US20070254325A1 - Method and device for the determination of platelet function under flow conditions - Google Patents

Method and device for the determination of platelet function under flow conditions Download PDF

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US20070254325A1
US20070254325A1 US11/790,853 US79085307A US2007254325A1 US 20070254325 A1 US20070254325 A1 US 20070254325A1 US 79085307 A US79085307 A US 79085307A US 2007254325 A1 US2007254325 A1 US 2007254325A1
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partition member
determination
antagonist
whole blood
mrs
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Andreas Rechner
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Siemens Healthcare Diagnostics Products GmbH
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Dade Behring Marburg GmbH
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

  • the invention lies in the area of platelet function diagnostics and relates to an in vitro method for the determination of platelet function under flow conditions as well as a device for the implementation of this method.
  • the method is particularly suitable for the determination of the effect of clopidogrel after oral intake and of other P2Y(12) antagonists with antithrombotic activity.
  • the blockade of the second platelet ADP receptor (P2Y(1) receptor) by specific antagonists can also be detected with the method.
  • thrombocytes thrombocytes
  • VWF von Willebrand factor
  • primary vessel wall occlusion (primary hemostasis) takes place which then is further stabilized by reactions of the plasmatic coagulation system (secondary hemostasis).
  • Dysregulation of these processes can lead to thrombophilia or a tendency towards hemorrhage, which dependent upon the degree of severity can have life-threatening consequences.
  • ASA acetylsalicylic acid
  • COX-1 cyclooxygenase-1
  • clopidogrel and ticlopidine belong to the class of P2Y(12) antagonists.
  • the purinergic P2Y(12) receptor is expressed on the platelet surface and can be activated by extracellular adenosine-5′-diphosphate (ADP).
  • ADP extracellular adenosine-5′-diphosphate
  • P2Y(12) antagonists block the purinergic P2Y(12) receptors on the platelet surface and thus possess an antithrombotic activity.
  • the second purinergic ADP receptor P2Y(1) is also expressed on the platelet surface and is activated by extracellular adenosine-5′-diphosphate.
  • extracellular adenosine-5′-diphosphate As a consequence of the activation of the purinergic P2Y(1) receptor intracellular processes are initiated in the platelets, for example an increase in intracellular calcium, that give rise to a platelet aggregation reaction.
  • P2Y(1) receptor antagonists act against this process and thus have an antithrombotic activity.
  • Clopidogrel resistance is present when the platelet function of a patient is only slightly influenced by the administration of a standard dose of clopidogrel, or not at all. On the one hand a test can be carried out to determine whether an adequate antithrombotic response is actually achieved with a selected dose by determination of platelet function. On the other hand, doses or responses of an antithrombotic medication that are too high can be determined and treated, which is necessary, for example, prior to surgery in order to exclude bleeding complications.
  • Bleeding time determination is a global in vivo test which records primary hemostasis. The bleeding time is determined wherein the patient is given a small cut or prick injury and the time for coagulation is measured. It is a poorly standardizable, coarsely informative test that is used primarily in an emergency situation in order to obtain an overview of primary hemostasis. Taking platelet aggregation inhibitors leads to an increase in bleeding time. The disadvantage of bleeding time determination is that platelet dysfunction cannot be excluded even with a normal bleeding time.
  • platelet aggregation is induced in a whole blood sample or in a sample of platelet-rich plasma (PRP) by the addition of an activator and the aggregation reaction is measured.
  • activators used for the induction of platelet activation are ADP (adenosine-5′-diphosphate), collagen, epinephrine (adrenaline), ristocetin and different combinations thereof as well as thrombin, TRAP (thrombin receptor activating protein) or serotonin.
  • light transmission aggregometry also known as Born platelet aggregation
  • the aggregation efficiency of platelets in platelet-rich plasma is measured photometrically in the presence of aggregation-inducing compounds in an aggregometer.
