WO2004086045A1 - Method of measuring platelet-derived microvesicles (pmv) using a time-resolved fluorescence immunoassay (tr-fia) - Google Patents

Method of measuring platelet-derived microvesicles (pmv) using a time-resolved fluorescence immunoassay (tr-fia) Download PDF

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WO2004086045A1
WO2004086045A1 PCT/NO2004/000083 NO2004000083W WO2004086045A1 WO 2004086045 A1 WO2004086045 A1 WO 2004086045A1 NO 2004000083 W NO2004000083 W NO 2004000083W WO 2004086045 A1 WO2004086045 A1 WO 2004086045A1
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pmv
fia
antibody
lanthanide
platelet
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PCT/NO2004/000083
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French (fr)
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Annika E. Michelsen
Ragnhild Wergeland
Oddvar Stokke
Frank Brosstad
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Forskningsparken As
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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the present invention relates to a method of diagnosing haemostatic disorders in a human or non-human subject.
  • the invention relates to a method for measuring platelet-derived microvesicles (PMV) in vitro by using a lanthanide in a time-resolved fluorescence immunoassay (TR-FIA).
  • TR-FIA time-resolved fluorescence immunoassay
  • the present invention also relates to a kit for measurements of PMV and the use of PMV and TR-FIA to diagnose haemostatic disorders.
  • Blood platelets are a normal component of blood and are known to participate actively in haemostatic processes. Through their interaction with the sub-endothelium of damaged blood vessels, for example, platelets may aggregate to form a primary haemostatic plug. This process is also usually accompanied by the production of various factors involved in haemostasis by the platelets.
  • Factor Va is a component of the so-called prothrombinase complex which produces thrombin from its precursor prothiOmbin.
  • platelets shed off microvesicles (also called microparticles, PMP) from their plasma membrane upon activation. This phenomenon was first described by Sandberg et al. (Biochem. J. 203: 303-311 (1982)).
  • microvesicles formed by platelets are an accurate indication of platelet activation and so reflect the activity of the haemostatic system as a whole.
  • George et al. (J. Clin. Invest. 78: 340-348 (1986)) monitored the change in glycoproteins appearing on the surface of platelets and on platelet-derived microvesicles following cardiopulmonary bypass surgery. It was noted that the number of microvesicles increased and it was postulated that this was due to the shear stresses that are experienced during such an operation. However it was not suggested that momtoring the concentration of microvesicles would be a good indicator of haemostatic disorders and could provide the basis for a method of diagnosing such conditions.
  • glycoproteins More than 40 glycoproteins (GP) are known to be present on the resting platelet surface. Many are routinely identified on PMV, chiefly GPIIbllla and GPIb since they are widely used as markers to identify PMV as such (platelet derived). See George JN, Pickett EB, Saucerman S, McEver RP, Kuniki TJ, Kieffer N, Newman PJ. Platelet surface glycoproteins: studies on resting and activated platelets and platelet membrane microparticles in normal subjects, and observations in patients during adult respiratory distress syndrome and cardiac surgery. Journal of Clinical Investigation 1986; 78: 340-8 and Fox JEB, Austin CD, Boyles JK, Steffen K. Role of the membrane cytoskeleton in preventing the shedding of procoagulantrich microvesicles from the platelet plasma membrane. Journal of Cell Biology 1990; 111: 483-93
  • GPUbllla is a platelet specific marker, and is widely used in flow cytometry studies. Patients suffering from Glanszmann thrombastenia are lacking this receptor which results in severe disorder of the haemostatic process.
  • PMV is a component of the platelet membrane, therefore similar proteins are found in the membranes of PMV and the platelet membrane.
  • the integrin receptor GPIIbllla is a component of the platelet plasma membrane and, therefore also of the PMV plasma membrane.
  • time-resolved fluorometry The principle of time-resolved fluorometry is well known in the art. (Wallac, Perkin Elmer and Kazuko Matsumoto et al. RIKEN Review No 35, May 2001; Fluorescent lanthanide labels for time-resolved fluoremetry in biological trace analysis). However, hithereto, no method is available for determining the presence or concentration of PMV by time-resolved fluorescence immunoassay.
  • the purpose of the present invention is to provide a new and improved method for assessing the presence and concentration of platelet-derived microvesicles in biological samples using a time-resolve fluorescence immunoassay.
  • the method according to the invention reveals a reliable method for quantification of circulating PMV, having excellent sensitivity and specificity. It is readily applicable and suitable for routine analysis.
  • the present invention also relates to a kit for measurements of PMV and the use of PMV and TR-FIA to diagnose haemostatic disorders.
  • the present invention relates to a method of diagnosing haemostatic disorders in a human or non-human subject.
  • the invention relates to a method for measuring platelet-derived microvesicles (PMV) in vitro by using a lanthanide in a time-resolved fluorescence immunoassay (TR-FIA).
  • TR-FIA time-resolved fluorescence immunoassay
  • the present invention also relates to a kit for measurements of PMV and the use of PMV and TR-FIA to diagnose haemostatic disorders.
  • the present invention relates to a method of measuring platelet-derived microvesicles (PMV) in a human or non-human subject wherein the amount of PMV in the specimen is analysed using a time-resolved fluorescence immunoassay (TR-FIA)
  • TR-FIA time-resolved fluorescence immunoassay
  • One aspect of the invention relates to a method comprising obtaining a sample containing platelet-derived microvesicles (PMV), obtaining a lanthanide labelled PMV specific antibody and measuring the amount of PMV in the specimen in a time-resolved fluorescence immunoassay (TR-FIA).
  • the method according to the invention may include further steps, and the conducting order is not essential.
  • the invention in yet another aspect relates to a method comprising: a) obtaining a sample containing platelet-derived microvesicles (PMV) b) obtaining a lanthanide labelled PMV specific antibody i c) mixing the lanthanide labelled antibody with another PMV specific biotinylated antibody d) applying the sample and antibody-mixture onto a streptavidin coated microtiter well e) addition of an enhancement solution to relocate the fluorescent lanthanide from the antibody to a micelle, and finally f) determination of the micelle fluorescence
  • the sample analysed is plasma.
  • a sample of any body fluid of an individual may be analysed such as whole blood, serum, lymph, spinal fluid and urine.
  • Microvesicles is not a specific feature of blood platelets. Rather, microvesicles are derived from apoptotic or activated cells of any type , such as monocytes, macrophages, granulocytes, lymphocytes and cell types associated with the circulatory system, such as the endothelial cells lining the blood vessels. Measurement of microvesicles derived from other elements than blood platelets are also included in the method according to the invention.
