EP1210101A1 - Verwendung der zusammenziehbaren kraft von blutplättchen und des elastischen modulus von blutgerinnseln als klinische marker - Google Patents

Verwendung der zusammenziehbaren kraft von blutplättchen und des elastischen modulus von blutgerinnseln als klinische marker

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Publication number
EP1210101A1
EP1210101A1 EP00957363A EP00957363A EP1210101A1 EP 1210101 A1 EP1210101 A1 EP 1210101A1 EP 00957363 A EP00957363 A EP 00957363A EP 00957363 A EP00957363 A EP 00957363A EP 1210101 A1 EP1210101 A1 EP 1210101A1
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EP
European Patent Office
Prior art keywords
measurement
patients
platelet
risk
clot
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00957363A
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English (en)
French (fr)
Other versions
EP1210101A4 (de
Inventor
Marcus E. Carr, Jr.
Ashok Krischnaswami
Erika Martin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hemodyne Inc
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Hemodyne Inc
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Filing date
Publication date
Application filed by Hemodyne Inc filed Critical Hemodyne Inc
Publication of EP1210101A1 publication Critical patent/EP1210101A1/de
Publication of EP1210101A4 publication Critical patent/EP1210101A4/de
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention is related to a method which uses platelet contractile force (PCF) measurements and/or clot elastic modulus (CEM) as clinical markers to allow rapid assessment of a patient's risk of atherosclerosis or a patient's bleeding risk during surgical procedures.
  • PCF platelet contractile force
  • CEM clot elastic modulus
  • Atherosclerosis and thrombosis The interplay between atherosclerosis and thrombosis is complex. Multiple local and systemic thrombotic risk factors have been shown to play a role in the destabilization of the vulnerable plaque and its clinical sequelae. Aside from local factors such as the degree of plaque erosion or stenosis, well known systemic risk factors include cholesterol, diabetes mellitus, tobacco, cocaine, hypertension, elevated fibrinogen, impaired fibrinolysis, activated platelets and products or by-products of the coagulation cascade.
  • Platelet activation occurs in the acute coronary syndrome 1 .
  • the acute coronary syndrome is a continuum from unstable angina to non-Q and Q-wave myocardial infarction depending on the extent and duration of ischemia.
  • Reduction in coronary blood flow occurs due to platelet aggregation, vasoconstriction at the site of coronary artery stenosis and endothelial injury.
  • Endothelial injury may result from plaque ulceration, hemodynamic factors, systemic arterial hypertension, cardiac catherization, balloon angioplasty, etc. 2 ' 3 ' 4 ' 5 . It is critical to recognize the acute coronary syndrome in patients who present to an emergency department with chest pain in order to prevent inappropriate discharge and adverse consequences 6 ' 7 .
  • Sensitive assays of individual components of the coagulation cascade have made laboratory evaluation of a biochemical hypercoagulable state possible.
  • Prospective studies have suggested that elevated levels of factor VII, fibrinogen and other markers are associated with the development of ischemic cardiac events.
  • traditional risk factors have not explained the increased cardiovascular risk in certain high risk groups such as diabetics.
  • the contribution of platelet activation in patients presenting with an acute coronary syndrome has been well established.
  • tests of platelet function have not reflected changes predictive of a hypercoagulable state.
  • Platelet aggregation may be a useful marker for predicting mortality in coronary events .
  • aggregation techniques that have been used to evaluate platelet dysfunction have been limited to a few non-cardiac clinical situations 13 .
  • Measurement of P-selectin 13 , ICAM-1 and/or E-selectin l4 as early markers of platelet activation is ill suited to an emergency department setting because the techniques of flow cytometry and ELISA are time consuming, require technical expertise and need substantial dedicated equipment. Newer methods to assess platelet function are needed.
  • the Hemodyne® Hemostasis Analyzer is an instrument which measures platelet activity (platelet contractile force, PCF) and clot strength (clot elastic modulus, CEM) in physical units of dynes & dynes/cm 2 respectively 15 16 .
  • PCF platelet contractile force
  • CEM clot elastic modulus
  • a blood sample obtained from a patient is deposited in a sample cup 10 using a syringe 12 or other suitable device.
  • the cup 10 is placed in a base 14, and a head piece 16 is inserted into the cup 10.
  • This causes the blood 18 to distribute itself along the surface of the head piece 16 and up the sides of the cup 10.
  • the force developed during contraction pulls the head piece 16 and base 14 closer together, and this force is measured using sensors connected to either or both the head piece 16 or base 14.
  • a force can be periodically applied to the blood 18 during clotting by the head piece 16.
  • PCF and CEM are potentially useful tools in a variety of clinical situations 17 18 19 .
  • PCF depends on thrombin production, platelet count, platelet viability and the degree of platelet inhibition 15 ' 20 ' 21 .