  • the light transmission of the PRP sample is increased due to aggregate formation so that the rate of aggregate formation, for example, can be determined by measurement of light transmission.
  • the therapeutic effects of platelet aggregation inhibitors used medically can also be determined with the aid of light transmission aggregometry.
  • a disadvantage of light transmission aggregometry is that only platelet-rich plasma can be used as sample material. Platelet-rich plasma lacks not only important blood components such as, for example, red and white blood cells, but also requires a time-consuming and error-prone sample preparation.
  • PFA-100® Platelet Function Analyzer
  • Dade Behring Marburg GmbH, Marburg, Germany Another test principle for the determination of platelet function is realized in the Platelet Function Analyzer (PFA-100®, Dade Behring Marburg GmbH, Marburg, Germany).
  • the PFA-100® is a global, automated and standardized in vitro whole blood test with which primary hemostasis is measured under flow conditions and thus in the presence of high shear forces.
  • a partial vacuum of about ⁇ 40 mbar is produced in a special test cartridge.
  • the citrated whole blood that is located in a sample reservoir is sucked through a capillary with a diameter of about 200 ⁇ m.
  • the capillary leads into a measurement chamber which is closed with a partition member, for example a membrane, which has a central capillary opening (aperture) through which the blood flows due to the partial vacuum.
  • a partition member for example a membrane, which has a central capillary opening (aperture) through which the blood flows due to the partial vacuum.
  • the membrane at least within the region surrounding the aperture, is coated with one or more activators that induce platelet aggregation so that the passing blood comes into contact with the aggregation-inducing substances in the region of the aperture.
  • a thrombus is formed in the region of the aperture which seals the membrane opening and stops the blood flow.
  • closure time correlates with the functional efficiency of the platelets.
  • test cartridge for use in a method for the determination of platelet function based on the closure time is described, for example, in patent specification WO 97/34698.
  • test cartridges that are equipped with a membrane that is coated with collagen (Col) and also with either ADP or epinephrine (Epi) are used in the method for the determination of closure time.
  • Different partition members as well as their preparation and use are described, for example in patent specification EP 716 744 B1.
  • Col/ADP test cartridges Normally a patient sample is first analyzed with the aid of a Col/Epi test cartridge. In the case of an abnormally prolonged Col/Epi closure time, which indicates a disorder of platelet aggregation, a Col/ADP measurement is subsequently carried out. If the Col/ADP closure time is likewise abnormally prolonged this is an indicator of platelet dysfunction or a disorder of the von Willebrand factor. If in contrast the Col/ADP closure time is normal this can indicate the presence of acetylsalicylic acid or the presence of an acquired or inherited thrombocytopathy such as, for example, storage pool disease.
  • a disadvantage of the PFA-100® system is that the available Col/ADP and Col/Epi test cartridges have only a limited sensitivity for the aggregation inhibitory effect of platelet aggregation inhibitors of the thienopyridine group (e.g. clopidogrel, ticlopidine).
  • a more reliable determination of the therapeutic effect of the medically used clopidogrel and ticlopidine, especially when the patient has also taken ASA (e.g. Aspirin®) is hitherto not possible with the help of the known Col/ADP and Col/Epi test cartridges in the PFA-100® system.
  • the patent specification WO 2005/007868 A2 describes an alternative method for the determination of platelet function that allows the detection of the therapeutic effect of clopidogrel and other P2Y(12) antagonists.
  • a whole blood sample of a patient is mixed with an anticoagulant and treated with ADP for the induction of platelet aggregation.
  • prostaglandin E1 PGE 1
  • Prostaglandin E1 a product of human arachidonic acid metabolism, is able to reduce the reactivity of platelets significantly, even in low doses, and is therefore also used for the inhibition of platelet activation.
  • PGE 1 is used to reduce the undesirable activation of the ADP receptor P2Y(1) and thus to increase the specificity of the test method for the P2Y(12) receptor and for P2Y(12) antagonists such as clopidogrel.