  • the lanthanide labels used are all distinguishable on the basis of wavelength and decaytime, and multiple labelling therefore permits simultaneous detection of several different as part of one single test.
  • the integrin receptor GPIIbllla is a component of the plasma membrane of the platelet and, therfore also of the plasma membrane of PMV. Based on this there is a direct correlation between the presence of GPIIbllla in a platelet free sample, and the presence of PMV in blood.
  • the method according to the invention provides a linear correlation between the quantity of GPUbllla and the amount of PMV in the sample and the fluorescence measured.
  • the PVM specific antibody is any antibody specific to GPIIbllla.
  • the PMV specific antibodies are antibodies against the GPlTb epitope (CD41) and antibodies against the GPIIIa epitope (CD61).
  • the PMV specific antibody may be any antibody, e.g. monoclonal, polyclonal, hybrid or fusion antibodies of any origin. Murine monoclonal antibodies are preferred.
  • the lanthanide is europium.
  • other elements belonging to the group lanthanide such as Terbiun (Tb), Samarium (Sm) and Dysprosium (Dy) may also be used.
  • the antibodies used according to the invention may be labelled with europium in the laboratory by the operator. However, commercially available Eu3+ labelled antibodies may also be used.
  • the invention in another aspect, relates to a kit comprising a lanthanide labelled PMV specific antibody together with reagents and equipment necessary to carry out a TR-FIA and detailed instructions for use for the measurement of PMV in a human or non-human subject. It is readily applicable and suitable for routine analysis determining the haemostatic condition in a subject.
  • the kit comprises a) a lanthanide labelled PMV specific antibody b) another PMV specific antibody labelled with biotin c) positive control d) assay buffer e) washing buffer f) enhancement buffer g) microtiter plates precoated with streptavidin, and f) instructions for use
  • the present invention also relates to the use of TR-FIA for measurement of PMV and the use of PMV and TR-FIA for diagnosing haematostatic disorders. Therefore, according to the invention a new and improved method for assessing the presence and concentration of platelet-derived microvesicles in biological samples using a time-resolve fluorescence immunoassay is provided.
  • the invention provides a reliable method for quantification of circulating PMV, having excellent sensitivity and specificity.
  • a readily applicable and suitable method for routine analysis and a ready to use kit for measurements of PMV are provided to diagnose haemostatic disorders.
  • haemostatic disorders includes any aberration or abnormality in the blood clotting cascade mechanism, in particular disorders which lead to increased likelihood of thrombus formation or to a decreased ability for blood clotting following injury for example congenital bleeding disorders, all forms of DIC (Disseminated Intravascular Coagulation) and atherosclerotic disease. Diagnosis of haemostatic disorders thus enables thromboembohc and/or cardiovascular diseases to be detected and/or monitored. This term also includes haemostatic disorders, which are a secondary phenomenon, such as for example as a result of cancer.
  • Figure 1 outlines the principle of the time-resolved fluorescent immunoassay, TR-FIA.
  • Figure 2 shows a standard curve revealing the linear correlation between quantity of GPIIbllla in the sample and the fluorescence measured (counts pr second; cps).
  • Figure 3 shows the quantity of GPIIbllla in plasma of patients undergoing PCI (percutaneous coronary intervention) compared to healthy volunteers.
  • Panel A shows the amount prior to PCI and panel B shows the amount after PCI.
  • Figure 4 shows a standard curve revealing the linear correlation between the quantity of GPLTbllla in the sample and the fluorescence measured (counts pr minute; cps).
  • Figure 5 shows the quantity of GPIIbllla in plasma of a sepsis patient compared to healthy volunteers.
  • Figure 6 shows a standard curve revealing the linear correlation between quantity of GPUbllla in the sample and the fluorescence measured (counts pr second; cps).
  • Figure 8 Correlation between assessment of PMV by TR-FIA (x-axis) and flow cytometry (y-axis) wherein TR-FIA analyses the content of PMV in plasma ( ⁇ g/L) and flow cytometry analyses the presentage of PMV in PRP.
  • a monoclonal antibody to the CD41 epitope on PMV (GPIIb) and a biotinylated monoclonal antibody to the CD61 epitope on PMV ( GPIIIa) were purchased from Diatec, Oslo, Norway and Immunotech/Beckman Coulter, respectively .
  • the Europium chelate was purchased from PerkinElmer Life Science.
  • the murine monoclonal anti-CD41 antibody (clone 96.2C1, #3120, id no Diatec, Oslo, Norway ) in a concentration of 1 mg/mL was dialysed against 0.15 mol L NaCl overnight to remove sodium azide.
  • the dialysis-bag was packed in PEG8000 powder to concentrate the solution.
  • Europium chelate (Eu-Nl-ITC labelling kit, #1244-302, PerkinElmer) was added in 12- fold molar excess to the antibody (anti- CD41 from above) in 0.15 mol/L NaCl, and then 0.1 mol/L sodium borate (pH 8.6) was added (1 volume borate buffer to 9 volumes of sample). The mixture was incubated dark at room temperature for 48 hours. Free europium chelate was removed by chromatography on a PD-10 column (Disposable PD10 desalting column, #17-0851-01, Amersham Bioscience).
  • Plasma was prepared by centrifuging 4.5 mL blood anticoagulated with 0.5 mL 0.129 M sodium citrate (Vacutainer, Becton Dickinson) at 2500g for 30 minutes at 4 °C. 150 ⁇ L of PMV-containing plasma was mixed with 50 ⁇ L of assay buffer containing 2% NP40 (Nonidet P-40, N-6507, Sigma- Aldrich)
  • Blood may be anticoagulated with any anticoagulant.
  • sodium citrate as described above is applicable, as well as CTAD (citrate in combination with the platelet inhibitors theophylline, adenosine and dipyridamole).
  • CTAD chloride in combination with the platelet inhibitors theophylline, adenosine and dipyridamole.
  • PMV GPIIbllla
  • a positive control was prepared by adding GPIIbllla (Enzyme Research) to a plasma pool of healthy blood donors, and stored as aliquots at -80°C.
  • Solubilized GPIIbllla (Enzyme Research) was diluted in Delfia Assay buffer (PerkinElmer Life Science) to concentrations corresponding to 10, 15, 20, 30, 60, 120, 240, 600, 1200 ⁇ g/L, respectively, and used as calibrators to generate a standard curve. The fluorescence measured being linearly correlated to the amount of GPIIbllla and further to the amount of PMV as described above.
