WO2002059623A2 - Methodes permettant de determiner l'activite des plaquettes a l'aide de compositions anti-plaquettes - Google Patents

Methodes permettant de determiner l'activite des plaquettes a l'aide de compositions anti-plaquettes Download PDF

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Publication number
WO2002059623A2
WO2002059623A2 PCT/US2002/002102 US0202102W WO02059623A2 WO 2002059623 A2 WO2002059623 A2 WO 2002059623A2 US 0202102 W US0202102 W US 0202102W WO 02059623 A2 WO02059623 A2 WO 02059623A2
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WIPO (PCT)
Prior art keywords
particles
aspirin
platelet
sample
agglutination
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PCT/US2002/002102
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English (en)
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WO2002059623A8 (fr
WO2002059623A3 (fr
Inventor
Victor Manneh
Lisa Swaim
Ted Lee
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Sendx Medical, Inc.
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Priority to EP02703225A priority Critical patent/EP1370874A2/fr
Publication of WO2002059623A2 publication Critical patent/WO2002059623A2/fr
Publication of WO2002059623A3 publication Critical patent/WO2002059623A3/fr
Publication of WO2002059623A8 publication Critical patent/WO2002059623A8/fr

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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
    • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
    • 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
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This invention relates to the field of diagnostic assays, and in particular to the determination of platelet function activity on blood samples to study effects of anti- platelet compositions.
  • the invention has particular application to the determination of platelet function activity on blood samples from patients undergoing therapy for platelet anti-aggregation.
  • platelets The role of platelets in mammalian physiology is extraordinarily diverse, but their primary role is in promoting hemostasis. In many situations, an evaluation of the ability of blood to clot is desired, a parameter that is frequently controlled by the ability of platelets to adhere and/or aggregate. Of interest, therefore, is the assessment of the adhesive functions of platelets. For example, questions of interest include whether to administer drugs that will block, or promote, clot formation, or whether to detect deficiencies in platelet function prior to surgical procedures. Also of interest is evaluating the effectiveness of a platelet inhibitor that is being tested as a new drug or is being used as approved clinical treatment in a patient.
  • Platelets are known to aggregate under a variety of conditions and in the presence of a number of different reagents. Platelet aggregation is a term used to describe the binding of platelets to one another. The phenomenon can be induced by adding aggregation-inducing agents to platelet-rich plasma (PRP) or to whole blood. Platelet aggregation in vitro depends upon the ability of platelets to bind fibrinogen to their surfaces after activation by an aggregation-inducing agent such as ADP or collagen.
  • an aggregation-inducing agent such as ADP or collagen.
  • Platelets play a critical role in the maintenance of normal hemostasis. When exposed to a damaged blood vessel, platelets will adhere to exposed sub-endothelial matrix. Following the initial adhesion, various factors released at the site of injury such as thrombin, ADP and collagen activate the platelets. Once platelets are activated, a conformational change occurs in the platelet glycoprotein GPIIb/IIIa receptor allowing it to bind fibrinogen and/or von Willebrand factor. It is this binding of the multivalent fibrinogen and/or von Willebrand factor molecules by GPIIb/IIIa receptors on adjacent platelets that results in the recruitment of additional platelets to the site of injury and their aggregation to form a hemostatic plug or thrombus.
  • In vitro platelet aggregation is the laboratory method used to assess the in vivo ability of platelets to form the aggregates leading to a primary hemostatic plug.
  • an aggregating agent such as ADP or collagen is added to whole blood or PRP and aggregation of platelets monitored.
  • Platelet aggregometry is a diagnostic tool that can provide insights difficult to obtain by other techniques, thus aiding in patient diagnosis and selection of therapy.
  • the CHRONO-LOG Model 530 and Model 540 use the optical method for PRP and the impedance method for whole blood aggregometry.
  • the impedance method has been shown to be substantially equivalent to the optical method for measuring platelet aggregation in PRP.
  • a rapid platelet function assay has recently been developed and is described in U.S. Patent No. 5,763,199 (Coller).
  • the assay determines glycoprotein (GP) Ilb/IIIa receptor blockade in whole blood. Agglutination of small polymeric beads coated with a GPIIb/IIIa ligand such as fibrinogen results when the beads are contacted with whole blood containing platelets with GPHb/IIIa receptors that are not blocked. Failure to agglutinate indicates that blockade of the GPIIb/IIIa receptors has been achieved.
  • the addition of a thrombin receptor activator results in an assay that is rapid and convenient enough to be performed at bedside and that results in agglutination of the small polymeric beads within a convenient, known period of time if the GPIIb/IIIa receptors are not blocked.
  • the assay includes the ability to transfer blood to be tested from a collection container to an assay device without opening the collection container. This platelet aggregation assay can be conducted at the same time as the activated clotting time (ACT), which is performed to assess the adequacy of heparinization.
  • ACT activated clotting time
  • aspirin is a relatively weak platelet anti-aggregation drug, it has been used as one of the first-line therapeutic options in the treatment of cardiovascular diseases.
  • Several clinical trials have proven its efficacy in primary and secondary prevention of occlusive cardiovascular events.
  • Aspirin exerts its effect by blocking only one of the pathways that lead to platelet aggregation. It acts by producing an irreversible inactivation of the enzyme prostaglandin G/H synthase that is necessary for the conversion of arachidonic acid to thromboxane A2 that ultimately stimulates platelet aggregation and vascular constriction.