  • microparticles to which a ligand for the GPIIb/IIIa receptor such as, for example, fibrinogen is coupled are added and the aggregation reaction is measured aggregometrically on the basis of the increasing light transmission.
  • a disadvantage of the previously described method is that as with light transmission aggregometry platelet function is not determined under the influence of flow conditions and shear forces.
  • the object forming the basis of the present invention is to provide a method for the determination of platelet function under flow conditions that allows the determination of the platelet aggregation inhibitory effect of P2Y(12) antagonists.
  • the solution to the object lies in the provision of the methods and materials according to the invention described in the claims.
  • the object of the present invention is an in vitro method for the determination of platelet function in a whole blood sample.
  • the whole blood sample is freshly drawn anticoagulated venous human or animal blood that is to be investigated within four hours after blood collection with the help of the method according to the invention.
  • the whole blood is preferably anticoagulated by the addition of an anticoagulant.
  • Suitable for use as anticoagulant are buffered calcium-binding citrate solutions such as, for example, 3.2 or 3.8% buffered sodium citrate solutions, as well as natural or synthetic direct thrombin inhibitors such as, for example, hirudin, PPACK (D-Phe-Pro-Arg-chloromethylketone, HCl), argatroban and melagatran, or natural or synthetic direct Factor Xa inhibitors such as, for example, antistasin, tick anticoagulant peptide, yagin, draculin, GGACK (H-Glu-Glu-Arg-chloromethylketone), diamidino Factor Xa inhibitors and monobenzamidine Factor Xa inhibitors.
  • buffered calcium-binding citrate solutions such as, for example, 3.2 or 3.8% buffered sodium citrate solutions
  • natural or synthetic direct thrombin inhibitors such as, for example, hirudin, PPACK (D-Phe-Pro-Arg-chloromethylketone,
  • the method according to the invention for the determination of platelet function comprises several methodological steps.
  • the blood that is initially located in a reservoir is passed though a capillary that preferably has a diameter of about 200 ⁇ m.
  • the capillary leads into a measurement chamber that is separated into two compartments by a partition member.
  • the partition member has an opening through which the blood is passed from the first into the second compartment.
  • the method of the invention is characterized in that the partition member used comprises an activator of purinergic receptors and an activator of intracellular adenylate cyclases whereby the blood flowing through the opening of the partition member is brought into contact with these substances contained in or on the partition member.
  • a thrombus forms at the opening of the partition member.
  • the time that is necessary for the formation of the thrombus at the opening of the partition member up to closure of the opening is measured.
  • the closure time is measured in that an apparatus is used that comprises a pressure sensor which determines the blood flow through the aperture during the test.
  • an apparatus is used that comprises a pressure sensor which determines the blood flow through the aperture during the test.
  • the initial flow rate is first determined. If the flow rate falls below 10% of this initial flow rate for more than 3 seconds the measurement is ended and the time passed until then is recorded as the so-called closure time.
  • This so-called closure time which is i.a. dependent on the aggregation reaction of the stimulated platelets, is a measure of platelet function.
  • the closure time that was measured for a whole blood sample of a patient is compared with a closure time reference range for whole blood samples of healthy subjects.
  • the blood flow through the capillary and through the opening of the partition member is produced by creating a partial vacuum in the measurement chamber, that is by suction.
  • the partial vacuum is produced by the combined action of a suitable test cartridge and an apparatus.
  • the partition member used in the method according to the invention comprises an activator of purinergic receptors, preferably from the group adenosine-5′-diphosphate (ADP), 2-methylthioadenosine-5′-diphosphate (2-MeSADP) and their derivatives.
  • ADP adenosine-5′-diphosphate
  • 2-MeSADP 2-methylthioadenosine-5′-diphosphate
  • a partition member is used that comprises an ADP salt or a 2-MeSADP salt.
  • a partition member is used that comprises 1 to 100 ⁇ g, especially preferred 5 to 50 ⁇ g, particularly preferred 20 to 25 ⁇ g ADP.
  • the partition member used comprises further an activator of intracellular adenylate cyclases, preferably from the group prostaglandin E1 (PGE 1), forskolin and its water-soluble derivatives, prostaglandin 12 and its stable derivatives, iloprost and cicaprost.
  • PGE 1 prostaglandin E1
  • a partition member is used that comprises 1 to 1000 ng, especially preferably 3 to 20 ng prostaglandin E1.
  • a partition member is used that comprises 0.1 to 10 ⁇ g, especially preferred 0.5 to 5 ⁇ g forskolin.
  • a partition member that comprises ADP and prostaglandin E1.
  • a partition member is used that also comprises calcium ions, preferably in the form of calcium chloride dihydrate.
  • a partition member is used that comprises 50 to 200 ⁇ g, especially preferred 100 to 150 ⁇ g, most especially preferred 125 ⁇ g calcium ions in the form of calcium chloride dihydrate.
  • ASA platelet aggregation inhibitory effect of acetylsalicylic acid
  • the whole blood sample to be investigated is anticoagulated with a non-calcium-binding anticoagulant such as, for example, with a direct thrombin or Factor Xa inhibitor
  • a non-calcium-binding anticoagulant such as, for example, with a direct thrombin or Factor Xa inhibitor
  • the calcium ion concentration contained endogenously in the sample is sufficient to reduce an ASA-induced platelet dysfunction.
  • a partition member that comprises calcium ions can also be used in these cases.
  • the method according to the invention is used most preferably for the determination of the antithrombotic (platelet aggregation inhibitory) effect of a P2Y(12) antagonist, especially for the determination of a P2Y(12) antagonist from the group clopidogrel, ticlopidine, prasugrel (synonym: CS-747) and other thienopyridines, AR-C67085MX (2-propylthio-D- ⁇ , ⁇ -dichloromethylene-adenosine-5′-triphosphate), cangrelor (synonym: AR-C69931 MX, N6-[2-methylthio)ethyl]-2-(3,3,3-trifluoropropyl)thio-5′-adenylic acid), C1330-7 (N1-(6-ethoxy-1,3-benzothiazol-2-yl-2-(7-ethoxy-4-hydroxy-2,2-dioxo-2H-2-6benzo[4,5][1,
  • the method according to the invention can also be used for the determination of the antithrombotic (platelet aggregation inhibitory) effect of a P2Y(1) antagonist.
  • the method can be used for the determination of the antithrombotic effect of P2Y(1) antagonists from the group MRS 2179 [2′-deoxy-N-6-methyladenosine-3′,5′-bisphosphate, diammonium salt], MRS 2279 [(N)-methanocarba-N-6-methyl-2-chloro-2′deoxyadenosine-3′,5′-diphosphate], MRS 2500 [2-iodo-N-6-methyl-(N)-methanocarba-2′-deoxyadenosine-3‘5’-diphosphate], A2P5P [adenosine-2′,5′-diphosphate], A3P5P [adenosine-3′,5′-diphosphate], A3P5PS [adenos
  • a further object of the present invention concerns a device, as for example a test cartridge, which is suitable for the determination of platelet function in a whole blood sample
  • the device comprises different elements: a) a reservoir for storing the sample; b) a capillary through which the blood is passed from the reservoir into a measurement chamber; c) a measurement chamber that is separated into two compartments by a partition member, wherein the first compartment receives the blood from the capillary; d) a partition member which divides the measurement chamber into two compartments and which has an opening through which the blood can flow from the first compartment into the second compartment.
  • the partition member comprises an activator of purinergic receptors and an activator of intracellular adenylate cyclases.
  • the partition member also comprises calcium ions, preferably in the form of calcium chloride dihydrate.
  • the partition member is a porous or nonporous support matrix for an activator of purinergic receptors and an activator of intracellular adenylate cyclases and optionally for calcium ions.
  • the partition member is constructed in the form of a membrane.