  • Plasma from 10 patients suffering from angina pectoris and 10 healthy volunteers were prepared as described above and analysed using a time-resolved fluorescence immunoassay according to the invention.
  • the results from the healthy volunteers and the patients are listed below in table 1 and 2, respectively.
  • Table 2 GPUbllla in plasma of patients suffering from stable angina pectoris prior to PCI and following PCI
  • the levels of GPIIbllla in healthy volunteers are ranging from 267 to 630 ⁇ g/L resulting in a mean value of 457 ⁇ 118 ⁇ g/L
  • the level of GPUbllla in the angina group are ranging from 228 to 1457 resulting in a mean value of 716 ⁇ 393 ⁇ g/L which is about a 2 fold increase in the level of GPIIbllla compared to healthy volunteers.
  • the level of GPUbllla in the same patients following PCI were ranging from 237 to 1313, resulting in a mean value of 879 ⁇ 399 ⁇ g/L. It is obvious that the medical condition angina pectoris is most often associated with pronounced haemostatic disorders.
  • the level of PMV is in all patients, except P33, significantly elevated compared to the normal healthy individuals. Most of the patients, i.e. P25, P28, P30 and P31, show an even larger increase in the PMV following PCI. P22, P29 and P32 showed a reduction of the level of GPIIbllla following PCI but the levels were significantly increased compared to healthy volunteers. P33 revealed a normal level of PMV both prior to PCI and following PCI.
  • Plasma from one sepsis patient and 3 healthy volunteers were prepared as described above and analysed using time-resolved fluorescence immunoassay. The results are listed table 3.
  • Table 3 GPIIbllla in plasma of 3 normal individuals (N1-N3) and one sepsis patient
  • the levels of GPIIbllla in healthy volunteers are 164, 254 and 134 ⁇ g/L revealing a mean value of 184 ⁇ 62 ⁇ g/L.
  • the level of GPIIbllla in the patient suffering from sepsis was 406 ⁇ g/L which is about a 2 fold increase in the level of GPIIbllla.
  • Europium chelate and Delfia reagents were from Perkin-Elmer Life Sciences (Boston, MA, USA) and solubilized GPIIbllla was from Enzyme Research (South Bend, IN, USA).
  • the thrombin receptor-mimicking peptide SFLLRN was a kind gift from Dr. Knut Sletten, The Biotechnology Centre of Oslo. All other reagents were from Sigma- Aldrich (St. Lois, MO, USA), unless other stated.
  • PRP platelet-rich plasma
  • concentration of platelets was adjusted to 300 000/ ⁇ L with autologous plasma.
  • PRP was incubated with 100 ⁇ M trombin- receptor mimicking peptide SFLLRN for 25 minutes at room temperature.
  • TS Tris-buffered saline
  • Ultracentrifugation 300 ⁇ L plasma was ultracentrifuged in a SW55Ti-rotor (Beckman Coulter, Hialeah, FL, USA) 35000rpm, 3 hours, 4 °C. The supernatant was stored at -20 °C until analysed.
  • Fluorescein isothiocyanate (FITC)- or R-phycoerythrin (RPE)-conjugated monoclonal antibodies were from BD Biosciences Pharmingen (San Diego, CA, USA). Five ⁇ L PRP was mixed with 5 ⁇ L FITC-conjugated anti-CD61 (GPIIIa) MAb and 5 ⁇ L RPE-conjugated anti-CD62P (P-selektin) MAb, and the volume was adjusted to 100 ⁇ L with PBS. After 25 minutes of incubation in the dark at room temperature, 1 mL PBS was added and the flow cytometric determination was performed immediately.
  • FITC Fluorescein isothiocyanate
  • RPE R-phycoerythrin
  • the labelled platelets and PMVs were analysed in a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) equipped with a 15 mW air-cooled 488 nm argon laser.
  • the light scatter and fluorescence channels were set at logarithmic gain.
  • acquisition was gated so as to include only positive events for GPIIIa. Consequently, a fluorescence threshold was set to analyze only platelets and MP. Ten thousand events positive for the fluorescent marker were analysed.
  • PMV and platelets were separated analytically on the basis of forward scatter. The number of PMV was expressed as per cent of total number of GPIIIa-positive events.
  • the GPIIbllla complex was solubilized out of any present PMV by mixing 150 ⁇ L plasma and 50 ⁇ L Delfia Assay buffer containing 2 % Igepal.
  • Europium chelate was added in 12-fold molar excess to antibody in 0.15 mol/L NaCl, and then 0.1 mol L sodium borate (pH 8.6) was added (1 volume borate buffer to 9 volumes of sample). The mixture was incubated dark at room temperature for 48 hours. Free europium chelate was removed by chromatography on a PD-10 column (Amersham Biosciences, Piscataway, NJ). Protein concentration and degree of conjugation were determined according to the manufacturer's protocol. The conjugated antibody was refrigerated in the presence of BS A treated with diethyleneamine pentaacetic (PerkinElmer Lifesciences, Boston, MA, USA).
  • Plasma was prepared by centrifuging 4.5 mL blood anticoagulated with 0.5 mL 0.129 M sodium citrate (Vacutainer, BD Biosciences) at 2500xg for 30 minutes at 4 °C. Plasma from 8 healthy volunteers was pooled. Plasma was stored as aliquots at -20°C and used for the calculation of intra- and inter-assay variation.
  • Solubilized GPIIbllla was diluted in Delfia Assay buffer to concentrations corresponding to 10, 15, 20, 30, 60, 120, 240, 600, 1200 ⁇ g/L, respectively, and used as calibrators to generate a calibration curve.
  • the Delfia streptavidin-coated plate was pre-washed twice with 400 ⁇ L Delfia Washing solution. 50 ⁇ L duplicate of plasma samples, 50 ⁇ L duplicate of calibrators and controls were pipetted into the steptavidin-coated microtitration wells, and 150 ⁇ L of the antibody-mixture was added. The plate was incubated for two hours at room temperature on a shaker, before washing each well 6 times with 700 ⁇ L Delfia Washing solution. Delfia Enhancement Solution (200 ⁇ L) was added to each well, shaked for 5 minutes, and the time-resolved fluorescence was measured in a Victor 2 1420 (PerkinElmer Life Sciences, Boston, MA, USA).
