Platelet analysis system

Abstract

A method for diagnosis of HIT (Heparin-induced thrombocytopenia) in a patient's serum or plasma sample and a system comprising kits for performing the method are provided, where the method involves incubating a patient's sample with and without heparin and normal control sample with and without heparin with a platelet-rich plasma (PRP) of an individual not having a platelet disorder and then incubating an aliquot of each sample with a first label for both heparin activated and non-heparin activated platelets and a second label for platelets activated by the heparin-immune complex formed in the patient sample. HIT is diagnosed when the difference between the amount of activated platelets from the patient's sample with and without heparin is substantially larger than the difference between the normal control sample with and without heparin.

1 - 19 . (canceled) 
     
     
         20 . A method for diagnosis of a platelet-related pathology, comprising at least one of the methods selected from the group consisting of:
 I. diagnosis of antiphospholipid syndrome (APS), comprising:
 A) incubating a sample of the patient's serum or plasma with a platelet reagent comprising exposed membrane phospholipids, to obtain a suspension of patient's sample with said platelet reagent (pSPEP); 
 B) incubating said pSPEP with a fluorescence labeled Annexin A5; 
 C) incubating a sample of serum or plasma from an individual not having a platelet-related pathology with a platelet reagent comprising exposed membrane phospholipids, to obtain a suspension of normal control sample with said platelet reagent (nSPEP); and 
 D) determining a degree of Annexin A5 binding to the exposed membrane phospholipids of the platelet reagent in said pSPEP and nSPEP, 
 whereby Annexin A5 binding to the exposed membrane phospholipids of the platelet reagent in pSPEP in a substantially lower degree than in nSPEP indicates the presence antiphospholipid antibodies in said patient's sample; 
   II. diagnosis of immune thrombocytopenia (ITP), comprising:
 A. preparing:
 1. patient's antibodies by:
 a) providing first microparticles covered with monoclonal antibodies (MoAb) against platelet specific receptors; 
 b) subsequently incubating the first covered microparticles with lysate from platelets from an individual not having a platelet-related pathology; 
 c) subsequently incubating the lysate-covered first microparticles with an aliquot of a sample of the patient's serum or plasma; and 
 d) subsequently adding a secondary fluorescence-labeled antibody (Ab) against human immunoglobulin; 
 
 2. reference normal antibodies by:
 a) providing second microparticles covered with MoAbs against platelet specific receptors; 
 b) subsequently incubating the covered second microparticles with lysate from platelets from an individual not having a platelet-related pathology; 
 c) subsequently incubating the lysate-covered second microparticles with an aliquot of serum or plasma from an individual not having a platelet-related pathology; and 
 d) subsequently adding a secondary fluorescence-labeled Ab against human immunoglobulin; 
 
