1. Field of the Invention
The present invention relates to a platelet/leukocyte interaction assay allowing for point of care assessment of interaction of platelets and leukocytes, and the reagents therefor.
2. Discussion of the Background
Platelets are known to interact with leukocytes both as a consequence of contact during normal blood flow (Stone and Nash, British Journal of Haematology, 105:514-22, 1999; Lorenz et al, Blood Coagulation and Fibrinolysis, 9:S49-59, 1998) and as a consequence of various pathological processes (Rinder et al, Journal of Cardiovascular Surgery, 118:460-6, 1999; Pevton et al, Journal of Vascular Surgery, 27:1109-15, 1998; Stuard et al, International Journal of Artificial Organs, 21:75-82, 1998; Gawaz et al, European Journal of Clinical Investigation, 25:843-51, 1995).
Pathological conditions such as Unstable Angina, Coronary Artery Disease (CAD), and Stroke are characterized by high levels of platelet and leukocyte activity. Measurement of platelet/leukocyte interaction can be predictive of these pathological states, particularly in combination with other diagnostic factors. Also, measurement of platelet/leukocyte interaction can be used as a means of monitoring therapy directed toward altering platelet and/or leukocyte function.
Exposure of flowing blood to artificial surfaces has been shown to enhance platelet/leukocyte interaction. The cell types involved and the extent of the interaction vary with the composition of the artificial surface in contact with the blood (Gawaz et al, Artificial Organs, 23:29-36, 1999).
Although platelet/leukocyte interactions have been quantified using various techniques (Hendricks et al, U.S. Pat. No. 5,503,982; Rinder et al, Blood, 78:1760, 1991;), assessment of the interaction has relied upon measurement of circulating platelet/leukocyte complexes. Measurements, to date, have taken the form of evaluating pre-existing platelet/leukocyte interactions in a blood sample.
In some pathological conditions (e.g. Acute Myocardial Infarction, AMI; post Angioplasty, PTCA; etc.) platelet/leukocyte complex formation is associated with interaction with damaged subendothelium, whether directly (plaque formation) or indirectly (release of biochemical markers such as ICAM-1, see Hendricks et al, U.S. Pat. No. 5,503,982).
Current assay systems used to assess platelet/leukocyte interactions, as exemplified by Hendricks et al (U.S. Pat. No. 5,503,982), evaluate pre-existing (circulating) platelet/leukocyte complexes and do not utilize a component representative of the vessel subendothelium (i.e. extracellular matrix) or other solid-phase stimulus. Moreover, the threshold at which discrete platelets and leukocytes interact could vary depending on the activation status of these cells at the time of testing. It is known in the art that platelet and/or leukocyte activation is a necessary prerequisite of platelet/leukocyte binding. It is also known in the art that certain pathological conditions are associated with upregulation of platelet and/or leukocyte activity. However, the upregulation in cellular activity associated with the pathological process may be insufficient to support platelet/leukocyte complex formation without additional stimulation and moreover, may not be detectable using conventional systems, which lack a stabilizing solid-phase support upon which the platelet/leukocyte complex could be maintained. (A solid-phase stimulus could be used as a means of localizing pre-existing platelet leukocyte complexes and/or inducing complex formation and localization in cells predisposed to do so.)
It is desirable that assay systems designed to incorporate the use of a solid-phase component such as immobilized subendothelial/extracellular matrix be facile, rapid and of reasonable cost to be useful in detecting platelet/leukocyte interaction in a clinical setting.
The present invention addresses shortcomings of previous methods and technologies by using microparticles of various compositions coated with plasma proteins and/or extracellular matrix proteins, either singly or in combination, to facilitate rapid assessment of platelet/leukocyte binding.
Platelets can interact with leukocytes through various mechanisms, such as contact during normal blood flow (Lorenz et al, Blood Coagulation and Fibrinolysis, 9:S49-S59, 1998), or as a consequence of a pathological process associated with platelet hyperactivity (Spanygenberg, Thrombosis Research, 74:S35-S44, 1994; Rinder et al, Journal of Cardiovascular Surgery, 118:460-6, 1999) or due to an inflammatory process (Gawaz et al, European Journal of Clinical Investigation, 25:843-51, 1995). Receptors found on the platelet surface interact with receptors found on various leukocytes through direct bridging or through an indirect linkage involving intermediary molecules (Weber and Springer, Journal of Clinical Investigation, 100:2085-93, 1997). Upregulation of platelet and/or leukocyte activity favors enhanced platelet/leukocyte interaction (Rinder et al, 1999; Stone and Nash, British Journal of Haematology, 105:514-22, 1999; Konstantopoulos et al, 1998; Gawaz et al, 1995; Spanenberg, 1994).
Individuals with Coronary Artery Disease (CAD), Diabetes or Cerebrovascular Ischemia demonstrate both platelet hyperactivity and an ongoing inflammatory process (Michelson and Furman, Current Opinion in Hematology, 6:342-8, 1999). Treatment of patients with CAD has involved the use of anti-platelet agents and anti-inflammatory medications (Vorchheimer et al, JAMA 281:1407-14, 1999; Mannaioni et al, Inflammation Research, 46:4-18, 1997).
