboplastin time reagent composed of synthetic phospholipids: usefulness for monitoring heparin therapy", CLINICAL CHEMISTRY, vol. 43, no. 7, 1 July 1997 (1997-07-01), pages 1215, XP055163919, ISSN: 0009-9147 * |
| ENRIQUEZ L.J.; SHORE-LESSERSON L., BR.J.ANAESTH., vol. 103, 2009, pages I14 - 122 |
| GANTER M.T.; HOFER C.K., ANESTH.ANALG., vol. 106, 2008, pages 1366 - 1375 |
| GOGSTAD G.O. ET AL., CLIN.CHEM., vol. 32, 1986, pages 1857 - 1862 |
| HOFFMANN J.J.M.L.; VERHAPPEN M.A.L., CLIN.CHEM., vol. 34, 1988, pages 2135 - 2140 |
| HUCKLE D., EXPERT REV.MOL.DIAGN., vol. 8, 2008, pages 679 - 688 |
| JACKSON C.M. ET AL., CLIN.CHEM., vol. 51, 2005, pages 483 - 485 |
| PERRY D.J. ET AL., BR.J.HAEMATOL., vol. 150, 2010, pages 501 - 514 |
| WEBSTER S., EUR.CLIN.LAB., vol. 25, 2006, pages 10 - 11 |
| WU H.M. ET AL., PER.MED., vol. 7, 2010, pages 65 - 73 |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017123266A1 (en) * | 2016-01-16 | 2017-07-20 | Hewlett-Packard Development Company, L.P. | Blood characteristic measurement |
| US11112400B2 (en) | 2016-01-16 | 2021-09-07 | Hewlett-Packard Development Company, L.P. | Blood characteristic measurement |
| JP2019507345A (en) * | 2016-02-18 | 2019-03-14 | ダイアグノスティカ スターゴ | Method for assaying specific D-dimers for venous thromboembolism and use thereof for diagnosing pulmonary embolism and deep vein thrombosis |
| CN111551701A (en) * | 2020-04-03 | 2020-08-18 | 深圳大学 | Flexible micro-column ring array for detecting clot contractility and preparation method and application thereof |
| CN112394183A (en) * | 2021-01-19 | 2021-02-23 | 潍坊库恩曼动力机械有限公司 | Intelligent blood detection system based on cross matching |
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|---|---|
| HUP1300542A2 (en) | 2015-03-30 |
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