  • prostaglandin G/H synthase that is necessary for the conversion of arachidonic acid to thromboxane A2 that ultimately stimulates platelet aggregation and vascular constriction.
  • aspirin has been the mainstay of anti-platelet therapy for several decades, it is acknowledged as a relatively weak inhibitor of platelet activity.
  • GPIIb/IIIa after platelet activation is quite interesting (Valettas, et al., Aspirin resistance using flow cytometry. Thrombosis, Thrombolytic Therapy and Anticoagulants. 1998; Abstract 3268), but again depends on very expensive instrumentation and very complicated procedures.
  • Clopidogrel and ticlopidine are thienopyridine derivatives that inhibit platelet aggregation. They are believed to inhibit the binding of adenosine-5-diphosphate (ADP) to its receptor.
  • Clopidogrel and ticlopidine are FDA approved anti-platelet drugs and inhibit platelet activation by blocking the P2Y12 ADP receptor.
  • the pharmacological activity of clopidogrel is very similar to the pharmacological activity of ticlopidine. However, the adverse effects of clopidogrel are far less severe than those of ticlopidine.
  • cPG cationic propyl gallate
  • U.S. Patent Nos. 5,700,634 and 5,709,889 cationic propyl gallate
  • cPG was shown to exhibit, in optical aggregometry, a very wide separation of rates of platelet aggregation between baseline and aspirin-treated PRP samples.
  • Propyl gallate/metal ion platelet activation has been used in various plasma assays to measure or assess platelet reserve, i.e., the qualitative and quantitative platelet excess necessary for normal coagulation, activated plasma clotting ti ⁇ fe (aPCT), and other plasma-based assays.
  • a considerable disadvantage of Jhese assays is that they require significant sample manipulation and take gpgst ⁇ f -than about thirty minutes to perform.
  • One embodiment of the present invention is a method for conducting an assay for platelet function activity on a blood sample.
  • a combination is provided to form a reaction medium.
  • the combination comprises the sample, a particle reagent comprising a GP Ilb/IIIa receptor ligand covalently attached to particles, a polyhydroxy aromatic compound and a metal cation.
  • the combination is incubated under conditions for agglutinating the particles.
  • the extent of agglutination of the particles is determined where the extent thereof is related to the platelet function activity of the sample.
  • compositions comprising (i) a particle reagent comprising fibrinogen covalently attached to particles, (ii) a lower alkyl ester of trihydroxybenzoic acid, and (ii) a divalent transition metal ion.
  • the composition is in the form of a lyophilized pellet.
  • Another embodiment of the present invention is an apparatus comprising a container for receiving a sample and a composition as described above, preferably, in lyophilized form.
  • the composition may comprise additional reagents such as, for example, buffers, lyophilization stabilizers and the like.
  • activator compositions comprising certain trihydroxy aromatic compounds and a metal cation are useful agonists in measuring inhibition of platelet aggregation by antiplatelet therapeutic agents such as aspirin, ADP antagonists, e.g., thienopyridines, and so forth, in whole blood samples.
  • antiplatelet therapeutic agents such as aspirin, ADP antagonists, e.g., thienopyridines, and so forth.
  • the aforementioned compositions may be employed to determine the effectiveness of antiplatelet therapy involving treatment of patients with aspirin or an ADP agonist such as a thienopyridine.
  • the above compositions may be employed in conjunction with particles coated with a GP Ilb/IIIa receptor ligand and any other reagents necessary for conducting an assay for the efficacy of aspirin or thienopyridines.
  • a lyophilized reagent composition may be used that comprises the aforementioned activator compositions and particles.
  • a metered volume of a sample to be measured such as whole blood is mechanically mixed with the lyophilized reagent.
  • a change in light transmission is monitored and an index of platelet activity is calculated.
  • a whole blood sample is combined in a cuvette or a unitized cartridge with the aforementioned lyophilized reagent.
  • An apparatus may be employed for carrying out the assay.
  • the apparatus comprises a well for receiving the sample where the well contains the lyophilized reagent and other reagents for conducting the assay.
  • the additional reagents may be various buffers and/or lyophilization stabilizers.
  • the present invention is directed to a method for conducting an assay for platelet function activity on a whole blood sample.
  • the sample is one that has been affected by an antiplatelet therapeutic agent.
  • antiplatelet therapeutic agent agents or drugs that interfere with the ability of platelets to aggregate and form a platelet plug.
  • antiplatelet therapeutic agents include aspirin or an adenosine-5 -phosphate (ADP) antagonist.
  • the sample may be from a patient undergoing treatment with aspirin or an adenosine-5 -phosphate (ADP) antagonist.
  • ADP adenosine-5 -phosphate
  • a combination is provided in an assay medium where the combination comprises the sample and a polyhydroxy aromatic compound in conjunction with a metal cation.
  • Suitable polyhydroxy aromatic compounds include, by way of illustration and not limitation, lower alkyl esters of polyhydroxybenzoic acids, tannin, and the like.
  • Lower alkyl means a straight or branched chain hydrocarbon moiety comprising from 1 to about 9, usually, about 2 to about 5, preferably, about 3 carbon atoms.
  • lower alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and any appropriate iso-, tert-, neo-, sec-, and other forms thereof such as, for example, isopropyl, isobutyl, sec-butyl, tert-butyl, and so forth.
  • number of hydroxy groups on the benzoic acid is about 1 to about 4, preferably, about 3.
  • a preferred polyhydroxy aromatic compound is propyl gallate.