  • CEM depends on the fibrinogen concentration, fibrin structure and platelet function 15 . Inhibition of f ⁇ brin(ogen) binding to GP Ilb/IIIa blockade either by disruption of GP Ilb IIIa or by competitive blockade, inhibits platelet mediated force development and results in clot structures which are substantially less resistant to deformation by outside forces 22 .
  • This invention provides a methodology where PCF and CEM are used to rapidly assess the risk of a patient for thrombotic events associated with atherosclerosis or with the risk of bleeding associated with deficient platelet function. Prior studies have not demonstrated that these measures could be used effectively as a screen for probable patient risk. In this invention, it is demonstrated that there is a statistically relevant correlation between PCF and/or CEM and thrombotic risk in patients with atherosclerosis. It is also demonstrated that there is a statistically relevant correlation between PCF and/or CEM and a patient's bleeding risk. In the emergency department, the measurement of PCF and CEM could be used to detect evidence of hyper-platelet function associated with atherosclerosis in patients presenting with chest pain.
  • Figure 1 is a schematic diagram of measurement system used to monitor platelet contractile force and clot elastic modulus during clot formation in whole blood.
  • Anticoagulated whole blood is placed in a shallow conical cup and clot formation is initiated by the addition of clotting agent.
  • a conical upper plate Prior to clot formation a conical upper plate is lowered onto the upper surface of the sample, trapping the sample between parallel surfaces separated by a known distance. Platelets within the sample attempt to collapse the clot resulting in a downward force on the upper platelet. This downward force is continuously monitored and the elastic modulus of the forming clot is intermittently measured.
  • FIG 2 is a graph which shows that preoperative platelet contractile force (PCF) is elevated in patients with documented coronary artery disease (CAD) who are undergoing coronary artery bypass grafting (CABG). The forces are higher in all such patients but are much higher in such patients who are not taking aspirin. Aspirin appears to decrease but does not normalize PCF values.
  • Figure 3 is a bar graph which shows the effect of aspirin on whole blood clot elastic modulus (CEM) in patients with documented CAD who are undergoing CABG. CEM were measured at the time of maximal clot retraction. Values for patients with CAD taking or not taking aspirin were significantly elevated over those of asymptomatic control volunteers (p ⁇ 0.0002).
  • PCF preoperative platelet contractile force
  • Figure 5 is a bar graph which shows PCF values increase with the severity of the patient's clinical presentation. While all groups of patients had significantly elevated PCF values, those patients with electrocardiographic evidence of cardiac ischemia (levels II and I) had the highest PCF levels.
  • Figure 6 is a bar graph which shows CEM is elevated in patients presenting in the emergency department with a complaint of chest pain. Upon presentation patient CEM values were significantly higher
  • Figure 15 is a line graph that shows that in the Italian study, PCF did not change with age in females under the age of 60.
  • Figure 16 is a line graph that shows that PCF increases with platelet count in all populations studied.
  • the slope of the regression line allows calculation of an average force per platelet number for varying populations. Patients with known arteriovascular disease have higher force per platelet values than asymptomatic age matched controls (see table 3).
  • the invention contemplates making PCF and/or CEM measurements on whole blood clots obtained from patient samples during clot formation, and then using these measurements as a screen to identify patient's at risk for an adverse vascular outcome.
  • Application of this technique to clinical samples confirmed that clots with low PCF and/or CEM were less hemostatic and placed the patient at risk for bleeding in conditions such as primary fibrinolysis, Glanzmann thrombasthenia and coronary artery bypass procedures.
  • PCF values less than 4 kilodynes after 720 seconds of clotting are abnormally low.
  • Patients with severe thrombasthenia typically have PCF values below 2 kilodynes.
  • CEM is affected by both fibrinogen concentration and platelet function.
  • CEM values less than 14 kilodynes per cm 2 are indicative of deficient clot formation.
  • application of this technique to clinical samples confirms that elevations of PCF and CEM are associated with arteriovascular disease and increased risk of arterial thrombosis.
  • patients with coronary artery disease, hypercholesterolemia, and diabetes mellitus have much higher PCF and CEM values than asymptomatic controls.
  • patients who present to the emergency department with complaints of chest pain have significantly elevated forces and the degree of elevation increases with increasing clinical risk.
  • PCF increases with age in males. However, while slightly higher in young females than in young males, PCF does not increase with age in females at least to the point of menopause.
  • Elevated whole blood PCF and CEM values should help identify patients at increased risk of arterial thrombosis due to atherosclerosis and enhanced platelet function. These measurements should prove useful during the triage of chest pain patients in the emergency department as well as the screening of asymptomatic patients with positive family histories or other documented risk factors for atherosclerosis. Since most therapeutic measures used to acutely treat arterial thrombosis reduce PCF and/or CEM, these parameters can also have applications as monitors of clinical response. Screening of asymptomatic individuals with PCF and CEM measurements could be useful in indentifying patients who might benefit from more invasive and expensive testing. This can be accomplished by testing a small sample of venous blood.