Platelet/monocyte (Hendricks et al, U.S. Pat. No. 5,503,982) and platelet/neutrophil (Gawaz et al, European Journal of Clinical Investigation, 25:843-51, 1995) interaction have been suggested to be predictive of acute myocardial infarction (AMI) and inflammation, respectively. A corollary to platelet/leukocyte interaction in various pathological conditions is involvement of the vessel wall, whether in plaque formation for example, or a localized inflammatory reaction.
Platelet function assessment using immobilized extracellular matrix proteins has been described by Shaw and Stewart (U.S. Pat. No. 5,427,913). The authors demonstrated that von Willebrand factor (VWF) immobilized on polystyrene beads could be used to activate platelets and thereby determine the functional status of platelets from patients with platelet function defects. In addition, the authors also demonstrated that the effects of agents designed to alter platelet function could be monitored using bead-immobilized VWF as a stimulus. The results of these studies underline the importance of evaluating a normal hematological interaction or the hematologic consequence of a pathological state in the presence of an agent that mimics components of the vessel wall.
Although Shaw and Stewart describe methods and compositions of determining platelet function, there is no description or suggestion of using their method for evaluating platelet/leukocyte interaction.
CVDI""s TAS(trademark) analyzer measures the kinetics of fibrin polymerization following activation of the coagulation pathway in a patient""s blood sample. The TAS(trademark) analyzer and disposable were designed for use with whole blood in a point-of-care setting. Paramagnetic iron oxide particles (PIOP) are an essential component of the detection system for each of the tests developed for the TAS(trademark) analyzer. The PIOP and other lyophilized ingredients for a particular test are located in the shallow reaction chamber of the TAS test card disposable. In addition to PIOP, the test reagent may contain buffers, stabilizers, fillers and specific coagulation pathway activator or agents. A test is initiated by insertion of a dry-chemistry test card into a slot of the TAS(trademark) analyzer that automatically positions the test card reaction chamber above an electromagnet. This chamber is also illuminated with infrared light from a light emitting diode. The instrument measures reflected infrared light from the surface of the test card by means of a solid state photodiode detector. A test is automatically initiated when the analyzer photodetector measures a change in reflected light intensity when blood or plasma is added to the sample well of the test card and, through capillary action, is pulled into the reaction chamber. The activators present in the reaction chambers stimulate the coagulation cascade in the patient""s sample to produce thrombin, which in turn catalyzes the formation of the fibrin clot.
During a clotting test the TAS(trademark) analyzer electromagnet oscillates on and off every second. The magnetic particles stand up when the electromagnet is on, causing more light to be reflected to the detector, and fall down when it is off, causing less light to be detected. This movement of PIOP produces an alternating current (AC) signal from the photodetector. As the test proceeds, more and more fibrin polymerization occurs and the PIOP movement is less. The analyzer in accordance with predetermined algorithms interprets the signal produced by the relative movement of the PIOP and reports an endpoint (clotting time) appropriate for each test.
Although PIOP is an integral component of the TAS(trademark) detection system, it does not participate directly in activation of the coagulation cascade or fibrin polymerization. To prevent undesired interactions between PIOP and activators within the reaction chamber of a test card, the PIOP is coated or blocked with bovine serum albumin (BSA). BSA is a protein commonly used by those skilled in assay development to prevent unwanted interactions between surface components of a test and its active ingredients. The TAS(trademark) system was designed to monitor fibrin polymerization and not interactions between platelets and leukocytes.
Accordingly, one object of the present invention is to provide an assay for platelet/leukocyte interaction that can be used with a variety of blood products as the sample, including whole blood, with ease of detection.
A further object of the present invention is to provide an assay for platelet/leukocyte interaction that can be used on the TAS(trademark) system for rapid determination of platelet/leukocyte interactions.
A further object of the present invention is to provide a platelet/leukocyte interaction assay reagent, preferably in a dry chemistry test card format that can be used in the assay of the present invention.
A further object of the present invention is to provide a method for diagnosis of conditions resulting in platelet/leukocyte interactions using the assay of the present invention.
A further object of the present invention is to provide a method for determination of platelet and/or leukocyte hyperactivity using the assay of the present invention.
These and other objects of the present invention have been satisfied by the discovery of a method of assessing platelet/leukocyte interaction, comprising:
contacting a whole blood or blood-derived sample with a solid-phase stimulus, wherein said solid-phase stimulus has bound to a surface thereof a ligand selective for binding platelets or leukocytes, and
detecting formation of one or more platelet/leukocyte/solid-phase stimulus complexes, and the reagents for performing the method, as well as the use of the method for detection of the occurrence of platelet/leukocyte interaction caused by various disease conditions and the predisposition of subjects to those conditions.