  • ⁇ -thromboglobulin ( ⁇ TG) was determined according to the manufacturer's protocol with a commercially available assay (Stago, Asnieres, France). Platelets were counted in a Coulter Particle Counter (Beckman Coulter, Hialeah, FL, USA). Results
  • PRP was subjected to stimulation by 100 ⁇ M of the thrombin-receptor mimicking peptide SFLLRN.
  • Tris-buffer (TS) was used as a control. The results are listed in table 4 below.
  • FC Flow cytometric
  • TR-FIA time-resolved fluorescence immunoassay
  • PMV was determined by TR-FIA.
  • the content of PMV in plasma was increased from 155 ⁇ 26 ⁇ g/L to 378 ⁇ 26 ⁇ g/L after stimulation.
  • TR-FIA correlated with the flow cytometric estimation of PMV in PRP demonstrating that TR-FIA can be used as a new an improved method for measuring PMV (figure 8).
  • PMV platelet-derived microparticles

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Abstract

The present invention relates to a method of diagnosing haemostatic disorders in a human or non-human subject. In particular, the invention relates to a method for measuring platelet-derived microvesicles (PMV) in vitro by using a lanthanide in a time-resolved fluorescence immunoassay (TR-FIA). The present invention also relates to a kit for measurements of PMV and the use of PMV and TR-FIA to diagnose haemostatic disorders.

Description

Method of measuring platelet-derived microvesicles (PMV) using a time-resolved fluoreseene immunoassay (TE-FIA)
Field of Invention
The present invention relates to a method of diagnosing haemostatic disorders in a human or non-human subject. In particular, the invention relates to a method for measuring platelet-derived microvesicles (PMV) in vitro by using a lanthanide in a time-resolved fluorescence immunoassay (TR-FIA). The present invention also relates to a kit for measurements of PMV and the use of PMV and TR-FIA to diagnose haemostatic disorders.
Background of the invention
Blood platelets are a normal component of blood and are known to participate actively in haemostatic processes. Through their interaction with the sub-endothelium of damaged blood vessels, for example, platelets may aggregate to form a primary haemostatic plug. This process is also usually accompanied by the production of various factors involved in haemostasis by the platelets.
Activation of blood platelets greatly enhances their catalysation of haemostatic reactions, and in particular activated platelets demonstrate an increased concentration and activity of factor Va. (Factor Va is a component of the so-called prothrombinase complex which produces thrombin from its precursor prothiOmbin.) In addition it has been found that platelets shed off microvesicles (also called microparticles, PMP) from their plasma membrane upon activation. This phenomenon was first described by Sandberg et al. (Biochem. J. 203: 303-311 (1982)).
Previous studies have endeavoured to monitor platelet activation by measuring the amounts of proteins secreted. Thus, assays for the measurement of platelet factor 4 (PF- 4), β-thromboglobulin (β-TG) and thrombospondin have been investigated (see Lane et al. Thromb. Haemostas. (Stuttgart) 52(2) pages 183-187 (1984)). However, none of these proteins were found to be suitable for routine monitoring. Thrombospondin was found to be non-specific for platelets and studies on β-TG and PF-4 revealed that their half-lives in the blood were too short, causing problems in distinguishing the sample concentration over the background level. Further, β-TG levels were found to be elevated in patients with renal failure and PF-4 levels were influenced by the presence of heparin (which is extensively used to treat patients with thrombotic disorders).
It is recognised within the art that the microvesicles formed by platelets are an accurate indication of platelet activation and so reflect the activity of the haemostatic system as a whole. George et al., (J. Clin. Invest. 78: 340-348 (1986)) monitored the change in glycoproteins appearing on the surface of platelets and on platelet-derived microvesicles following cardiopulmonary bypass surgery. It was noted that the number of microvesicles increased and it was postulated that this was due to the shear stresses that are experienced during such an operation. However it was not suggested that momtoring the concentration of microvesicles would be a good indicator of haemostatic disorders and could provide the basis for a method of diagnosing such conditions.
More than 40 glycoproteins (GP) are known to be present on the resting platelet surface. Many are routinely identified on PMV, chiefly GPIIbllla and GPIb since they are widely used as markers to identify PMV as such (platelet derived). See George JN, Pickett EB, Saucerman S, McEver RP, Kuniki TJ, Kieffer N, Newman PJ. Platelet surface glycoproteins: studies on resting and activated platelets and platelet membrane microparticles in normal subjects, and observations in patients during adult respiratory distress syndrome and cardiac surgery. Journal of Clinical Investigation 1986; 78: 340-8 and Fox JEB, Austin CD, Boyles JK, Steffen K. Role of the membrane cytoskeleton in preventing the shedding of procoagulantrich microvesicles from the platelet plasma membrane. Journal of Cell Biology 1990; 111: 483-93
GPUbllla is a platelet specific marker, and is widely used in flow cytometry studies. Patients suffering from Glanszmann thrombastenia are lacking this receptor which results in severe disorder of the haemostatic process. PMV is a component of the platelet membrane, therefore similar proteins are found in the membranes of PMV and the platelet membrane. The integrin receptor GPIIbllla is a component of the platelet plasma membrane and, therefore also of the PMV plasma membrane.
Prior art also describe quantification of PMV by using conventional ELISA techniques. See S Miyamoto, Marcinkiewicz C, Edmunds LH, Niewiarowski S: "Measurement of Platelet Microparticles during Cardiopulmonary Bypass by Means of Captured ELISA for GPIIbllla" Thrombosis and HAemostasis 1998; 80: 225-30) and Nomura et al. (K Osumi, Ozeki Y, Saito A, Nagamura Y, Ito H, Kimura Y, Ogura H, Nomura S: "Development and Assessment of Enzyme Immunoassay for Platelet-derived Microparticles" Thrombosis and Haemostasis 2001; 85: 326-30. Also US 5.811.250 describes a method of diagnosing haemostatic disorders by assessing the presence or concentration of platelet-derived microvesicles (PMV) by using an immunoassay.
However, prior art does not describe or suggest use of a time-resolved fluorescent immunoassay for measurement of PMV.
The principle of time-resolved fluorometry is well known in the art. (Wallac, Perkin Elmer and Kazuko Matsumoto et al. RIKEN Review No 35, May 2001; Fluorescent lanthanide labels for time-resolved fluoremetry in biological trace analysis). However, hithereto, no method is available for determining the presence or concentration of PMV by time-resolved fluorescence immunoassay.