  • PCF value is greater than one standard deviation above the mean of normals, greater than 8.5 kilodynes and the patient has a positive family history or other risk factors (diabetes, cigarette smoking, hypercholesterolemia, etc.), then they should undergo additional testing. If the PCF is normal, 6.9 ⁇ 0.7 kilodynes, no additional testing is needed. If the PCF is above 7.6, testing at intervals to assess whether the force is increasing would be appropriate.
  • Noncardiac CP with As appropriate clear-cut diagnosis o The hospital course for admitted patients was followed for pre-selected endpoints.
  • Platelet aggregation was measured utilizing a Chrono-Log® whole blood lumi-aggregometer. 450 ⁇ L of citrated whole blood was mixed with 450 ⁇ L of saline and placed in an aggregometer cuvette equipped with a stirring bar. Platelet aggregation was induced by the addition of collagen (3 mg/ml, Chronolog, Havertown, PA) and the change in impedance was monitored for six minutes.
  • Human thrombin greater than 90% alpha, was purchased as a lyophilized powder from Sigma Chemical Co. (St. Louis, MO). The material with a specific gravity of 3000 NIH units/ml was dissolved in water, diluted with 0.10 M NaCl to a final concentration of 225 units/ml, divided into 50 ⁇ L lots and frozen at 80° C. Thrombin was free of plasmin and plasminogen. Nanopure water was used in the preparations of all solutions. Clotting was initiated by adding thrombin (1 NIH unit/ml) and calcium chloride (lOmM) to 700 ⁇ L of whole blood. Force development was measured for 900 seconds.
  • the Hemodyne® RM-2 hemostasis analyzer measures forces generated by platelets within a clot formed between two parallel cone-shaped plates ( Figure 1).
  • the temperature of the sample is held constant via thermal control of the bottom cone, which serves as the sample cup.
  • the upper cone is centered above the cup and lowered into the clotting solution. As the clot forms, it attaches to the inner walls of the cup and upper cone. The entire sample volume is contained between the upper and lower surfaces.
  • Clot Elastic Modulus is obtained simultaneously with the PCF.
  • stress equals the applied force (F app
  • strain is the degree of shape change induced by the applied force.
  • the strain induced by F apphed is measured as the change in gel thickness, which is the same as the change in the gap between the two cones. Strain is recorded as the ratio of the change in gap distance (d,) to the original gap distance (d 0 ).
  • Chest pain patients were injected with ⁇ 20mCi sestamibi in the emergency department (not more than 6 hours after the last episode of chest pain) as per the chest pain protocol.
  • Perfusion images were evaluated by an experienced nuclear medicine attending physician and all data were made available to the physicians treating the physician. For purposes of this study, images were classified as either positive or negative for acute myocardial infarction (MI) or ischemia.
  • MI myocardial infarction
  • a positive study required a discrete perfusion defect with associated abnormalities in wall motion and thickening. Studies visually interpreted as normal, equivocal or consistent with cardiomyopathy were considered negative for acute coronary syndromes. Normal studies had normal perfusion and systolic function without regional wall motion or thickening abnormalities. Studies consistent with cardiomyopathy showed reduced systolic function on cinematic replay with either normal perfusion or perfusion defects without accompanying segmental wall motion abnormalities.
  • Endpoints Patients who were admitted to the hospital were followed for specific endpoints.
  • the primary endpoints were myocardial infarction, death, or urgent revascularization (coronary artery bypass graft surgery (CABG), or percutaneuous transluminal coronary angioplasty (PTCA) during the initial evaluation or within 5 days of admission.
  • CABG coronary artery bypass graft surgery
  • PTCA percutaneuous transluminal coronary angioplasty
  • Myocardial infarction was defined as CK-MB mass >_8.0 ng/dl with a relative index (CK-MB mass/total CK x 100) > 4.0.
  • MI Magnetic resonance
  • Anginal symptoms were considered typical if they were described as pressure, tightness, squeezing, burning, heaviness, crushing, or indigestion, or were similar to prior symptoms of angina.
  • the baseline demographics in the patients with chest pain and control patients are given in Table 2.
  • the mean age was 52.8 + 13.9 (23-87) in the chest patients as compared to 37.7 + . 10.1 (19-62) which was statistically significant.
  • a significant difference in race and sex were also present.
  • the chest pain patients had a greater number of traditional risk factors as compared to the control population.
  • Platelet Force Per Platelet (FPP). PCF is dependent upon and increases with increasing platelet concentration (Figure 16). However, the increased PCF values in chest pain patients were not due to elevated platelet counts (Table 2). Instead, the slope of the force versus platelet concentration plot ( Figure 16) was increased in similar plots for CAD and DM patients. Such plots allows the calculation of a new parameter - force per platelet (FPP). Table 3 shows FPP was highly significantly elevated in CAD and
  • Carr ME Measurement of platelet force: the Hemodyne® hemostasis analyzer. Clin Lab Management Rev 9:312-319,1995. 17.
  • Carr ME Zekert SL. Abnormal clot retraction, altered fibrin structure, and normal platelet function in multiple myeloma. Am J Physiol (Heart
  • