WO2009073861A1 - Procédés et dispositifs servant à neutraliser l'héparine dans un échantillon - Google Patents

Procédés et dispositifs servant à neutraliser l'héparine dans un échantillon Download PDF

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Publication number
WO2009073861A1
WO2009073861A1 PCT/US2008/085745 US2008085745W WO2009073861A1 WO 2009073861 A1 WO2009073861 A1 WO 2009073861A1 US 2008085745 W US2008085745 W US 2008085745W WO 2009073861 A1 WO2009073861 A1 WO 2009073861A1
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Prior art keywords
test strip
sample
reagent
heparin
heparin neutralizing
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PCT/US2008/085745
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English (en)
Inventor
Alfred Greenquist
Ulka Parikh
Hyejung Lee
Conan Li
Ji LIN
Terrence Andreasen
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Hemosense Inc
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Hemosense Inc
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Publication of WO2009073861A1 publication Critical patent/WO2009073861A1/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
    • G01N33/483—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

Definitions

  • Heparin is a negatively charged polymeric molecule ranging in molecular weight from 3 to 40 kDa. It is commonly used therapeutically as an anticoagulant to reduce the risk of blood clotting in various coagulation disorders and during surgical procedures such as coronary bypass, coronary intervention, and heart valve implantation. Its biological mechanism of action involves binding to antithrombin (AT) and thereby amplifying the ability of AT to inhibit the function of other coagulation enzymes, particularly thrombin and factor Xa.
  • AT antithrombin
  • Heparin as unfractionated heparin or low molecular weight heparin (LMWH), may be used as the sole anticoagulant in therapy or during surgery, but is often used together with other anticoagulants such as warfarin (Coumadin®), aspirin or platelet inhibitors.
  • LMWH low molecular weight heparin
  • Anticoagulation therapy requires striking a balance between administering an effective amount of anticoagulant and simultaneously keeping the risk of bleeding low. For this reason, patients who are on anticoagulation therapy benefit greatly from being able to accurately monitor the coagulation properties of their blood. Convenient and reliable tests to allow monitoring the coagulation properties of blood are thus highly desirable.
  • Several tests have been established to measure coagulation including the prothrombin time test (PT), the activated clotting time test (ACT), the activated partial thromboplastin time test (aPTT), and platelet function tests such as platelet aggregometry, VerifyNow, and the Dade PFA-100. More recently, point of care versions of some of these tests have been developed. Point of care tests allow more rapid turnaround time of test results, enable more convenient and frequent testing to help assure that patients are in the therapeutic range, and could reduce the number of inconvenient trips to a care provider.
  • PT prothrombin time test
  • ACT activated clotting time test
  • aPTT activated partial thromboplastin
  • the PT test may be used to monitor the coagulation of patients taking a vitamin K antagonist (VKA).
  • VKAs may interfere with synthesis of factors in the extrinsic coagulation pathway and thereby reduce the risk of unwanted clotting.
  • VKA is typically a longer term anticoagulant, and its effects may continue after shorter term anticoagulants such as heparin are discontinued and decrease in physiological circulation. Because of this, it may be important to determine the specific effect of VKA-induced inhibition of coagulation as opposed to the activity of other anticoagulants. For example, in the treatment of venous thrombosis, or when anticoagulant therapy is administered after mechanical heart valve replacement, patients may receive heparin at the same time as VKA.
  • heparin may prolongs the PT such that the inhibition of coagulation caused by VKA treatment can be overestimated.
  • heparin can also affect platelets, such that the response of platelets to certain agonists may not provide an accurate indication of platelet function. Because of the importance of obtaining accurate measurements of specific blood coagulation properties, reliable removal or quantitative reduction of the heparin content of a sample would be highly desirable.
  • the invention provides devices, methods, test strips and kits for measuring the properties of a sample comprising heparin.
  • Figure 1 illustrates a device and test strips of the present invention
  • Figures 2 A-D illustrate exemplary configurations of test strips according to the present invention
  • Figures 3 A-K illustrate exemplary localization of heparin neutralizing reagent(s) on test strips according to the present invention
  • Figures 4 A-C illustrate a method of preparing test strips of the present invention
  • Figure 5 describes the results of measurements of prothrombin time in the presence and absence of heparin neutralizing reagent
  • the present invention encompasses a device, test strips, methods and kits for measuring the properties of a sample comprising hepa ⁇ n
  • the measurements to be perfomed on a sample are coagulation or lysis assays
  • Such assays include, but are not limited the prothrombin time (PT), the activated clotting time (ACT), the thrombin clotting time (TCT) or the auto hemolysis test (AHT) Specific examples of tests that can be used m the measurement areas are disclosed m more detail below
  • the PT test was first described by Quick in 1935 The PT test measures the tissue factor-induced coagulation time of blood or plasma It is used as a screening test to evaluate the integrity of the extrinsic coagulation pathway and is sensitive to coagulation factors II, V, VII, and X A prolonged clotting time suggests the presence of an inhibitor to, or a deficiency in, one or more of the coagulation factors of the extrinsic pathway The PT time can also be prolonged for patients on anticoagulant therapy, for example, couma ⁇ n drug therapy, or for those with vitamin K deficiency or liver dysfunction Thus the PT test can provide an assessment of the extrinsic coagulation pathway PT test results can be converted to International Normalized Ratio (INR) values according to the following formula
  • INR International Normalized Ratio
  • INR (PT ratio) Isl , and PT ratio-Patient's PT/Mean Normal PT, where ISI is the International sensitivity index
  • a device used to test PT may include, within one or more of its measurement areas, reagents appropriate for measuring PT, such as thromboplastin (e g , recombinant or purified)
  • the ACT test is used primarily to monitor a patient's coagulation status in connection with clinical procedures that involve the administration of high doses of hepa ⁇ n or other anticoagulants (e g , coronary bypass surgery and percutaneous transluminal coronary angioplasty) Prolongation of ACT is directly proportional to the concentration of heparin or other anticoagulants in the blood
  • a device used to test ACT may include within one or more of its measurement areas reagents that use appropriate for measuring ACT, such as kaolin or celite
  • the TCT test measures the rate of a patient's clot formation compared to that of a normal plasma control
  • the TCT test can be used as an aid in the diagnosis of disseminated intravascular coagulation (DIC) and liver disease
  • a device of the invention may include, within one or more of its measurement areas, reagents that are appropriate for measuring TCT, such as thrombin
  • the invention provides a test strip comprising at least one channel connecting at least one sample measurement area to at least one sample input area, wherein a heparin neutralizing reagent is deposited within at least one of the channels.
  • the test strip can contain one, two, three or more sample measurement areas.
  • a test strip is provided according to the invention containing three sample measurement areas, each connected by channels to a sample input area, wherein a heparin neutralizing reagent is deposited within at least one of the channels.
  • the heparin neutralizing reagent is deposited outside of the sample measurement area. In still other embodiments, the heparin neutralizing reagent is deposited in a junction region connecting two or more channels. Alternatively, the heparin neutralizing reagent is deposited within a channel connecting a sample input region and a junction region linking two or more additional channels. In another embodiment, the heparin neutralizing reagent is deposited within the sample input area.
  • At least one of the sample measurement areas comprises electrodes allowing a DC or AC potential to be applied to the sample.
  • a test strip is provided comprising two sample measurement areas, wherein one of the sample measurement areas is used for quality control purposes.
  • An alternative embodiment provides for a test strip comprising three sample measurement areas, wherein two of the sample measurement areas are used for quality control purposes.
  • the invention provides for test strips comprising a heparin neutralizing reagent and a reagent for increasing blood viscosity, wherein the heparin neutralizing reagent is located proximal to the reagent for increasing blood viscosity.
  • the test strips described herein can be used in combination with a device to measure the properties of a sample comprising heparin.
  • the test strips of the invention are configured to measure a coagulation curvea time course of coagulation of a sample.