  • the amount of the polyhydroxy aromatic compound employed in the reaction medium is that which is effective in achieving activation of the platelets so that an accurate assessment may be made of the activity of the platelets.
  • the amount of the polyhydroxy compound is dependent on the type of sample. For example, the optimal propyl gallate concentration in platelet rich plasma (PRP) optical aggregometry was determined to be about 30 ⁇ M.
  • the optimal propyl gallate concentration for whole blood RPFA-PG differs for at least two reasons: (i) the sample is different and red cells in whole blood might adsorb some propyl gallate, which is hydrophobic, and (ii) propyl gallate was specially prepared to remove extra unwanted salts (which is in the regular SPAT, Slide Platelet Aggregation Test, reagent) so as to not cause aggregation in a human fibrinogen-coated bead dispense reagent.
  • the range of the amount of the polyhydroxy aromatic compound for all types of samples is about 20 to about 5000 ⁇ M, more usually, about 100 to about 1000 ⁇ M, preferably, about 200 to about 450 ⁇ M.
  • the metal cation employed in the present compositions is any suitable cation that in conjunction with the polyhydroxy aromatic compound achieves the desired platelet activation effect.
  • a preferred metal cation is a divalent metal cation, usually, a divalent transition metal ion such as, for example, Ni(II), Cu(II), Co(II), and the like.
  • the amount of the metal cation employed in the reaction medium is that which is effective, in conjunction with the polyhydroxy aromatic compound, in achieving activation of the platelets so that an accurate assessment may be made of the activity of the platelets.
  • the amount of the metal cation is about 1 to about 5000 ⁇ M, more usually, about 5 to about 1000 ⁇ M, preferably, about 10 to about 40 ⁇ M.
  • a reagent comprising particles coated with a compound that can result in the specific agglutination of platelets, i.e., the agglutination of platelets by the specific interaction between a receptor on the platelets and the compound on the particles.
  • a compound that can result in the specific agglutination of platelets, i.e., the agglutination of platelets by the specific interaction between a receptor on the platelets and the compound on the particles.
  • Such compounds include, by way of illustration and not limitation, antibodies to a platelet receptor and GPIIb/IIIa receptor ligands, which may be a small organic molecule, polypeptide, protein, monoclonal antibody or nucleic acid that binds, complexes or interacts with GPIIb/IIIa receptors on the platelet surface.
  • GPIIb/IIIa receptors on the surface of platelets bind, complex or otherwise interact with the GPHb/IIIa receptor ligands on the particles.
  • Typical GPIIb/IIIa ligands include fibrinogen, monoclonal antibody 10E5 (Coller, et al, J. Clin. Invest. 72:325 (1983)), monoclonal antibody c7E3 (The EPIC investigators, N.E.
  • the particles to which the compound is attached are at least about 0.1 microns and not more than about 20 microns. In one embodiment the particles are about 0.1 microns to about 10 microns. In another embodiment the particles are at least about 1 micron and less than about 8 microns.
  • the particles can be virtually any shape, but are generally spherical with uniform diameters.
  • the particle may have any density, but preferably of a density approximating water, generally from about 0.7 to about 1.5g/ml.
  • the particles may or may not have a charge on the surface, either positive or negative, preferably negative.
  • the particles are functionalized or functionalizable so as to covalently bind or attach sbp members at their surface, either directly or indirectly.
  • the particles may be solid ⁇ e.g., comprised of organic and inorganic polymers or latex), oil droplets (e.g., hydrocarbon, fluorocarbon, silicon fluid), or vesicles (e.g., synthetic such as phospholipid or natural such as cells and organelles).
  • the solid particles are normally polymers, either addition or condensation polymers, which are readily dispersible in a liquid medium. Examples of suspendable particles are polymeric materials such as latex, lipid bilayers, oil droplets, cells and hydrogels.
  • Other particle compositions include polymers, such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, polyacrylate, polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, poly( vinyl butyrate), polysaccharides such as dextrans and modified dextrans, etc.; either used by themselves or in conjunction with other materials.
  • the solid particles can be comprised of polystyrene, polyacrylamide, homopolymers and copolymers of derivatives of acrylate and methacrylate, particularly esters and amides, silicones and the like.
  • the compound is coated on the particles.
  • the compound is covalently attached to particles.
  • Such covalent attachment can be accomplished by well-known techniques, commonly available in the literature. See, for example, “Immobilized Enzymes,” Ichiro Chibata, Halsted Press, New York (1978) and Cuatrecasas, J. Biol. Chem., 245:3059 (1970).
  • the surface of the particle may be polyfunctional or be capable of being polyfunctionahzed.
  • a wide variety of functional groups are available or can be incorporated. Functional groups include carboxylic acids, aldehydes, amino groups, cyano groups, ethylene groups, hydroxyl groups, mercapto groups and the like.
  • the manner of linking a wide variety of compounds to surfaces is well known and is amply illustrated in the literature (see above).
  • the attachment of the sbp member may be directly by a bond or indirectly through the intermediacy of a linking group.
  • the length of a linking group may vary widely, depending upon the nature of the sbp member and of the particle.
  • the ratio of molecules of compound to particle is controlled in the attachment of the molecules of compound to the particle.
  • the number of functionalized sites on the surface of the particle may be controlled by adjusting the number of such sites introduced on the surface of the particle.
  • the ratio of molecules of compound to particle may be controlled by adjusting the concentration of the compound in the reaction medium for the attachment.