Thus the purpose of the present invention is to provide a new and improved method for assessing the presence and concentration of platelet-derived microvesicles in biological samples using a time-resolve fluorescence immunoassay. The method according to the invention reveals a reliable method for quantification of circulating PMV, having excellent sensitivity and specificity. It is readily applicable and suitable for routine analysis. The present invention also relates to a kit for measurements of PMV and the use of PMV and TR-FIA to diagnose haemostatic disorders.
Detailed description of the invention
The present invention relates to a method of diagnosing haemostatic disorders in a human or non-human subject. In particular, the invention relates to a method for measuring platelet-derived microvesicles (PMV) in vitro by using a lanthanide in a time-resolved fluorescence immunoassay (TR-FIA). The present invention also relates to a kit for measurements of PMV and the use of PMV and TR-FIA to diagnose haemostatic disorders.
In particular, the present invention relates to a method of measuring platelet-derived microvesicles (PMV) in a human or non-human subject wherein the amount of PMV in the specimen is analysed using a time-resolved fluorescence immunoassay (TR-FIA)
One aspect of the invention relates to a method comprising obtaining a sample containing platelet-derived microvesicles (PMV), obtaining a lanthanide labelled PMV specific antibody and measuring the amount of PMV in the specimen in a time-resolved fluorescence immunoassay (TR-FIA). The method according to the invention may include further steps, and the conducting order is not essential.
In yet another aspect the invention relates to a method comprising: a) obtaining a sample containing platelet-derived microvesicles (PMV) b) obtaining a lanthanide labelled PMV specific antibody i c) mixing the lanthanide labelled antibody with another PMV specific biotinylated antibody d) applying the sample and antibody-mixture onto a streptavidin coated microtiter well e) addition of an enhancement solution to relocate the fluorescent lanthanide from the antibody to a micelle, and finally f) determination of the micelle fluorescence
In one aspect of the invention the sample analysed is plasma. However, a sample of any body fluid of an individual may be analysed such as whole blood, serum, lymph, spinal fluid and urine.
Microvesicles is not a specific feature of blood platelets. Rather, microvesicles are derived from apoptotic or activated cells of any type , such as monocytes, macrophages, granulocytes, lymphocytes and cell types associated with the circulatory system, such as the endothelial cells lining the blood vessels. Measurement of microvesicles derived from other elements than blood platelets are also included in the method according to the invention.
The lanthanide labels used (Eu3+, Sm3+, Tb3+, Dy3"1") are all distinguishable on the basis of wavelength and decaytime, and multiple labelling therefore permits simultaneous detection of several different as part of one single test. Here, one might use different monoclonal antibodies labelled to different lanthanides to quantify microvesicles of different origin.
The integrin receptor GPIIbllla is a component of the plasma membrane of the platelet and, therfore also of the plasma membrane of PMV. Based on this there is a direct correlation between the presence of GPIIbllla in a platelet free sample, and the presence of PMV in blood. The method according to the invention provides a linear correlation between the quantity of GPUbllla and the amount of PMV in the sample and the fluorescence measured.
According to one aspect of the invention the PVM specific antibody is any antibody specific to GPIIbllla. In a preferred embodiment, the PMV specific antibodies are antibodies against the GPlTb epitope (CD41) and antibodies against the GPIIIa epitope (CD61). The PMV specific antibody may be any antibody, e.g. monoclonal, polyclonal, hybrid or fusion antibodies of any origin. Murine monoclonal antibodies are preferred.
In another aspect of the present invention the lanthanide is europium. However, other elements belonging to the group lanthanide such as Terbiun (Tb), Samarium (Sm) and Dysprosium (Dy) may also be used.
The antibodies used according to the invention may be labelled with europium in the laboratory by the operator. However, commercially available Eu3+ labelled antibodies may also be used.
In another aspect, the invention relates to a kit comprising a lanthanide labelled PMV specific antibody together with reagents and equipment necessary to carry out a TR-FIA and detailed instructions for use for the measurement of PMV in a human or non-human subject. It is readily applicable and suitable for routine analysis determining the haemostatic condition in a subject.
hi a preferred embodiment according to the invention the kit comprises a) a lanthanide labelled PMV specific antibody b) another PMV specific antibody labelled with biotin c) positive control d) assay buffer e) washing buffer f) enhancement buffer g) microtiter plates precoated with streptavidin, and f) instructions for use
The present invention also relates to the use of TR-FIA for measurement of PMV and the use of PMV and TR-FIA for diagnosing haematostatic disorders. Therefore, according to the invention a new and improved method for assessing the presence and concentration of platelet-derived microvesicles in biological samples using a time-resolve fluorescence immunoassay is provided. The invention provides a reliable method for quantification of circulating PMV, having excellent sensitivity and specificity. By this invention a readily applicable and suitable method for routine analysis and a ready to use kit for measurements of PMV are provided to diagnose haemostatic disorders.
Definitions: As used herein, the term "haemostatic disorders" includes any aberration or abnormality in the blood clotting cascade mechanism, in particular disorders which lead to increased likelihood of thrombus formation or to a decreased ability for blood clotting following injury for example congenital bleeding disorders, all forms of DIC (Disseminated Intravascular Coagulation) and atherosclerotic disease. Diagnosis of haemostatic disorders thus enables thromboembohc and/or cardiovascular diseases to be detected and/or monitored. This term also includes haemostatic disorders, which are a secondary phenomenon, such as for example as a result of cancer.
Short description of the figures:
Figure 1 outlines the principle of the time-resolved fluorescent immunoassay, TR-FIA.
Figure 2 shows a standard curve revealing the linear correlation between quantity of GPIIbllla in the sample and the fluorescence measured (counts pr second; cps).
Figure 3 shows the quantity of GPIIbllla in plasma of patients undergoing PCI (percutaneous coronary intervention) compared to healthy volunteers. Panel A shows the amount prior to PCI and panel B shows the amount after PCI.
Figure 4 shows a standard curve revealing the linear correlation between the quantity of GPLTbllla in the sample and the fluorescence measured (counts pr minute; cps).
Figure 5 shows the quantity of GPIIbllla in plasma of a sepsis patient compared to healthy volunteers.
Figure 6 shows a standard curve revealing the linear correlation between quantity of GPUbllla in the sample and the fluorescence measured (counts pr second; cps). Figure 7. The effect of ultracentrifugation. GPIIbllla in plasma was quantified before and after ultracentrifugation analysed by TR-FIA. *Significant difference p=0,011
Figure 8. Correlation between assessment of PMV by TR-FIA (x-axis) and flow cytometry (y-axis) wherein TR-FIA analyses the content of PMV in plasma (μg/L) and flow cytometry analyses the presentage of PMV in PRP.