  • the preferred material is liquid absorbing so that the aforementioned substances can be applied in solution.
  • Especially preferred materials are cellulose esters, ceramic, nylon, polypropylene, polyether sulfone, and polyvinylidene fluoride (PVDF).
  • the partition member wetted or soaked with the desired substances is dried. By contact of the blood with the partition member the substances are dissolved from the partition member and mix with the blood sample.
  • the partition member preferably has a circular opening that is produced in the support matrix by punching.
  • the diameter of the opening in the partition member is so dimensioned that a thrombus can form under the conditions of the respective method which closes the opening and can thus stop the blood flow.
  • the opening in the partition member has a diameter between approximately 100 ⁇ m and approximately 200 ⁇ m. Particularly preferably the diameter of the opening in the partition member is about 100 ⁇ m.
  • the device according to the invention is preferably so constructed that a partial vacuum that brings about a blood flow from the reservoir through the capillary into the measurement chamber and through the opening of the partition member is produced in the device with the help of an apparatus that is integrated with components of the device.
  • the present invention further relates to the use of a device according to the invention in a method for the determination of platelet function.
  • a preferred use of a device according to the invention relates to the use for the determination of the antithrombotic effect of a P2Y(12) antagonist.
  • Another preferred use of a device according to the invention relates to the use for the determination of the antithrombotic effect of a P2Y(1) antagonist.
  • FIG. 1 A first figure.
  • FIG. 1 shows by way of example how a device for the determination of platelet function according to the invention can be constructed.
  • a test cartridge in accordance with WO 97/34698 in longitudinal section that is placed in a suitable apparatus for implementing the method according to the invention and into which extends a vacuum apparatus ( 15 ) that is responsible for the generation of the partial vacuum
  • the vacuum apparatus ( 15 ) has a ring gasket ( 27 ) which is located as a seal on the circumferential edge ( 12 ) of the sample container ( 10 ).
  • the test cartridge has a housing that forms a reservoir ( 61 ) and a test chamber ( 63 ).
  • the test chamber ( 63 ) is constructed to accept a sample container ( 10 ) the cavity of which can also be referred to as measurement chamber.
  • the sample container ( 10 ) supports a partition member ( 6 ) treated with reagents and with a central opening (aperture) and a capillary attachment ( 30 , 31 ) that connects the capillary ( 40 ) with the sample container ( 10 ).
  • Reservoir ( 61 ) and test chamber ( 63 ) are separated by a penetrable element ( 70 ).
  • the figure shows a phase of the test cycle after the vacuum apparatus ( 15 ) is in contact with sample container ( 10 ) and has moved downwards so that the base of the sample container ( 10 ) is in contact with the support ( 71 ) and the capillary ( 40 ) has penetrated the penetrable element ( 70 ) and penetrated into the sample ( 11 ).
  • the apparatus produces a partial vacuum in the sample container ( 10 ) by means of which the sample ( 11 ) is pulled through the capillary ( 40 ) into the first compartment ( 18 ) of the measurement chamber and then through the opening in the partition member ( 6 ).
  • a comparison of the two types of test cartridge shows that with use of an ADP/PGE1 test cartridge according to the invention, closure times that lie significantly above the upper reference value (cut-off) were measured with samples that were treated with the P2Y(12) antagonist MRS 2395, whereas the same samples with the use of a Col/Epi test cartridge lie to a greater extent below the upper reference value (cut-off). That means that the method for determination of platelet function according to the invention allows a more sensitive determination of platelet dysfunction induced by a P2Y(12) antagonist than the comparison method from the prior art.
  • Whole blood samples from 10 healthy donors anticoagulated with PPACK were used.
  • the mean values and the standard deviations of the closure times that were determined with the ADP/PGE1 test cartridges according to the invention cut-off: 90 seconds).