  • the particle reagent employed in the present invention may be treated with a sufficient amount of material to block areas of adsorption on the particles. Such materials will not affect the functioning of the particles for their intended purpose in the present invention.
  • the blocking materials include proteins such as bovine serum albumin, bovine gamma globulin, etc., polysaccharides such as dextran, etc., and the like.
  • particles are employed wherein the number of functionalized sites for attachment substantially reduce the adsorption area on the surface of the particles.
  • the particles usually comprise a label, either attached thereto or incorporated therein.
  • the label may be any moiety that may be used for the purpose of detection.
  • the label is often a member of a signal producing system.
  • the label is capable of being detected directly or indirectly.
  • the label can be isotopic or nonisotopic, usually non- isotopic, and can be a dye, fluorescent molecule, chemiluminescent molecule, a catalyst, such as an enzyme, a polynucleotide coding for a catalyst, promoter, coenzyme, enzyme substrate, radioactive group, a small organic molecule, amplifiable polynucleotide sequence, and so forth.
  • the particles contain one or more dyes that absorb in the infrared.
  • “Infrared” means electromagnetic radiation at wavelengths longer than the red end of visible light and shorter than microwaves. Visible light has wavelengths of about 400 to about 700 nm. Thus, light with a wavelength longer than about 700 nm and shorter than about 1000 nm is generally recognized as near infrared. In a preferred embodiment, the wavelength is about 800 nm.
  • Such dyes include bacteriochlorin, bacteriochlorophytin, meropolymethine dyes, benzoannulenes, vinylogous porphorins, polymethine dyes, cyanines and merocyanines, and the like.
  • Specific dyes of interest are Copper(fl)-tetra-tert-butyl- tetrakis(dimethylamino)-29H-3 lH-phthalocyanine and Vanadyl-tetra-tert-butyl- tetrakis(dimethylamino)-29H-31H-phthalocyanine.
  • the particular dye that is selected is one of convenience, availability, stability, compatibility with the particle and the like. These dyes may be incorporated directly into the particle itself, through polymerization or passive adsorption. The dyes may be loaded individually (i.e., sequentially) or in combination (i.e., simultaneously).
  • the dyes may be linked to the bead in combination with the linking component, such that they do not leach from the surface.
  • the conditions are such that the particle surface is unaffected with respect to the ability to agglutinate under appropriate conditions.
  • the dyes absorb light in the range of about 750 nm - 900 nm, particularly in the range of about 750 - 850 nm. For samples with high levels of red blood cells, the light is at about 800 nm ⁇ 10 nm, which is the isobestic point for oxyhemoglobin and reduced hemoglobin.
  • the amount of dye employed with the particles varies with the extinction coefficient of the dye in the light range of interest, the required sensitivity of the assay, the size of the particles, the mode of binding of the dye to the particles, compatibility of the dye with the particle matrix, and the like. Usually, the amount of dye incorporated is in the range of about 1 to 20 weight percent, more usually 5 to 15 weight percent.
  • Dyes which find a particular use in the present invention are phthalocyanines.
  • transition metals and phthalocyanines are chemically very stable to light and heat. They are formed by condensation of opthalodinitriles in the presence of an appropriate metal.
  • metals used in the formation of the metalophthalocyanines besides the copper (Cu) and the Vanadium (V) are magnesium (Mg), zinc (Zn), and cobalt (Co).
  • carboxylated microparticles with a flat absorption maximum are employed. These microparticles are prepared by incorporating multiple dyes that have distinct absorption maximum close to 805 nm. This results in a flat maximum absorption spectrum across a broad range wavelength from 780-820 nm.
  • the sample may be any solution, synthetic or natural, to be analyzed where the sample has been subject to an effect from an antiplatelet therapeutic agent.
  • sample includes biological tissue, including body fluids, from a host, and so forth.
  • the sample can be examined directly or may be pretreated, usually.
  • the present invention has particular application to samples that comprise platelets, including body fluids such as, for example, whole blood, platelet-containing blood fractions such as plasma, and the like. In one embodiment the invention has particular application to whole blood samples.
  • the amount of the sample depends on the nature of the sample. For fluid samples such as whole blood, the amount of the sample is usually about 30 ⁇ l to 5000 ⁇ l, preferably, about 100 to 300 ⁇ l.
  • sample includes unprocessed samples directly from a patient or samples that have been pretreated and prepared in any convenient liquid medium, usually an aqueous medium.
  • the medium for conducting the assays in accordance with the present invention is an aqueous medium.
  • Other polar cosolvents may also be employed in the medium, usually oxygenated organic solvents of from 1-6, more usually from 1-4 carbon atoms, including alcohols, ethers and the like. Usually, such cosolvents are present in less than about 70 weight percent, more usually, in less than about 30 weight percent.
  • various ancillary materials are frequently employed in the method in accordance with the present invention.
  • buffers are normally present in the assay medium, as well as stabilizers for the assay medium and the assay components; surfactants, particularly non-ionic surfactants; binding enhancers, e.g., polyalkylene glycols; or the like.
  • the pH for the medium is usually in the range of about 2 to about 11, preferably, about 4 to about 9.
  • Various buffers may be used to achieve the desired pH and maintain the pH during the method.
  • Illustrative buffers include HEPES, borate, phosphate, carbonate, Tris, barbital, and the like.
  • the particular buffer employed is not critical to the method but one buffer may be preferred over others in certain circumstances. In some circumstances HEPES is preferred and is present at a concentration of about 0.05M to about 0.001M but generally at a concentration of about 0.01M.