  • the test strips are configured to measure prothrombin time.
  • the present invention also provides a method for measuring blood properties by contacting a blood sample on a test strip with a heparin neutralizing reagent and subsequently contacting the sample with reagents for increasing blood viscosity.
  • the reagent for increasing blood viscosity is thromboplastin.
  • the reagent for increasing blood viscosity is recombinant tissue factor.
  • the invention is also directed to a method for measuring blood properties by contacting a blood sample with a heparin neutralizing reagent and subsequently contacting the sample with reagents that initiate blood coagulation, followed by measurement of an electrical property of the sample.
  • the method will include measurement of one of the properties selected from the group consisting of impedance, resistance or capacitance.
  • PT prothrombin time test
  • ACT activated clotting test
  • the sample is smaller than 20 ⁇ L.
  • Samples suitable for measurement according to the invention comprise whole blood or citrated blood samples. Samples may be extracted from a subject in various ways known to one skilled in art, including without limitation by an ear prick, a finger prick, and by drawing blood from a vein. Suitable samples may be collected by a health care worker, such as a nurse or medical technician, the patient, or others.
  • the volume of collected sample may vary. In some embodiments, the volume collected may be as low as about 1, 2, 5, 10, 15, 20, 25 ⁇ L. In other embodiments, the volume may be as high as about 100, 250, 1000, 2000, 5000, or 10000 ⁇ L.
  • the devices herein are used to measure the ability of a sample to coagulate or lyse.
  • a portion of the sample e.g. plasma or whole blood
  • a heparin neutralizing reagent deposited within the channel.
  • the measurement area that includes one or more reagent compositions that activate the intrinsic and/or extrinsic coagulation cascade, the time until clot formation is measured.
  • the heparin neutralizing reagent is deposited outside of said sample measurement area.
  • the reagent composition(s) may comprise thromboplastin, which can be of human and/or recombinant origin.
  • at least two sample measurement areas are provided.
  • one or more of the sample measurement areas may be used as a quality control.
  • Sample measurement areas may also comprise electrodes.
  • the device may be configured to measure an electrical property.
  • the electrical property is impedance, resistance or capacitance.
  • the sample tested in the device may be smaller than about 3, 10, 15, 20, or 25 ⁇ L.
  • the device may be configured to measure a time course of coagulation.
  • the device may also be configured to perform a blood test, such as a prothrombin time test.
  • the heparin neutralizing reagent comprises polycationic reagents, enzymes, metal salts, polypeptides or synthetic molecules.
  • the heparin neutralizing reagent comprises polybrene, polylysine, heparinase, protamine, calixarene, zinc salt or copper salt.
  • the device may be configured to provide a response within about 5 minutes from initiation of coagulation.
  • the device may comprise a test strip which is disposable.
  • different coagulation or lysis promoting reagents may be provided within different measurement areas to allow for the simultaneous measurement of two or more different coagulation times.
  • PT assays might be carried out simultaneously each in a different measurement area.
  • the PT test is in a delayed measurement area and the other is in a non-delayed measurement area.
  • the reagent compositions for the specified test are applied to the test strip during the manufacturing process of the test strip and using various types of micro- dispensing techniques which include, but is not limited to, ink jet, striper and sprayer deposition methods, or dip coating, and air dried in situ during the manufacturing process.
  • a sample may be assayed for the thrombolytic effect of a drug, and lysis of a blood sample is monitored.
  • the effect of plasminogen activators can be monitored by assaying the rate of coagulation of a sample from a patient receiving a thrombolytic drug, such as urokinase or tPA.
  • the clotting or lysis time of the patient sample may then be compared to the clotting or lysis time of pooled normal plasma or whole blood to provide a standard measurement of the patient's hemostatic status.