 
 B. comparing a first quantity of secondary-labeled antibody adsorbed on said incubated first microparticles, indicating the quantity of the patient's antibodies against platelet specific receptors of the patient, with a second quantity of secondary-labeled antibody adsorbed on said incubated second microparticles, indicating the quantity of human immunoglobulin attached to platelets of the individual not having a platelet-related pathology, wherein when the first quantity is substantially more than the second quantity, indicating the presence of antibodies against platelet specific antigens in said patient's sample; 
   III. assessing response to platelet stimulation, comprising:
 A. simultaneously incubating platelet-containing samples of a patient and of an individual not having a platelet-related pathology, each with a first labeled MoAb against platelet CD41a or CD61 glycoproteins, and a second labeled MoAb against activated CD41a and/or CD62p, to provide labeled platelets of the patient and platelets of the individual not having a platelet-related pathology, wherein said first labeled MoAb has a measurement in a first band and said second labeled MoAb has a measurement in a second band; 
 B. subsequently adding to each of said samples a platelet stimulant selected from the group consisting of adenosine-di-phosphate (ADP), arachidonic acid and thrombin receptor agonist/activating peptide (TRAP) to provide stimulated labeled platelets of patient and of an individual not having a platelet-related pathology; 
 C. subsequently identifying labeled platelets in said samples by measuring in said first band; and 
 D. subsequently measuring activation state of said identified labeled platelets of the patient and said identified labeled platelets of the individual not having a platelet-related pathology, by measuring in said second band; 
 wherein the measurement of said identified labeled platelets of the patient obtained by measuring in said second band that is significantly less than the measurement of said labeled platelets of the individual not having a platelet-related pathology indicates reduced platelet reactivity in said sample of patient; 
   IV. assessing platelet procoagulant activity, comprising:
 A. simultaneously incubating platelet-containing samples of a patient and of an individual not having a platelet-related pathology, each with labeled MoAb against platelet glycoproteins, and labeled Annexin A5; 
 B. subsequently identifying labeled platelets in said samples by measuring labeled MoAb against platelet glycoproteins; 
 C. subsequently measuring fluorescence of Annexin A5 on said labeled platelets; 
   wherein an Annexin A5 fluorescence of the patient's platelets significantly greater than the Annexin A5 fluorescence of the platelets of the individual not having a platelet-related pathology indicates platelet procoagulation in said sample of patient;   V. diagnosis of Bernard-Soulier syndrome, comprising:
 A. simultaneously incubating platelet-containing samples of a patient and of an individual not having a platelet-related pathology, each with a-first labeled MoAb against platelet specific glycoproteins excluding CD42, and with a second labeled MoAb against CD42; 
 B. subsequently identifying labeled platelets in said samples by measuring the first labeled MoAb, and 
 C. subsequently measuring said second MoAb from said identified labeled platelets of the patient and said second MoAb from said identified labeled platelets of an individual not having a platelet-related pathology, 
   wherein a measurement of said second MoAb binding by said identified labeled platelets of the patient that is significantly less than the measurement of said second MoAb binding by said identified labeled platelets of an individual not having a platelet-related pathology, indicates Bernard-Soulier syndrome,   VI. diagnosis of a patient for a carrier state of Bernard-Soulier syndrome, comprising:
 A. simultaneously incubating platelet-containing samples of a patient, of an individual not having a platelet-related pathology, and of an individual known to have Bernard-Soulier syndrome, each with a-first labeled MoAb against platelet specific glycoproteins excluding CD42, and with a second labeled MoAb against CD42 (GP Ib-IX-V complex); 
 B. subsequently identifying labeled platelets in said samples by measuring the first labeled MoAb, and 
 C. subsequently measuring said second MoAb from said identified labeled platelets of each of the samples, 
   wherein a measurement of said second MoAb binding of said identified labeled platelets of the patient that is less than that of an individual not having a platelet-related pathology but greater than that of the individual known to have Bernard-Soulier syndrome, indicates a carrier state in the patient;   VII. diagnosis of Glanzmann thrombasthenia syndrome, comprising:
 A. simultaneously incubating platelet-containing samples of a patient and of an individual not having a platelet-related pathology, each with a first labeled MoAb against platelet glycoproteins excluding CD41a, and with a second labeled MoAb against CD41a; 
 B. subsequently identifying labeled platelets in said samples by measuring MoAb against platelet glycoproteins excluding CD41a, and 
 C. subsequently measuring MoAb against CD41a of said identified labeled platelets of the patient and MoAb against CD41a of said identified labeled platelets of an individual not having a platelet-related pathology, 
   wherein a measurement of binding to said labeled platelets of MoAb against CD41a in the patient's sample is less than the measurement of MoAb against CD41a binding to said platelets of said individual not having a platelet-related pathology, indicates Glanzmann thrombasthenia syndrome in said sample of the patient;   VIII. diagnosis of being in a carrier state for Glanzmann thrombasthenia syndrome, comprising:
 A. simultaneously incubating platelet-containing samples of a patient, of an individual not having a platelet-related pathology, and of an individual known to have Glanzmann thrombasthenia syndrome, each with a first labeled MoAb against platelet glycoproteins excluding CD41a, and with a second labeled MoAb against CD41a; 
 B. subsequently identifying labeled platelets in said samples by measuring MoAb against platelet glycoproteins excluding CD41a, and 
 C. subsequently measuring said second MoAb from said identified labeled platelets of each of the samples, 
   wherein a measurement of binding to said labeled platelets of MoAb against CD41a in a patient's sample that is less than that of an individual not having a platelet-related pathology but greater than that of an individual known to have Glanzmann thrombasthenia syndrome, indicates a carrier state of Glanzmann thrombasthenia syndrome;   IX. diagnosis of a drug inhibitory effect in a platelet-containing sample of a patient, comprising:
 A. simultaneously incubating platelet-containing samples of the patient and of an individual not having a platelet-related pathology and not taking a platelet inhibitory drug, each with a first labeled MoAb against platelet glycoproteins CD41a or CD61, a second labeled MoAb against CD62p and a third labeled MoAb against activated CD41a; 
 B. subsequently incubating said samples with a stimulator (platelet agonist) selected from the group consisting: ADP, arachidonic acid and TRAP, and any combination thereof, in a single or in multiple concentrations; 
 C. identifying labeled platelets in said samples by measuring MoAb against platelet glycoproteins CD41a or CD61; and 
 D. subsequently measuring MoAb against platelet glycoproteins CD62p and activated CD41a of said identified labeled platelets of patient and said identified labeled platelets of an individual not having a platelet-related pathology, 
   wherein either a measurement of MoAb against platelet glycoproteins CD62p or a measurement of MoAb against activated CD41a of the patient that is significantly less than the measurement of MoAb against platelet glycoproteins CD62p or activated CD41a of said individuals not having a platelet-related pathology and not taking a platelet inhibitory drug, indicates a drug inhibitory effect in said sample of the patient; and   X. determination of on-going real-time in vivo, prothrombotic activity by detecting activation labels of circulating platelet or platelet-related particles, comprising:
 A. detecting platelets and platelet-related particles in aliquots of a patient's blood or PRP sample and control blood or PRP sample from an individual not having a platelet-related pathology by incubation with MoAb against platelet glycoproteins CD41a (GPIIb/IIIa) or CD61; and detecting activation labels of circulating platelet-related particles in said blood or PRP samples by incubation with one or more of fluorescence-labeled receptor specific MoAb or labeling protein selected from the group consisting of (a) anti-activated CD41a (GPIIb/IIIa) antibody; (b) anti-CD62p (P-selectin) antibody or anti CD63 antibody; and (c) fluorescently labeled Annexin A5 protein against platelet anionic-phospholipids for detection of platelets and platelet-related particles procoagulant activity; and 
 B. following incubation for 15-30 minute at room temperature, diluting said samples with buffer and measuring a fluorescent signal level in said samples, wherein the signal level of the patient's sample is significantly greater than the signal level of a sample from an individual not having a platelet-related pathology indicates activation of circulating platelets or platelet-related particles. 
   