  • the volume of assay medium is about 25 to about 500 microliters, usually about 75 to about 250 microliters.
  • the assays may be carried out in any suitable container.
  • the container is a cuvette or cartridge that is used with the instrument for carrying out the assay and measuring the assay results.
  • the reaction container usually contains the particle reagent in accordance with the present invention in dry lyophilized form together with other reagents such as an activation initiator and the like, stabilizers and so forth.
  • the amount of the particle reagent is about 1 x 10 6 to about 5 x 10 9 particles/milliliter.
  • sample and particle reagent is incubated under conditions for agglutinating the particles.
  • Moderate temperatures are normally employed for carrying out the method.
  • the temperature may be constant or may vary. Usually, a constant temperature is employed during the reaction step.
  • the temperature employed is usually about 10 to about 80°C, more usually, about 15 to about 45°C, preferably, the temperature should be at least 25°C, more preferably in the range of about 30 to about 40°C, usually about 37°C.
  • the extent of agglutination of the particles is determined and is related to the presence and/or amount of the member in the sample.
  • the presence of agglutination may be determined visually by observing clumping of the particles, which would indicate agglutination.
  • the particles may be colored to aid in visualizing agglutination or clumping of the matrix.
  • the extent of agglutination may be measured spectrophotometrically, turbidimetrically, nephelometrically, etc., by observing the rate of change of optical density of the medium, and so forth.
  • an assay for platelet function activity is conducted on a whole blood sample from a patient undergoing treatment with an antiplatelet therapeutic agent.
  • the sample is combined in a suitable container, e.g., reaction cuvette, with fibrinogen coated particles, a lower alkyl ester of a trihydroxybenzoic acid, e.g., propyl gallate, and a metal cation, e.g., Ni(II) ion, to form an assay medium.
  • the particles of the particle reagent have one or more infrared dyes incorporated therein.
  • the combination is subjected to agglutination conditions.
  • the medium is irradiated with light in the infrared region.
  • the transmission of infrared light from the assay mixture is determined where the level of transmission is related to platelet function activity.
  • the agglutination medium is selected to have high absorption at about 800 nm.
  • the ratio between the agglutination medium absorption coefficient and whole blood absorption coefficient should preferably be greater than about 4:1 at about 800 nm.
  • the absorption ratio for a particular assay is a function of both the absorption coefficient of the agglutination medium and the concentration of the agglutination medium in the assay sample.
  • the sample After the sample has been combined with the reagents, desirably it will be heated to a temperature above room temperature, but below that which would interfere with the assay, so as to insure that the temperature can be controlled without adversely affecting the assay result. Desirably, the temperature should be at least 25°C, preferably in the range of 30-40°C, more preferably about 37°C.
  • the reaction medium is usually gently agitated upon combining of the reagents with the sample and during the period of the reaction. Agitation is sufficient to achieve and maintain homogeneity in the assay samples.
  • the total time of the readings from the zero time may range from about 10 sec. to 5 min., more usually about 30 sec. to 5 min., and preferably about 30 sec. to 2 min.
  • the data may be analyzed by any convenient means, particularly using an algorithm that can manipulate the data in relation to calibrators and/or controls.
  • the level of agglutination is an indication of the platelet function activity of the sample tested.
  • the level of agglutination may be compared against a standard of known platelet function activity. Usually, the result will be compared to a calibrator, which may be performed concomitantly or have been performed previously or may be provided as a standard curve.
  • the method of the present invention may be employed in conjunction with an assay for platelet count such as that described in U.S. Patent Application Serial No. 09/177,884 filed October 23, 1998 (the '884 application), the relevant disclosures of which are incorporated herein by reference.
  • the above assays preferably may be conducted in a device, which allows the reactions in accordance with the present invention to occur and which measures the results thereof.
  • the instrument should assess platelet function based upon the ability of activated platelets to bind fibrinogen. As activated platelets bind and agglutinate fibrinogen-coated particles, there is an increase in light transmittance.
  • an instrument to measure the result of the assay is one that can measure agglutination.
  • the instrument measures a change in optical signal due to agglutination.
  • Suitable instruments include, by way of illustration and not limitation a kinetic spectrophotometer, ULTEGRA SYSTEM® instrument (commercially available from Accumetrics, San Diego, CA and employed for rapid platelet function activity measurements on normal samples), or the like.
  • the ULTEGRA SYSTEM® instrument is a turbidometric based optical detection system, which measures platelet induced aggregation as an increase in light transmittance.
  • the system consists of an analyzer, disposable cartridge and controls.
  • the cartridge contains reagents based on microparticle agglutination technology.
  • the quality control system includes an electronic control, two levels of assayed "wet" controls (WQC), an in-cartridge humidity sensor, an in-packaging temperature indicator, and a test for concurrence of two assay channels.
  • the analyzer controls assay sequencing, establishes the assay temperature, controls the reagent-sample mixing for the required duration, determines the degree of platelet function, displays the result and performs self-diagnostics.
  • the test cartridge of the system contains a lyophilized preparation comprising particles with covalently attached GPIIa/IIIb receptor ligand, a polyhydroxy aromatic compound, a metal cation, preservative and buffer.
  • the patient sample is usually citrated whole blood, which is automatically dispensed from the blood collection tube into the cartridge by the analyzer, with no blood handling required by the user.
  • the interaction is monitored by the infra red absorbency characteristics of the particles.