Figure 9. The effect of stimulation on PMV content (μg/L) analysed by TR-FIA (panel A) and by flow cytometry (panel B). PRP was stimulated with 100 μM SFLLRN or TS as control. *Signifϊcant difference between values in control and stimulated samples A) p=0,0236 and B) p=0,0081
Embodiments
The following non-limiting examples illustrate the invention.
A monoclonal antibody to the CD41 epitope on PMV (GPIIb) and a biotinylated monoclonal antibody to the CD61 epitope on PMV ( GPIIIa) were purchased from Diatec, Oslo, Norway and Immunotech/Beckman Coulter, respectively . The Europium chelate was purchased from PerkinElmer Life Science.
1. Labelling of antibody with a lanthanide
The murine monoclonal anti-CD41 antibody (clone 96.2C1, #3120, id no Diatec, Oslo, Norway ) in a concentration of 1 mg/mL was dialysed against 0.15 mol L NaCl overnight to remove sodium azide. The dialysis-bag was packed in PEG8000 powder to concentrate the solution.
Europium chelate (Eu-Nl-ITC labelling kit, #1244-302, PerkinElmer) was added in 12- fold molar excess to the antibody (anti- CD41 from above) in 0.15 mol/L NaCl, and then 0.1 mol/L sodium borate (pH 8.6) was added (1 volume borate buffer to 9 volumes of sample). The mixture was incubated dark at room temperature for 48 hours. Free europium chelate was removed by chromatography on a PD-10 column (Disposable PD10 desalting column, #17-0851-01, Amersham Bioscience).
Preparation of antibody mixture The catching biotinylated murine monoclonal anti-CD61 antibody (clone SZ21, #IM0721, Beckman Coulter) and the tracing Eu-labelled antibody from above was mixed in Delfia Assay buffer (#1244-111, PerkinElmer Life Science) to 1 μg/mL and 0,4 μg/mL, respectively.
3. Sample preparation
Plasma was prepared by centrifuging 4.5 mL blood anticoagulated with 0.5 mL 0.129 M sodium citrate (Vacutainer, Becton Dickinson) at 2500g for 30 minutes at 4 °C. 150 μL of PMV-containing plasma was mixed with 50 μL of assay buffer containing 2% NP40 (Nonidet P-40, N-6507, Sigma- Aldrich)
Blood may be anticoagulated with any anticoagulant. However, sodium citrate as described above is applicable, as well as CTAD (citrate in combination with the platelet inhibitors theophylline, adenosine and dipyridamole). Any PMV (GPIIbllla) present in a platelet free plasma, is derived from apoptotic or activated platelets.
A positive control was prepared by adding GPIIbllla (Enzyme Research) to a plasma pool of healthy blood donors, and stored as aliquots at -80°C.
4. Preparation of standard curves
Solubilized GPIIbllla (Enzyme Research) was diluted in Delfia Assay buffer (PerkinElmer Life Science) to concentrations corresponding to 10, 15, 20, 30, 60, 120, 240, 600, 1200 μg/L, respectively, and used as calibrators to generate a standard curve. The fluorescence measured being linearly correlated to the amount of GPIIbllla and further to the amount of PMV as described above.
5. Measurements of platelet-derived microvescicles
50 μL triplicate of plasma samples, 50 μL duplicate of calibrators and controls were pipetted into the Delfia steptavidin-coated microtitration strips (#4009-0010, PerkinElmer Life Science), and 150 μL of the antibody-mixture was added. The plate was incubated for two hours at room temperature before washing each well 6 times with 700 μL Delfia Washing solution (#1244-114, PerkinElmer Life Science).200 μL of Delfia Enhancement Solution (#1244-105, PerkinElmer Life Science) was added to each well and the time-resolved fluorescence was determined in a Victor 1420 (Perkin- Elmer Life Science). 6. Results
Experiment 1: Measurement of platelet-derived microvesicles in plasma of patients suffering from angina pectoris
Plasma from 10 patients suffering from angina pectoris and 10 healthy volunteers were prepared as described above and analysed using a time-resolved fluorescence immunoassay according to the invention. The results from the healthy volunteers and the patients are listed below in table 1 and 2, respectively.
Figure imgf000011_0001
Table 2: GPUbllla in plasma of patients suffering from stable angina pectoris prior to PCI and following PCI
Figure imgf000011_0002
Figure imgf000012_0002
As shown in table 1 and 2 the levels of GPIIbllla in healthy volunteers are ranging from 267 to 630 μg/L resulting in a mean value of 457 ± 118 μg/L The level of GPUbllla in the angina group are ranging from 228 to 1457 resulting in a mean value of 716 ± 393 μg/L which is about a 2 fold increase in the level of GPIIbllla compared to healthy volunteers. The level of GPUbllla in the same patients following PCI were ranging from 237 to 1313, resulting in a mean value of 879 ± 399 μg/L. It is obvious that the medical condition angina pectoris is most often associated with pronounced haemostatic disorders. And as can be seen from above the level of PMV is in all patients, except P33, significantly elevated compared to the normal healthy individuals. Most of the patients, i.e. P25, P28, P30 and P31, show an even larger increase in the PMV following PCI. P22, P29 and P32 showed a reduction of the level of GPIIbllla following PCI but the levels were significantly increased compared to healthy volunteers. P33 revealed a normal level of PMV both prior to PCI and following PCI.
These results show that the method of diagnosing haematostatic disorders according to the invention are applicable on patient suffering from angina pectoris.
Experiment 2: Measurement of platelet-derived microvesicles in plasma of patients suffering from sepsis
Plasma from one sepsis patient and 3 healthy volunteers were prepared as described above and analysed using time-resolved fluorescence immunoassay. The results are listed table 3.
Table 3: GPIIbllla in plasma of 3 normal individuals (N1-N3) and one sepsis patient
Figure imgf000012_0001
Figure imgf000012_0003
Figure imgf000013_0001
As shown in table 3 the levels of GPIIbllla in healthy volunteers are 164, 254 and 134 μg/L revealing a mean value of 184 ± 62 μg/L. The level of GPIIbllla in the patient suffering from sepsis was 406 μg/L which is about a 2 fold increase in the level of GPIIbllla.
This result shows that the method of diagnosing haematostatic disorders according to the invention are applicable on patient suffering from sepsis.
Experiment 3: Comparison between TR-FIA and flow cytometric examination following in vitro stimulation of normal platelets.