  • the performance evaluation shows that with use of an ADP/PGE1 test cartridge according to the invention, closure times that lie significantly above the reference value (cut-off) were measured with samples that were treated with the P2Y(12) antagonist MRS 2395 or the P2Y(1) antagonist MRS 2179, whereas the samples treated with COX-1 inhibitor acetylsalicylic acid show no prolongation of closure times and thus lie below the cut-off.
  • a polyether sulfone filter membrane (Supor®) membrane, Pall GmbH, Dreieich, Germany) was cut into strips. 1 ⁇ L of a solution comprising 7 ⁇ g/ ⁇ L ADP (adenosine-5′-diphosphate potassium salt.2H 2 O, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and 5 ng/ ⁇ L PGE1 (prostaglandin E1, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) and 367.5 ⁇ g/ ⁇ L CaCl 2 .2H 2 O (equivalent to 100 ⁇ g/ ⁇ L Ca 2+ ions) were pipetted punctiform onto the membrane and the membrane was dried.
  • ADP adenosine-5′-diphosphate potassium salt.2H 2 O
  • PGE1 prostaglandin E1
  • CaCl 2 .2H 2 O equivalent to 100 ⁇ g/ ⁇ L Ca 2+ ions
  • ASA COX-1 inhibitor acetylsalicylic acid
  • the blood samples were incubated at room temperature for 5 minutes.
  • platelet rich (PRP) and platelet poor (PPP) plasma was prepared from aliquots of the untreated and MRS 2395-treated whole blood samples described under Example 2a), and the samples were then treated with 2 ⁇ M ADP.
  • the PPP samples were used as blank controls.
  • the photometric measurement of the aggregation reaction was carried out in the automated coagulation apparatus BCT® (Dade Behring Marburg GmbH, Marburg, Germany) under continuous stirring (600 rpm).
  • the platelet aggregation of the samples treated with MRS 2395 was reduced by a mean of 27% compared with the platelet aggregation of the untreated samples.
  • Example 2a To determine the closure time as a measure of platelet function the whole blood samples described under Example 2a) were investigated with the aid of the ADP/PGE1/calcium test cartridge according to the invention described in Example 1 in a PFA-100® apparatus (Platelet Function Analyzer-100, Dade Behring Marburg GmbH, Marburg, Germany). For this purpose 700 ⁇ L of a blood sample were placed in the reservoir of the temperature equilibrated test cartridge (+37° C.) and incubated at +37° C. for 3 minutes.
  • a partial vacuum of ⁇ 40 mbar was generated by the apparatus by which means the blood was sucked through a capillary from the reservoir (diameter 200 ⁇ m) and finally through an opening (aperture) of the partition member in the measurement chamber.
  • the time required up to the closure of the aperture by formation of a blood clot was determined as closure time. Every sample investigated was determined in duplicate and the mean value of a duplicate determination was used as the measurement value.
  • Example 2a For comparison purposes the whole blood samples described under Example 2a) were investigated in parallel with a known Col/Epi PFA-100® test cartridge (2 ⁇ g collagen and 10 ⁇ g epinephrine on the membrane; 150 ⁇ m aperture diameter; Dade Behring Marburg GmbH, Marburg, Germany) in the PFA-100® apparatus.
  • a known Col/Epi PFA-100® test cartridge (2 ⁇ g collagen and 10 ⁇ g epinephrine on the membrane; 150 ⁇ m aperture diameter; Dade Behring Marburg GmbH, Marburg, Germany
  • the method according to the invention has a very low sensitivity for acetylsalicylic acid.
  • an abnormally reduced platelet aggregation is measured with the aid of the method according to the invention, whereas, in contrast, with the conventional Col/Epi test cartridge 8 of the 11 samples treated with acetylsalicylic acid were determined as abnormal.
  • Samples that are treated with MRS 2395 and acetylsalicylic acid are classified 100% as abnormal with the aid of the conventional method, whereas only 9 of the 11 samples (as with sole addition of MRS 2395) are classified as abnormal with the method according to the invention.
  • the method according to the invention is suitable for differentiation of the two classes of antithrombotics.