  • the agglutination of the particles is measured through the optical system of the ULTEGRA SYSTEM® instrument.
  • the agglutination is detected as an increase in the transmission of infrared light through the sample.
  • the reaction kinetics are analyzed and translated into "Platelet Aggregation Units", PAU.
  • kits comprising in packaged combination a lyophilized preparation comprising particles with covalently attached fibrinogen, a lower alkyl ester of trihydroxybenzoic acid, a divalent transition metal ion, preservative and buffer.
  • the lyophilized preparation may be present in a reaction container such as a cartridge used in the instrument of analysis.
  • the lyophilized preparation may be placed in the outer wells of the four- well cartridge used in the analyzer.
  • the kit may also include a sample collection container and/or a device for carrying out the present method.
  • the relative amounts of reagents may be varied widely to provide for concentrations in solution of the reagents that substantially optimize the sensitivity of a determination.
  • the reagents can be placed in an airtight package in order to maintain the activity of any reagents.
  • the package may be, for example, a bag, pouch, or the like fabricated from a material that is substantially non-permeable to moisture. Such materials include, by way of example and not limitation, plastic, aluminum foil, and the like.
  • the kit may also include an article for piercing a person's skin, disinfectant or sterilizing pads and so forth.
  • the kit may also include calibrators and standards.
  • the kit may also include one or more reagents for conducting an assay for platelet count.
  • aspirin resistance or “aspirin resistant” means that the rate of platelet aggregation in the presence of aspirin is substantially unaffected compared to the rate of aggregation in the absence of aspirin.
  • a blood sample is assayed by three methods (Aggregometry- PG, Aggregometry-EPI and RPFA-PG) in duplicate, if at least half of the test results (total of 6; 3 methods in duplicate) are positive, i.e., above the cut-off for the respective assay, then the platelets in the sample are said to be "aspirin resistant.”
  • aspirin sensitivity means that an extremely low steady state platelet activity is maintained in response to aspirin administration.
  • Aspirin irreversibly inactivates cyclooxygenase (COX).
  • COX cyclooxygenase
  • the liver keeps making more of the enzyme to replenish it.
  • a steady state is reached in which the platelet activity is kept at a certain low level.
  • this steady state platelet activity becomes extremely low, causing bleeding problems.
  • the optimal PG concentration for whole blood RPFA-PG was determined. Rates of agglutination were obtained from running baseline and aspirin-treated whole blood (3.2% citrate) samples with increasing concentrations of spiked PG using cartridges, cuvettes or the like containing all necessary ingredients but any activators. The optimal PG concentration (or range) should be where there is maximum differential in rates of agglutination between baseline (higher rates) and aspirin-treated samples (lower rates).
  • Typical time courses of PG-induced RPFA agglutination in pre- and post-aspirin blood samples at two PG concentrations are shown in Figure 1.
  • the optimal PG concentration for RPFA in whole blood was significantly higher than that for aggregometry in PRP (about 30 ⁇ M).
  • the RPFA PG-activation time - the time at which maximum slope is found - decreases with increasing PG concentration; lower PG concentration resulted in unnecessarily long activation time and, therefore, long assay time.
  • higher PG concentration caused higher rate of post-aspirin aggregation and, therefore, less differentiation between pre- and post- aspirin rates of platelet aggregation.
  • the reagent cartridges were prepared in a manner similar to that for preparation of reagent cartridges for RPFA-iso-TRAP assays on the ULTEGRA SYSTEM® instrument except PG was used instead of iso-TRAP and no HEPES saline was added to the dispense reagent.
  • the detection well of the RPFA-PG cartridges contained PG, fibrogen-coated infrared dyed particles, buffers and lyophilization stabilizers.
  • a preferred buffer was HEPES. HEPES was added to a final concentration of 10-20 mM after reconstitution with the whole blood sample.
  • ASA Acetyl Salicylic Acid
  • the algorithm used was a moving window linear regression (with a width of 40 seconds).
  • the maximum rates of agglutination were obtained in three consecutive time zones: Zone I (40-180 seconds), Zone II (180-360 seconds) and Zone III (360-510 seconds). If either the maximum rate from Zone I was greater than 95 mOD/min or that from Zone II was greater than 70 mOD/min, then the maximum of the three maximum rates was obtained. If neither held, the maximum rate from Zone III was used. The results reported herein were obtained using the maximum rate from Zone III unless otherwise noted. For purposes of comparison, two PRP aggregometry methods were studied. The study of the rates of agglutination or platelet aggregation as a function of increasing ASA concentrations provided the basis of comparison.
  • a stock solution of 15 mM ASA in PBS was prepared. Appropriate amounts were then added to blood pools to final ASA concentrations at 0, 1.5, 3, 6, 15 and 150 ⁇ M.
  • PRP samples were then prepared for the aggregometry assays: for aggregometry-PG, 44.4 ⁇ L of SPAT Reagent was added to 400 ⁇ L of PRP to attain a final [PG] of 30 ⁇ M; for aggregometry-EPI, 44.4 ⁇ L of a 500 ⁇ M epinephrine solution was added to 400 ⁇ L of PRP to attain a final [EPI] of 50 ⁇ M.
  • Blood was first drawn from a donor who had not taken aspirin or any NS AID medicines in the past 10 days; this was the pre-aspirin (or baseline) sample. A 500 mg tablet of Bayer aspirin was then given to the donor and ingested. A minimum of 2 hours later, blood was drawn again; this was the post-aspirin sample. All three methods - aggregometry-PG, aggregometry-EPI and RPFA-PG - were used to assess platelet activities.