Material and Methods
Reagents
Europium chelate and Delfia reagents were from Perkin-Elmer Life Sciences (Boston, MA, USA) and solubilized GPIIbllla was from Enzyme Research (South Bend, IN, USA). The thrombin receptor-mimicking peptide SFLLRN was a kind gift from Dr. Knut Sletten, The Biotechnology Centre of Oslo. All other reagents were from Sigma- Aldrich (St. Lois, MO, USA), unless other stated.
Preparation of PMVs
Blood from healthy donors were drawn by venipuncture into 1:10 volume of 0.129 M tri-sodium citrate (Vacutainer, BD, Franklin Lakes, NJ USA) and centrifuged at 250 x g for 8 minutes at 20 °C to obtain platelet-rich plasma (PRP). The concentration of platelets was adjusted to 300 000/μL with autologous plasma. To stimulate platelets, and generate PMVs, PRP was incubated with 100 μM trombin- receptor mimicking peptide SFLLRN for 25 minutes at room temperature. As a control, PRP was incubated with Tris-buffered saline (TS). To remove platelets from the PMV- containing PRP, centrifugation was performed twice at 11000 x g for 4 minutes. The plasma, PPP (platelet poor plasma) was stored at -20 °C until analysed.
Filtration: Any PMV, larger than 0,1 μm, was separated from the plasma by filtration (Ultra-free- MC Filter Units, Millipore, Billerica, MA, USA). The filtrate was stored at -20 °C until analysed.
Ultracentrifugation: 300 μL plasma was ultracentrifuged in a SW55Ti-rotor (Beckman Coulter, Hialeah, FL, USA) 35000rpm, 3 hours, 4 °C. The supernatant was stored at -20 °C until analysed.
Immunolabelling and performance of flow cytometry
Fluorescein isothiocyanate (FITC)- or R-phycoerythrin (RPE)-conjugated monoclonal antibodies (MAb) were from BD Biosciences Pharmingen (San Diego, CA, USA). Five μL PRP was mixed with 5 μL FITC-conjugated anti-CD61 (GPIIIa) MAb and 5 μL RPE-conjugated anti-CD62P (P-selektin) MAb, and the volume was adjusted to 100 μL with PBS. After 25 minutes of incubation in the dark at room temperature, 1 mL PBS was added and the flow cytometric determination was performed immediately. The labelled platelets and PMVs were analysed in a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) equipped with a 15 mW air-cooled 488 nm argon laser. The light scatter and fluorescence channels were set at logarithmic gain. To study PMV formation, and in order to resolve PMV from background light scatter, acquisition was gated so as to include only positive events for GPIIIa. Consequently, a fluorescence threshold was set to analyze only platelets and MP. Ten thousand events positive for the fluorescent marker were analysed. PMV and platelets were separated analytically on the basis of forward scatter. The number of PMV was expressed as per cent of total number of GPIIIa-positive events.
Preparation of samples for measurement of PMV by TR-FIA
The GPIIbllla complex was solubilized out of any present PMV by mixing 150 μL plasma and 50 μL Delfia Assay buffer containing 2 % Igepal.
Conjugation of monoclonal antibody with europium The labelling was performed essentially as described by Bjerner et al. (Bjerner et al. 2002). Monoclonal anti-CD41 antibody (Diatec, Oslo, Norway) 1 mg/mL was transferred to a dialysis-bag (Pierce Biotechnology, Rockford, IL, USA) and dialysed against 0.15 mol L NaCl overnight to remove sodium azide. The dialysis-bag was packed in polyethylene glycol 8000 powder to concentrate the solution. The dialysed, concentrated antibody solution was transferred to a reaction tube for conjugation. Europium chelate was added in 12-fold molar excess to antibody in 0.15 mol/L NaCl, and then 0.1 mol L sodium borate (pH 8.6) was added (1 volume borate buffer to 9 volumes of sample). The mixture was incubated dark at room temperature for 48 hours. Free europium chelate was removed by chromatography on a PD-10 column (Amersham Biosciences, Piscataway, NJ). Protein concentration and degree of conjugation were determined according to the manufacturer's protocol. The conjugated antibody was refrigerated in the presence of BS A treated with diethyleneamine pentaacetic (PerkinElmer Lifesciences, Boston, MA, USA).
Controls
Plasma was prepared by centrifuging 4.5 mL blood anticoagulated with 0.5 mL 0.129 M sodium citrate (Vacutainer, BD Biosciences) at 2500xg for 30 minutes at 4 °C. Plasma from 8 healthy volunteers was pooled. Plasma was stored as aliquots at -20°C and used for the calculation of intra- and inter-assay variation.
Performance of the TR-FIA The catching biotinylated monoclonal anti-CD61 antibody (Immunotech, Marseille, France) and the tracing Eu-labelled antibody was mixed in Delfia Assay buffer to 1 μg/mL and 0,4 μg/mL, respectively.
Solubilized GPIIbllla was diluted in Delfia Assay buffer to concentrations corresponding to 10, 15, 20, 30, 60, 120, 240, 600, 1200 μg/L, respectively, and used as calibrators to generate a calibration curve.
The Delfia streptavidin-coated plate was pre-washed twice with 400 μL Delfia Washing solution. 50 μL duplicate of plasma samples, 50 μL duplicate of calibrators and controls were pipetted into the steptavidin-coated microtitration wells, and 150 μL of the antibody-mixture was added. The plate was incubated for two hours at room temperature on a shaker, before washing each well 6 times with 700 μL Delfia Washing solution. Delfia Enhancement Solution (200 μL) was added to each well, shaked for 5 minutes, and the time-resolved fluorescence was measured in a Victor21420 (PerkinElmer Life Sciences, Boston, MA, USA).
β-thromboglobulin (βTG) was determined according to the manufacturer's protocol with a commercially available assay (Stago, Asnieres, France). Platelets were counted in a Coulter Particle Counter (Beckman Coulter, Hialeah, FL, USA). Results
The standard curve, generated by serial dilution of GPUbllla, was linear over an extended range of concentrations (10- 1200 μg/L corresponding to 40-4800 pmol/L) and are presented in figure 6. The detection limit was calculated to 2.14 μg/L (sign ≠O), functional sensitivity to 2.98 (CV<20%), intra-assay CV < 6.5% and inter-assay CV < 12%.
PRP was subjected to stimulation by 100 μM of the thrombin-receptor mimicking peptide SFLLRN. Tris-buffer (TS) was used as a control. The results are listed in table 4 below.