  • a sample is collected from a patient before the patient is administered a drug (e g urokinase or tPA) and after drug administration
  • a drug e g urokinase or tPA
  • a difference in coagulation level, lysis, presence or absence or amount of an analyte in a sample can be determined usmg the devices herein This can be used to determine effectiveness of drug treatment
  • the present mvention provides devices used to measure the properties of a sample comprising heparin
  • a representative example of the devices of the present invention is illustrated m Fig. 1
  • the device includes a housing 100 having an inlet port 101 for receiving a disposable fluidic test strip 123 for at least partial insertion therethrough
  • the test strip 123 is preferably disposable after performing the desired assay Test strips can include any assay strip, cartridge, adapted and configured to receive a fluid sample and support one or more reagents and electrodes as described herein, as well as any other device suitable to achieve that purpose available to a person of skill in the art Additionally the test strips and/or the device itself may assume any convenient geometric shape as long as the electronics and chemistry described herein are cost effectively contamed with acceptable performance
  • the device is configured such that all of the components are mounted in the interior space of the housing 100, including a power supply 121 to conduct the assay
  • the device may provide a plug for an AC or DC adaptor
  • the test strip 123 is adapted and configured to be inserted into the device and is positioned in thermal proximity to a heater 102 which is used to warm the sample on the test strip to a target temperature
  • the target temperature can be pre-deterrmned and can be above room temperature, i e , greater than 35°C
  • Any conventional heater of the appropriate size and heating capacity for the anticipated sample size is suitable
  • the heater is mounted between or below a substrate such as aluminum or other thermally conductive material for efficient and uniform transfer of heat to the test strip.
  • the temperature is maintained at 37 0 C so that the test results can readily compared to other standardized test results without interpolation
  • a temperature sensor 103 is mounted in proximity to the detection or sampling area 109, 110, 111 where the sample is applied or transported to in order to detect the temperature of the system and provide ambient temperature information for calibration adjustment at temperature extremes It is suitable to locate the temperature sensor 103 anywhere m or on the device For example, the temperature sensor 103 maybe located on the test strip
  • the power supply 121 has a lead from its negative pole connected to one side of an electrode pair 115, 116, and 117 and a lead from its positive pole for delivering electric potential to the electrodes and detection of the impedance or resistance signal, which is relayed by electrical connection to an analog to digital converter 118 and display 120
  • a processor and memory component 119 is connected to the analog to digital converter 118 and the display 120
  • External ports 122 are connected to the analog to digital converter 118 for receiving assay calibration information, interfacing with a computer, or downloading test results
  • the distal end of the receiver electrodes 112, 113, and 114 that are m contact with corresponding contact pads 115, 116, and 117 of the measuring device which provide connection to the processor 119 and the power source 121
  • the connection between the electrodes 112, 113, and 114 and the contact pads 115, 116, and 117 is made when the test strip 123 is inserted into the inlet port 101
  • the processor 119 can be any common or custom integrated circuit with memory
  • the power supply 121 can be any convenient device including, but not limited to, a battery, AC or DC adapter, or a solar cell
  • the display 120 preferably is a liquid crystal device LCD or any conventional, inexpensive display device
  • the number size in the display should be sufficiently large to allow most people to read the assay value, even if they have poor vision.
  • the display height can be about 2 cm.
  • the number of digits in the display can be anywhere from 1 to about 10 digits, however, a 3 to 5 digit display is usually sufficient.
  • the display may show messages relating to the assay result or processing or give error messages.
  • the converter 118 can include a multiplexer to integrate the signal from the electrodes and provide the digital signal to the processor 119.
  • the processor can be used to count the time required for the integral to reach a fixed voltage comparator threshold. The time is proportional to the average signal over the sampling period.
  • the device can be of any convenient size with the optimal dimensions determined by several factors including convenience of use to the consumer.
  • the device has a volume range of about 5 cm 3 to about 500 cm 3 .
  • the inserted test strip 123 is positioned in proximity of a heater 102.
  • the sample is maintained at a target temperature.
  • a heater can be included within the test strip.
  • the temperature of the sample is maintained constant by signaling from a temperature sensor 103 to the heater.