     
     
         21 . The method of  claim 20 , wherein after incubating the platelet suspension a Ca2+ ionophore, the method further comprises:
 incubating the platelet suspension with paraformaldehyde.   
     
     
         22 . The method of  claim 21 , wherein the incubating the platelet suspension with paraformaldehyde is for substantially an hour. 
     
     
         23 . The method of  claim 21 , wherein the concentration of paraformaldehyde is substantially 1% (v/v). 
     
     
         24 . The method of  claim 21 , wherein the incubating the platelet suspension with paraformaldehyde is at room temperature. 
     
     
         25 . The method of  claim 20 , wherein the platelet suspension is lyophilized. 
     
     
         26 . The method of  claim 25 , wherein before incubating a sample of the patient's serum or plasma with a platelet reagent comprising exposed membrane phospholipids, the method further comprises:
 resuspending a lyophilized platelet suspension in a buffer.   
     
     
         27 . The method of  claim 26 , wherein the resuspending a lyophilized platelet suspension in a buffer is to a concentration of 250,000 platelets/□l. 
     
     
         28 . The method of  claim 20 , wherein the fluorescence label of the fluorescence labeled Annexin A5 is fluorescein isothiocyanate (FITC). 
     
     
         29 . A method for diagnosis of immune thrombocytopenia (ITP), comprising:
 A. preparing:
 1. patient's antibody-platelet specific antigen complex by:
 a) providing first microparticles covered with MoAbs against platelet specific receptors; 
 b) subsequently incubating the covered first microparticles with lysate from the patient's platelets; and 
 c) subsequently adding a secondary fluorescence-labeled antibody (Ab) against human immunoglobulin; 
 
 2. normal antibody-platelet specific antigen complex by:
 a) providing second microparticles coated with MoAbs against platelet specific receptors; 
 b) subsequently incubating the covered second microparticles with lysate from platelets from an individual not having a platelet-related pathology; and 
 c) subsequently adding a secondary fluorescence-labeled Ab against human immunoglobulin; and 
 
   B. comparing a first quantity of secondary-labeled antibody adsorbed on said incubated first microparticles, indicating the quantity of immunoglobulin attached to antibody-platelet specific antigen complexes in the patient, with a second quantity of secondary-labeled antibody adsorbed on said incubated second microparticles, indicating the quantity of immunoglobulin attached to antibody-platelet specific antigen complexes in the individual not having a platelet-related pathology, wherein when the first quantity is substantially more than the second quantity, indicating the presence of antibodies against platelet specific antigens in said patient's sample.   
     
     
         30 . A system for diagnosing platelet-related pathology, comprising at least one of the kits selected from the group consisting of:
 Kit I for diagnosis of APS, comprising:
 A. a platelet reagent comprising exposed membrane phospholipids; and 
 B. a fluorescently labeled Annexin A5; 
   Kit II for diagnosis of ITP, comprising:
 A. microparticles covered with MoAbs against platelet specific receptors and/or microparticles covered with normal platelet lysate; 
 B. a secondary fluorescence-labeled Ab against human immunoglobulin; 
   Kit III, comprising:
 A. A. labeled MoAbs against CD41a, and/or labeled MoAbs against CD62p; 
 B. labeled MoAbs against activated CD41; and 
 C. platelet stimulants selected from the group consisting of one or more of adenosine-di-phosphate (ADP), arachidonic acid and thrombin receptor agonist/activating peptide (TRAP); 
   Kit IV, comprising:
 A. a labeled MoAb against platelet glycoproteins; and 
 B. a labeled Annexin A5; 
   Kit V for diagnosing Bernard-Soulier Syndrome, comprising:
 A. a first labeled MoAb against platelet specific glycoproteins excluding CD 42; and 
 B. a second labeled MoAb against CD42 (GP Ib-IX-V complex); 
   Kit VI for diagnosing Glanzmann trombasthenia syndrome, comprising:
 A. a labeled MoAb against platelet glycoproteins excluding CD41a; and 
 B. a labeled MoAb against CD41a; 
   Kit VII for diagnosing drug inhibitory effect, comprising:
 A. labeled MoAbs against platelet glycoproteins CD41a or CD61; 
 B. labeled MoAbs against CD62p; 
 C. labeled MoAbs against activated CD41a; and 
 D. platelet stimulants selected from the group consisting of one or more of adenosine-di-phosphate (ADP), arachidonic acid and thrombin receptor agonist/activating peptide (TRAP); 
   Kit VIII for determination of on-going real-time in vivo, prothrombotic activity, comprising:   A. a MoAb against platelet glycoproteins CD41a (GPIIb/IIIa) or CD61;   B. anti-activated CD41a (GPIIb/IIIa) antibody;   C. anti-CD62p (P-selectin) antibody, or anti CD63 antibody, and   D. fluorescently labelled Annexin A5 protein.   
     
     
         31 . The system of  claim 30 , wherein Kit I for diagnosis of APS further comprises:
 An Ab for platelet identification.