  • RPFA-PG rates, Aggregometry-PG and Aggregometry-EPI slopes were obtained for pre- and post-aspirin blood samples from normal, healthy donors (11 males, of which 3 were tested twice; 6 females, of which one was tested twice).
  • Two kinds of RPFA-PG cartridges were used, with final PG concentration in blood at 250 and 417 ⁇ M, respectively (3 of the 11 male donors were not tested using the former). The latter did seem to increase the RPFA-PG rate of post-aspirin samples relative to that of pre-aspirin ones and, therefore, exhibited less specificity.
  • the RPFA-PG rate differentiation between pre- and post-aspirin samples seemed to be somewhat better using cartridges with the lower PG concentration.
  • the word "maturing” refers to the settling of PG-activation time to a minimum with concomitant settling of RPFA rate to a maximum. Frequently, when replicate blood samples were run, the latter ones tended to have shorter PG activation times and higher RPFA-PG rates.
  • Donor 16 was a female, only pre-aspirin blood was studied; Donor 4 was a male, both pre- and post-aspirin blood samples were studied.
  • IRB approval in a clinical setting was obtained to run pre- and post-aspirin studies on 30 normal donors and to run RPFA-PG on 270 CAD patients that are on aspirin regimen.
  • Two 4.5 mL Becton Dickinson Vacutainer tubes and two 2 mL Greiner tubes were filled in each drawing.
  • the 4.5 mL tubes were processed to obtain PRP and PPP for running the aggregometry-PG and -EPI assays and the 2 mL tubes were used directly to run RPFA-PG assays.
  • a pre- and post-aspirin RPFA-PG rate distribution was constructed from the data.
  • a number of aspirin-resistant CAD patients were identified. It should be noted that this study was carried out in mid-December in Salt Lake City with a mean altitude of around 4500 ft. In general, it was found that all rates of platelet aggregation were significantly slower there than in the warmer climates and lower elevations of San Diego using the same ULTEGRA SYSTEM® instrument (the Chronolog aggregometer was different) and the same lots of reagents.
  • the mean pre- aspirin aggregometry slopes were 185 (agg.-PG) and 88 (agg.-EPI) and RPFA-PG rate was 195 in San Diego, whereas the corresponding numbers were 148 (agg.-PG), 62 (agg.-EPI) and 152 (RPFA-PG) in SLC.
  • One subject had aggregometry slopes of 209 (agg.-PG) and 95 (agg.-EPI) and RPFA-PG rate of about 200 when tested in San Diego, but the slopes were 142 (agg.-PG) and 40 (agg.-EPI) and the RPFA-PG rate was about 100 when tested three days after arrival in Salt Lake City.
  • Bar graphs of pre- and post-aspirin RPFA-PG rates for the normal donors are shown in Figures 19. Genders of donors can be found below the donor ID's of the top graph. Distributions of pre- and post- aspirin RPFA-PG rates along with those of patients are shown in Figures 20 and those for Aggregometry-PG and Aggregometry-EPI slopes are shown in Figures 21. If 60 was used as cutoff in RPFA-PG, the following aspirin-resistant patients were identified: # 1, 2, 3, 4, 5, 8, 9, 18, 21, 23, 25 and 28, of which # 8, 18 and 28 were resistant in one of two replicates.
  • the aspirin-resistant patients identified were # 1, 2, 3, 4, 6, 11, 12, 18, 20, 21, 24 and 25 as aspirin- resistant, of which # 21 was resistant in one of two replicates.
  • the aspirin-resistant patients identified were # 1, 2, 3, 4, 5, 15, 21, 23, 25 and 28 as aspirin-resistant, of which # 1, 15, 23 and 25 were resistant in one of two replicates. Since there is no single reference assay at present for aspirin resistance, it is perhaps more reliable to identify a patient as aspirin-resistant if at least half of the test results (total of 6: 3 methods x 2 replicates) are positive. Application of the latter approach identified, as true aspirin-resistant, the following 10 patients: # 1, 2, 3, 4, 5, 18, 21, 23, 25 and 28. The sensitivities, specificities and accuracy of the three methods were then calculated and are shown in the following table (Table 1). Table 1
  • Carboxylated particles (Interfacial Dynamics (IDC), Portland OR) with an absorbency spectrum of 780-830nm were covalently linked to fibrinogen to facilitate measurements of bead agglutination in whole blood.
  • the conjugation procedure started with activation of the carboxylated particles.
  • the particles were first reacted with an ethyl-carbodiimide (EDC) solution at lOmg/mL and an n-hydroxysuccinimide (NHS) solution at 50mg/mL during a 30-minute incubation. Following the incubation, the particles underwent several centrifugation steps with 0.01M MES, pH 6.0 (Calbiochem- Novabiochem Corporation; La Jolla, CA) buffer, to remove excess EDC and NHS.
  • EDC ethyl-carbodiimide
  • NHS n-hydroxysuccinimide
  • the activated particles were incubated with fibrinogen for 2 hours. Fibrinogen became covalently linked to the particle through the attached EDC and produces a NHS-ester leaving group. The final fibrinogen conjugate was then washed several times with 20mmol/L HEPES, pH7.4
  • the fibrinogen conjugate was combined with 20mmol/L HEPES, pH 7.4, containing trehalose, bovine serum albumin (BSA), mannitol, sodium azide, and the propyl gallate reagent (American Control System, Fishers IN, containing 250 ⁇ M propyl gallate and corresponding metal cation) as an agonist to produce the pellet dispense reagent.