  • the sensor is mounted in proximity to the detection area of the test strip, or it may be mounted anywhere on the device, and it provides temperature information for calibrating the device and ensuring that the assays are conducted at a predetermined constant temperature to eliminate interassay temperature variations.
  • a sample is applied to the sample input area 104 of the test strip 123 and is transported by capillary action through the microfluidic channel 105 to the junction area 124, where it contacts a heparin neutralizing reagent.
  • the sample further travels through the branch channels 106, 107 and 108 by to the measurement areas 109, 110, and 111.
  • the volume of blood that is applied to the test strip can be between 5 and 20 ⁇ L, or between 10 and 15 ⁇ L.
  • Sample flow is stopped within each of the measurement area(s) by air vents 109, 110, and 111 that act as stop junctions.
  • the fluid sample reacts with the reagent(s) in the measurement area and bridges the electrodes, thereby changing the impedance, resistance or capacitance across the electrodes and signaling the change to the measuring device, thereby initiating the assay.
  • the electrodes are adapted to receive and pass a predetermined signal into the sample. The electrodes generate an electrical signal corresponding to the impedance of the sample.
  • the change in impedance is signaled through the distal end of the receiver electrodes 112, 113, and 114 that are in contact with corresponding contact pads 115, 116, and 117 of the measuring device to an analog to digital converter 118, which integrates the signal from the electrodes and conveys it to a processor and memory component 119, and a display 120.
  • the device is driven by a source 121 that can be any power source such as a battery, AC or DC adaptor, or a solar powered cell.
  • a test strip of the device can comprise a heparin neutralizing reagent and a reagent for inducing blood coagulation wherein the heparin neutralizing reagent is located in a different location than the reagent for inducing blood coagulation.
  • the reagent for inducing blood coagulation may be thromboplastin, including human and/or recombinant thromboplastin.
  • the test strip may comprise one channel or more than one channel.
  • the heparin neutralizing reagent may be located at a junction formed by the channels, or it may be located near a junction formed by said channels.
  • the heparin neutralizing reagent comprises polycationic reagents, enzymes, metal salts, polypeptides or synthetic molecules.
  • the heparin neutralizing reagent comprises polybrene, polylysine, heparinase, protamine, calixarene, zinc salt or copper salt.
  • the test strip may be configured to measure a time course of coagulation. In come embodiments, the test strip is configured to provide a response within about 5 minutes from initiation of coagulation.
  • Electrolytic conductance is a measure of the ability of electrolytes in a solution to carry an electrical current by virtue of the mobility of the ions in the solution.
  • Reagents in the measurement areas can comprise any combination of cations, salts, lipids, phosphatides, sulfatides, proteins or nucleic acids.
  • at least one of the measurement areas comprises a reagent for inducing blood coagulation.
  • at least one of the measurement areas comprises thromboplastin.
  • the thromboplastin used in the present invention can be of mammalian or bacterial origin, and can be purified from a natural source or obtained through recombinant methods. Coagulation or lysis can also be measured through the quantitation of the activity or activities of a specific coagulation factor or factors of interest.
  • Such activity can be measured through a physical properties of the sample, such as viscosity, optical extinction, electrical impedance, electrical capacitance, or electrical conductivity.
  • the activity of one or more specific coagulation factors can be measured by detectable reaction processes or products that are caused or generated by the factors of interest. Such reaction products may be detected by a measurable label, whether the label be one that is measured through optical extinction, optical fluorescence, optical dichroism, optical polarization, fluorescence polarization, luminescence, paramagnetism, electrochemical properties, electrical impedance, electrical conductance, or electrical capacitance.
  • test strips to be used for measuring the properties of samples comprising heparin.
  • the test strips comprise a sample input area 201 in fluid connection (202) with a junction region 227 comprising a heparin neutralizing reagent. Additional microfluidic channels link the junction area 227 with the sample measurement areas 207, 208 and 209.
  • the test strips may be configured to have one, two, or more measurement areas. Each measurement area also contains a detector for detecting an electrical property of the sample comprising a driver and a receiver electrode.