  • Venous blood was taken from healthy donors and anticoagulated with sodium citrate (3.2% buffered Na citrate). The closure time determination was carried out for each whole blood sample in the PFA-100® apparatus. Samples from 186 donors were determined in duplicate with a Col/Epi PFA-100® test cartridge [see Example 2c)]. Samples from 159 donors were determined in duplicate with an ADP/PGE1/calcium test cartridge according to the invention [see Examples 1 and 2c)].
  • the reference ranges (normal range) for the Col/Epi closure time and the ADP/PGE1 closure time were established in that the measurement value ranges in which 90% of the measurement values found for the healthy subjects lay were determined (90% central interval of the normal distribution of all measurements). This gave the following reference ranges for the closure times:
  • the upper reference limit of the reference range was defined as cut-off, i.e. as threshold value, for a platelet dysfunction. If the closure time of a patient sample deviates from the reference range it can indicate a platelet dysfunction. This means Col/Epi closure times that are greater than 158 seconds and ADP/PGE1 closure times that are greater than 81 seconds indicate the presence of a platelet dysfunction within the sense of a reduced aggregation efficiency.
  • Venous blood was taken from 13 patients suffering from peripheral arterial obstructive disease and who had been treated with a daily dose of 75 mg clopidogrel (Plavix®, Sanofi-Aventis) as sole antithrombotic for a period of at least 4 weeks and the blood was anticoagulated with sodium citrate (3.8% buffered Na citrate). The samples were investigated with the aid of different methods to determine platelet function.
  • clopidogrel Plavix®, Sanofi-Aventis
  • Table 2 presents in detail with which of the four methods in which of the 13 patient samples an antithrombotic effect of clopidogrel could be detected. “+” means an antithrombotic effect could be detected. “ ⁇ ” means no antithrombotic effect could be detected. “0” means that the duplicate determination gave contradictory results, i.e. one value above and one value below the cut-off.
  • the method according to the invention is more sensitive towards the platelet dysfunction induced by clopidogrel than the standard method of ADP-induced light transmission aggregometry according to Born and more sensitive than the method with which a test cartridge is used that comprises ADP but no PGE1.
  • Venous blood was taken from 10 healthy donors and anticoagulated with 75 ⁇ M PPACK.
  • ASA COX-1 inhibitor acetylsalicylic acid
  • the blood samples were incubated at room temperature for 5 minutes.
  • Example 4a For the determination of the closure time as a measure of the platelet function the whole blood samples described under Example 4a) were investigated with the aid of a ADP/PGE1 test cartridge according to the invention in a PFA-100® apparatus (Platelet Function Analyzer-100, Dade Behring Marburg GmbH, Marburg, Germany).
  • the ADP/PGE1 test cartridge according to the invention used was prepared essentially as described in Example 1 but without the partition member having been treated with CaCl 2 .2H 2 O.
  • the test cartridge thus comprised 7 ⁇ g ADP and 5 ng PGE1 but no calcium ions.
  • the method according to the invention is suitable for differentiation of the two classes of antithrombotics.
  • the reference range for the ADP/PGE1 closure time from the 10 samples of the healthy donors treated with PPACK was calculated by the determination of the 90% central interval of the normal distribution of the mean values of the duplicate determinations. This gave the following reference ranges for the closure times:
  • the upper limit of the 90% central interval was defined as cut-off, i.e. as threshold value for a platelet dysfunction.