  • the pellet dispense reagent was used to produce lyophilized pellets. These pellets were used as platelet function reagents to measure the activity of the platelets in whole blood samples from a patient being treated with clopidogrel. Pellets were produced by dispensing 0.0125mL of the pellet dispense reagent into liquid nitrogen.
  • the pellets froze and sunk to a collecting tray in the bottom of a nitrogen reservoir.
  • the frozen pellets were transferred to a frozen lyophilizer tray and freeze dried over a 24- hour cycle. Once the cycle was complete, the pellets were stored in a moisture-free environment at room temperature.
  • the pelletized reagent was transferred to the mixing chamber of the cartridge of the Ultegra® System together with one steel-mixing ball.
  • the cartridge contained four mixing chambers. One pellet and one mixing ball were placed in the two outer mixing chambers of the cartridge.
  • the mixing chamber was welded to the cartridge manifold and a needle was inserted onto the cartridge. Cartridges were sealed in a pouch containing one desiccant packet.
  • Blood samples from a patient under going anti-platelet therapy with clopidogrel (in anti-coagulant) were introduced into a cartridge for the Ultegra® System analyzer.
  • the cartridge volume was 160 microliters and contained the particle reagents prepared as described above.
  • the cartridges were agitated during reconstitution and during the assay.
  • the Ultegra System analyzer performed the operations of incubating, agitating and recording absorption readings according to its intended purpose.
  • the apparatus measured the rate of change of light transmittance.
  • Blood was drawn from normal, healthy subjects into blood collection tubes containing 3.2% citrate following a 3.0 mL discard tube. Baseline platelet function was tested using RPFA assay with Propyl Gallate as an agonist. An additional 2.0 mL EDTA tube was collected to determine baseline hematocrit and platelet count using Coulter Act-8 counter. After determining baseline platelet function, hematocrit and platelet count, the donor was given a 300 mg loading dose of clopidogrel. Blood was collected and processed in the same manner as above approximately 24 hours after ingestion of clopidogrel. Platelet function, hematocrit and platelet count was measured for post clopidogril sample. Percent inhibition of platelet function due to clopidogril was calculated as [(1-post clopidogril/baseline)*100]
  • the difference in values is expressed as a percent of the baseline value:

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Abstract

L'invention concerne des méthodes, des compositions et des kits permettant de déterminer l'activité des plaquettes sur des échantillons, par exemple de sang entier, affectés par l'exposition à un agent thérapeutique anti-plaquettes tel que, par exemple, l'aspirine ou une thienopyridine. Une combinaison est générée dans un milieu de réaction, ladite combinaison comprenant l'échantillon ainsi qu'un réactif particulaire comprenant un ligand récepteur GP IIb/IIIa tel qu'un fibrinogène lié par covalence aux particules. Le milieu comprend également un composé aromatique polyhydroxy et un cation métallique. La combinaison est incubée dans des conditions d'agglutination des particules. Le niveau d'agglutination des particules est déterminé et est associé à l'activité fonctionnelle des plaquettes de l'échantillon. Ces méthodes et ces compositions sont appliquées, en particulier, dans la détection de la sensibilité des plaquettes à une thérapie grâce à l'aspirine ou à une thienopyridine, et l'évaluation de l'efficacité d'une telle thérapie.
PCT/US2002/002102 2001-01-24 2002-01-23 Methodes permettant de determiner l'activite des plaquettes a l'aide de compositions anti-plaquettes WO2002059623A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005113006A3 (fr) * 2004-05-13 2007-02-08 Boehringer Ingelheim Int Utilisation de dipyridamole pour le traitement de la resistance a des inhibiteurs plaquettaires
CN104792982A (zh) * 2015-03-10 2015-07-22 山东盛百灵医药科技有限公司 一种阿司匹林耐药性监测试剂
US20160320415A1 (en) * 2013-12-16 2016-11-03 Xen Biofluidx, Inc. Fast reconstituting reagent pellets
EP4118219A4 (fr) * 2020-03-11 2024-04-17 Shanghai Belief-Delivery Biomed Co., Ltd. Nouvelle utilisation d'un composé d'aspirine pour augmenter l'expression d'acides nucléiques

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WO2000073792A1 (fr) * 1999-05-28 2000-12-07 Sendx Medical, Inc. Temoin pour methodes de determination de numeration des plaquettes et de fonction plaquettaire

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005113006A3 (fr) * 2004-05-13 2007-02-08 Boehringer Ingelheim Int Utilisation de dipyridamole pour le traitement de la resistance a des inhibiteurs plaquettaires
JP2007537184A (ja) * 2004-05-13 2007-12-20 ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング 抗血小板剤に対する耐性を治療するためのジピリダモールの使用
US20160320415A1 (en) * 2013-12-16 2016-11-03 Xen Biofluidx, Inc. Fast reconstituting reagent pellets
CN104792982A (zh) * 2015-03-10 2015-07-22 山东盛百灵医药科技有限公司 一种阿司匹林耐药性监测试剂
EP4118219A4 (fr) * 2020-03-11 2024-04-17 Shanghai Belief-Delivery Biomed Co., Ltd. Nouvelle utilisation d'un composé d'aspirine pour augmenter l'expression d'acides nucléiques

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