  • the driver electrodes 217, 223 and 225 deliver an electric potential to the measurement areas 206, 207, and 208, and the receiver electrodes 218, 224, and 226 deliver an electrical signal reflective of the the electrical properties, such as impedance or conductance, of the sample present in the measurement areas to a measuring device that translates the impedance signal into a digital output.
  • an AC electric potential is applied to the electrodes through the fluid sample and measured signal is impedance.
  • heparin neutralizing reagents can be used to carry out the present invention.
  • Reagents suitable for use include substances that directly bind, precipitate, immobilize or degrade heparin, or alternatively interfere with the binding of heparin to blood clotting proteins.
  • Heparin neutralizing reagents can be used alone or in combination to carry out the described invention.
  • heparin neutralizing reagents includes enzymes and other compounds that degrade heparin. This category comprises natural and recombinant heparinases of various origins, e.g. mammalian or bacterial origin. Examples of such heparinases include heparinase I, II, and III.
  • polycationic reagents may be used as heparin neutralizing reagents. Polycationic reagents include positively charged compounds or polymers. The cationic residues of such reagents can be tertiary or quaternary amino residues, urea, guanidine, histidine or other positively charged moieties, such as polybrene and polylysine.
  • heparin inactivating reagents suitable for use according to the invention comprise natural or synthetic polypeptides and their analogs.
  • Reagents in this category include, but are not limited to, natural or recombinant protamine or protamine analogs.
  • Yet another category of heparin neutralizing reagents encompasses synthetic molecules such as calixarene, calixarene analogs and derivatives thereof. These can be used as heparin neutralizing reagents according to the present invention.
  • metal salts such as zinc and copper salts may be used to inactivate heparin in a sample.
  • the heparin neutralizing reagents may be present in solid form, or as a semisolid gel, a powder, a liquid solution, or liquid suspension. Heparin neutralizing reagents may also be layered on top of another surface or may be shaped in the form of a filter or membrane contacting a sample. The reagent may be deposited in liquid or gel form by spraying, droplet deposition, brushing, layering, or blotting onto a substrate. The amount of the heparin neutralizing reagent will vary depending on type of molecule and charge density. The amount can range from 0.1 to 10 micrograms for a sufficient amount to neutralize therapeutic amounts of heparin in an approximately 15 microliter sample of whole blood.
  • Typical maximum therapeutic levels of heparin in plasma are approximately 1 IU/mL unfractionated heparin or 2 anti-Xa units/mL low molecular weight heparin, and whole blood levels are usually about half of these concentrations.
  • a person skilled in the art will know how to derive an appropriate amount of heparin neutralizing reagent based on the expected concentration of heparin in the samples to be measured.
  • Suitable heparin neutralizing reagents of the invention additionally include substrates functionalized or associated with heparin neutralizing reagents. Such substrates can be any solid matter amenable for carrying the heparin neutralizing reagent, including but not limited to non-porous or porous media, hollow or solid fibers, polymeric membranes or beads. Any of the heparin neutralizing reagents noted previously may be used to functionalize the substrate.
  • Neutralization of heparin in a sample according to the present invention can be complete or incomplete, and the extend of neutralization will vary based on the specific desired goal of each embodiment. In some applications, it may be desirable to remove most or all of the heparin present in the sample, such as for prothrombin time tests performed on blood samples. In other instances, partial removal is preferred, for example if the goal is to measure the response of a sample to a known quantity of heparin through quantitative removal of heparin from the sample.
  • Figure 3 illustrates several configurations for the location of the heparin neutralizing reagent deposited within the test strips.
  • Figs. 3A-D and Fig. 3I-J show the heparin neutralizing reagent positioned proximal to a junction formed by the microfluidic channels on the test strip.
  • Fig. 3A shows the heparin inactivating reagent 301 deposited along the microfluidic channel between the sample input area and the channel junction area.
  • the heparin neutralizing reagent 301 can be deposited along the channel between the junction area and each sample measurement area.