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US11/790,853 2006-04-28 2007-04-27 Method and device for the determination of platelet function under flow conditions Abandoned US20070254325A1 (en)

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US20070254324A1 (en) * 2006-04-28 2007-11-01 Dade Behring Marburg Gmbh Method for determination of platelet function under flow conditions
US20100267066A1 (en) * 2007-11-26 2010-10-21 Fujimori Kogyo Co., Ltd. Microchip and blood monitoring device
US20130224777A1 (en) * 2012-02-28 2013-08-29 Siemens Healthcare Diagnostics Products Gmbh Screening method for finding samples having antiphospholipid antibodies
US8796031B2 (en) 2010-02-10 2014-08-05 Fujimori Kogyo Co., Ltd. Microchip for platelet examination and platelet examination device using same
US9052258B2 (en) 2011-07-07 2015-06-09 Siemens Healthcare Diagnostics Products Gmbh Method for standardizing measured results in a system for measuring thrombocyte function
EP2937695A4 (en) * 2012-12-18 2016-06-29 Daiichi Sankyo Co Ltd METHOD FOR MEASURING THROMBIN GENERATION
CN111190000A (zh) * 2020-03-02 2020-05-22 美高怡生生物技术(北京)有限公司 血小板功能判断方法及装置
EP2352025B1 (en) * 2008-08-11 2020-07-15 Fujimori Kogyo Co., Ltd. Blood-platelet test method

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EP2525228A1 (de) * 2011-05-18 2012-11-21 Siemens Healthcare Diagnostics Products GmbH Verfahren zur Bestimmung des Risikos einer Clopidogrel-Resistenz
EP2562542B1 (de) 2011-08-22 2015-01-28 Siemens Healthcare Diagnostics Products GmbH Dynamischen Bestimmung der Thrombozytenfunktion
US10761082B2 (en) 2016-04-29 2020-09-01 Neotek Bioscience Co., Ltd. Bracket, thrombelastography device, and support system
US10885478B2 (en) * 2016-07-06 2021-01-05 Palo Alto Research Center Incorporated Computer-implemented system and method for providing contextually relevant task recommendations to qualified users
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US11477302B2 (en) 2016-07-06 2022-10-18 Palo Alto Research Center Incorporated Computer-implemented system and method for distributed activity detection

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Publication number Priority date Publication date Assignee Title
US20070254324A1 (en) * 2006-04-28 2007-11-01 Dade Behring Marburg Gmbh Method for determination of platelet function under flow conditions
US8895259B2 (en) 2006-04-28 2014-11-25 Siemens Healthcare Diagnostics Products Gmbh Method for determination of platelet function under flow conditions
US20100267066A1 (en) * 2007-11-26 2010-10-21 Fujimori Kogyo Co., Ltd. Microchip and blood monitoring device
US8425840B2 (en) 2007-11-26 2013-04-23 Fujimori Kogyo Co., Ltd. Microchip and blood monitoring device
EP2352025B1 (en) * 2008-08-11 2020-07-15 Fujimori Kogyo Co., Ltd. Blood-platelet test method
US8796031B2 (en) 2010-02-10 2014-08-05 Fujimori Kogyo Co., Ltd. Microchip for platelet examination and platelet examination device using same
US9052258B2 (en) 2011-07-07 2015-06-09 Siemens Healthcare Diagnostics Products Gmbh Method for standardizing measured results in a system for measuring thrombocyte function
US20130224777A1 (en) * 2012-02-28 2013-08-29 Siemens Healthcare Diagnostics Products Gmbh Screening method for finding samples having antiphospholipid antibodies
EP2937695A4 (en) * 2012-12-18 2016-06-29 Daiichi Sankyo Co Ltd METHOD FOR MEASURING THROMBIN GENERATION
US10266871B2 (en) 2012-12-18 2019-04-23 Daiichi Sankyo Company, Limited Measurement method for thrombin production
CN111190000A (zh) * 2020-03-02 2020-05-22 美高怡生生物技术(北京)有限公司 血小板功能判断方法及装置

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US20150093770A1 (en) 2015-04-02
JP2007298511A (ja) 2007-11-15
DE102006020385A1 (de) 2007-10-31
US9759730B2 (en) 2017-09-12
CA2586268A1 (en) 2007-10-28
EP1850134A1 (de) 2007-10-31
EP1850134B1 (de) 2013-01-30
JP4989292B2 (ja) 2012-08-01
CA2586268C (en) 2015-04-14

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