Table 4: Flow cytometric (FC) and time-resolved fluorescence immunoassay (TR-FIA) examination of PMV.
Figure imgf000016_0001
PMV; platelet-derived micro vescicles, PRP; platelet-rich plasma, β-TG; β- tromboglobulin. Values are means±SEM. * = sign. difference after filtration, p= 0,0204
To confirm that the platelets in PRP responded normally to stimulation, PRP was stimulated . As shown in table 4 platelet activation was confirmed in that the percentage of PMV in PRP increases from 4,6±1,0 to 9.7±0,8 (p=0,0081), the percentage of CD62P positive cells increased from 24,9 ±4,8 to 95,0 ±1,2 and βTG was elevated from 191=1= 29 IU/ml to 8010 ± 405 IU/ml.
After elimination of platelets from plasma by centrifugation, PMV was determined by TR-FIA. The content of PMV in plasma was increased from 155±26μg/L to 378±26μg/L after stimulation.
Filtration of plasma significantly reduced the PMV in stimulated samples from 378±69μg/L to 167± 21μg/L , but not in untreated samples 155± 26μg/L vs 145± 35μg/L, indicating that untreated samples from healthy volunteers does not contain significant amounts of large PMV, and that the GPIIbllla-positive material in the plasma is part of particles.
In separate experiments shown in figure 7, ultracentrifugation decreased the PMV in plasma, verifying that GPIIbllla in plasma was a component of particles.
TR-FIA correlated with the flow cytometric estimation of PMV in PRP demonstrating that TR-FIA can be used as a new an improved method for measuring PMV (figure 8).
The effect of stimulation of PRP on PMV content (μg/L) in plasma analysed by TR-FIA (A) and in PRP analysed by flow cytometry (%PMPin PRP) is shown in fig. 9. PRP was stimulated with 100 μM SFLLRN and TS was used as a control. As appearant from figure 9, there is a good correlation between TR-FIA and flow cytometry demonstrating that TR-FIA can be used as a new and improved method to measure PMV in plasma, the level of which is indicative of the haematostatic disorders.
Experiment 4: Increased plasma concentration of platelet-derived microparticles in young survivors of myocardial infarction
Prothrombotic mechanisms are of major importance for the formation of an occlusive thrombus in young patients with myocardial infarction (MI). Experimental studies have shown that platelet-derived microparticles (PMV) possess procoagulant properties suggesting a pivotal role in the pathogenesis of arterial thrombotic diseases. The aim of this study was to determine the concentrations of PMV in plasma from young MI patients and study their relation to coagulation activation, assessed by thrombin-antithrombin (TAT) complexes.
Material and methods: A population based case (n=61)-control (n=61) study matched for age and gender was conducted in Mi-patients between 40 and 60 years of age and more than a year after the coronary event. An ELISA assay was developed to quantify PMV by the concentration of activated GPIIbllla in plasma and plasma was filtered (0.1 μm size exclusion) to discriminate between small (< 0.1 μm) and large (> 0.1 μm) PMV.
Results: Young survivors of MI had significantly higher mean platelet volume (MPV) (p=0.012) and PMV (314.3 μg/L, 273.1-361.4 vs. 225.8 μg/L, 186.8-273.1, p=0.009) (geometric mean and 95%CI) than age- and gender matched healthy controls. In simple regression analysis, the total amount of PMV positively associated with platelet count (r=0.44, p<0.001) and soluble CD40L (r=0.50, p<0.001), whereas large PMV was correlated with platelet count (r=0.28, ρ<0.05), sCD40L (r=0.37, pO.Ol) and TAT (r=0.35, p<0.01). To study the predictive role of PMV for MI, logistic regression models were conducted. In simple regression models, MPV (1.70, 1.09-2.67) (OR, 95%CI), total PMV (2.39, 1.20-4.78), small PMV (2.52, 1.10-5.74) and large PMV (1.65, 1.06-2.72) were significant predictors for MI. The predictive role of total, small and large PMV for MI, remained significant even after adjustment for age, sex, BMI and main serum lipids.
Conclusions: The present study suggests that PMV is an independent risk factor for MI. The positive correlation found between large PMV and thrombin generation support the concept that formation of PMV are of crucial importance for increased coagulation activation in MI patients.

Claims

C l a i m s
1. Method of measuring platelet-derived microvesicles (PMV) in a human or non- human subject wherein the amount of PMV in the specimen is analysed using a time- resolved fluorescence immunoassay (TR-FIA)
2. Method according to claim 1, comprising: a) obtaining a sample containing platelet-derived microvesicles (PMV) b) obtaining a lanthanide labelled PMV specific antibody c) measuring the amount of PMV in the specimen in a time-resolved fluorescence immunoassay (TR-FIA)
3. Method according to claim 1, comprising: a) obtaining a sample containing platelet-derived microvesicles (PMV) b) obtaining a lanthanide labelled PMV specific antibody c) mixing the lanthanide labelled antibody with another PMV specific biotinylated antibody d) applying the sample and antibody-mixture onto a streptavidin coated microtiter well e) addition of an enhancement solution to relocate the fluorescent lanthanide from the antibody to a micelle, and finally f) determination of the micelle fluorescence
4. Method according to claim 1 - 3, wherein the sample is a body fluid such as blood, serum, plasma, lymph, urine and spinal fluid.
5. Method according to claim 1 - 3, wherein the sample is a cell type capable of shedding microvesicles, such as blood cells and endothelial cells.
6. Method according to claim 1-3, wherein the PVM specific antibody is any antibody specific to GPIIbπia, preferentially CD41 (GPIIb) and CD61 (GPIIIa).
7. Method according to claim 1, wherein the lanthanide is Terbium, Samarium, Dysprosium or Europium, preferably Europium.
8. A kit comprising a lanthanide labelled PMV specific antibody together with reagents and equipment necessary to carry out a TR-FIA and detailed instructions for use for the measurement of PMV in a human or non-human subject.
9 A kit according to claim 10, comprising a) a lanthanide labelled PMV specific antibody b) another PMV specific antibody labelled with biotin c) positive control d) assay buffer e) washing buffer f) enhancement buffer g) microtiter plates precoated with streptavidin, and f) instructions for use
10. Use of TR-FIA for measurement of PMV.
11. Use of PMV and TR-FIA for diagnosing haematostatic disorders.
PCT/NO2004/000083 2003-03-28 2004-03-24 Method of measuring platelet-derived microvesicles (pmv) using a time-resolved fluorescence immunoassay (tr-fia) WO2004086045A1 (en)

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