  • One, two or all of the channels leading from the junction to the sample measurement areas can contain heparin neutralizing reagents.
  • Another embodiment described in Fig. 3E shows the heparin neutralizing reagent 301 located within the sample input area.
  • the heparin neutralizing reagent 301 is positioned within the channel junction area.
  • the invention is applicable to test strips containing one or two sample chambers as shown in Figs. 3G, H, or to test strips in which the length, volume or position of individual channels or sample chambers is different (Figs. 3I-K).
  • a bibulous membrane may comprise assay reagents in one part of the membrane and additional heparin neutralizing reagents in the same or a different area of the membrane.
  • Reagents including assay reagents and heparin neutralizing reagents, may be deposited on the membrane, adsorbed to the membrane, impregnated into the membrane, or otherwise associated with the membrane.
  • the heparin neutralizing reagent is deposited on one face or close to one face of the membrane, while other assay reagents are deposited on the opposite face or close to the opposite face of the membrane.
  • test strip 400 is obtained by first assembling the top layer 401 and middle layer 402 to form a top-middle layer 404 (illustrated in Figure 4C), and then combining the top-middle layer 404 with the electrode layer 403.
  • the fluidic path 405, which transports the sample from the sample well 406 through the microfluidic feeder 407 and branch channels 408 to the measurement areas 409 of the assembled strip can be formed by microfabricating a cutout on a two-sided adhesive middle layer 402.
  • the top layer 401 which is made of nonporous material is perforated to provide a circular opening 410 that aligns with the portion of the micro fludic path 405 that forms the sample well 406 in the assembled test strip 400. Additional cutouts of the top layer 401 can be made to provide the air vents 411, which act as stop junctions during use of the strip.
  • the top layer houses the reagents or compositions that enable the coagulation or lysis of a sample during use of the test strip.
  • the reagents are deposited in the reagent areas 412 that are below the cut of the air vent, and on the side of the top layer that is adjacent to the middle layer. Additionally, the heparin neutralizing reagents are deposited on reagent area 416.
  • the adhesive is removed from the a first side of the middle layer 402 that is adjacent to the top layer 401, and the top 401 and middle layer 402 are assembled to form the top-middle layer 404 ( Figure 4C).
  • the electrodes are formed on the electrode layer 403 by depositing a suitable inert conductor using a pump or any other means of deposition, such as vacuum or sputtering. Electrodes may also be produced by the printing of an ink using methods known in the art, such as screen printing. Suitable conductor materials that can be used to form the electrodes include but are not limited to silver, carbon, gold or platinum.
  • the electrodes have a proximal end 413 and a distal end 414, which respectively span the region of the electrode layer 403 that is complementary to that defining the measurement area 409 and the signaling edge 415 of the assembled test strip 400.
  • the adhesive is removed from the second side of the middle layer 402 of the top-middle layer assembly 404, and is adhered to the electrode layer 403 to form the assembled strip.
  • Example 1 Construction of a test strip for measuring prothrombin time.
  • a test strip is constructed comprising a sample input area linked through a capillary channel to a junction region.
  • the junction region is connected via three separate capillary channels to three independent sample measurement chambers.
  • Test strips were produced with deposition of 70 nanoliters of a reagent containing the heparin-neutralizing reagent in the channel in the region indicated as Fig. 4B (407, 416).
  • the deposited reagent contained 0.32 micrograms of hexadimethrine bromide (HMB) as the heparin neutralizing reagent, 0.021 micrograms of sodium chloride, and 0.7 micromolar sorbitol for facilitating resolubilization of the reagent.
  • HMB hexadimethrine bromide
  • the deposited reagent was dried on the strip by evaporation.
  • the test strip of Example 1 is inserted into an INRatioTM PT/INR monitor (HemosenseTM).
  • INRatioTM PT/INR monitor HemosenseTM
  • Non- anticoagulated venous whole blood is obtained from a human subject taking a vitamin K antagonist.
  • the obtained blood is applied to the test strip.
  • the blood is drawn into the test strip by capillary action where it first mixes with the polybrene deposited in the junction of the test strip, and subsequently mixes with reagents that initiate coagulation in the measurement chambers.
  • the monitor performs the PT test and two quality control tests (normal and therapeutic) simultaneously, and determines whether the controls are within preset limits. If they are, strip integrity is verified, and the monitor reports the PT test result. If they are not, the monitor displays an error message.
  • the test results are displayed in less than 2 minutes as an INR or as PT seconds and INR.
  • Example 3 Measurement of prothrombin time in the presence and absence of heparin neutralizing reagent.

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Abstract

L'invention concerne des procédés et des dispositifs servant à neutraliser les effets de l'héparine dans le sang total, le plasma ou un autre échantillon biologique, afin de réaliser un essai diagnostique clinique à partir des processus de coagulation sanguine en évitant les interférences de l'héparine. L'invention concerne également l'exécution d'un tel test à l'aide d'un dispositif au chevet du patient.
PCT/US2008/085745 2007-12-05 2008-12-05 Procédés et dispositifs servant à neutraliser l'héparine dans un échantillon Ceased WO2009073861A1 (fr)

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US99254207P 2007-12-05 2007-12-05
US60/992,542 2007-12-05

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WO2009073861A1 true WO2009073861A1 (fr) 2009-06-11

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015178924A1 (fr) 2014-05-23 2015-11-26 Nova Biomedical Corporation Procédé et système de détection d'hémolyse
US20230341424A1 (en) * 2020-07-31 2023-10-26 Micropoint Biotechnologies, Co., Ltd. Device for detecting blood coagulation characteristics and application of device

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050233466A1 (en) * 2004-04-19 2005-10-20 Wright David W Blood coagulation test cartridge, system, and method
US20060016701A1 (en) * 2004-05-17 2006-01-26 Wei Qin Point of care heparin determination system
US20060035298A1 (en) * 2003-02-01 2006-02-16 James Hill System and method for determining a coagulation parameter
US20070082372A1 (en) * 2005-09-30 2007-04-12 Jyotsna Ghai Membrane system for blood coagulation testing

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060035298A1 (en) * 2003-02-01 2006-02-16 James Hill System and method for determining a coagulation parameter
US20050233466A1 (en) * 2004-04-19 2005-10-20 Wright David W Blood coagulation test cartridge, system, and method
US20060016701A1 (en) * 2004-05-17 2006-01-26 Wei Qin Point of care heparin determination system
US20070082372A1 (en) * 2005-09-30 2007-04-12 Jyotsna Ghai Membrane system for blood coagulation testing

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015178924A1 (fr) 2014-05-23 2015-11-26 Nova Biomedical Corporation Procédé et système de détection d'hémolyse
KR20170010364A (ko) * 2014-05-23 2017-01-31 노바 바이오메디컬 코포레이션 용혈 검출 방법 및 시스템
CN107003296A (zh) * 2014-05-23 2017-08-01 诺尔生物医药有限公司 溶血检测方法和系统
EP3146332A4 (fr) * 2014-05-23 2017-11-22 Nova Biomedical Corporation Procédé et système de détection d'hémolyse
CN107003296B (zh) * 2014-05-23 2019-12-03 诺尔生物医药有限公司 溶血检测方法和系统
US10809246B2 (en) 2014-05-23 2020-10-20 Nova Biomedical Corporation Hemolysis detection method and system
KR102215174B1 (ko) * 2014-05-23 2021-02-10 노바 바이오메디컬 코포레이션 용혈 검출 방법 및 시스템
US20230341424A1 (en) * 2020-07-31 2023-10-26 Micropoint Biotechnologies, Co., Ltd. Device for detecting blood coagulation characteristics and application of device
EP4184170A4 (fr) * 2020-07-31 2024-08-28 Micropoint Biotechnologies, Co., Ltd. Dispositif de détection de caractéristiques de coagulation sanguine et application de dispositif

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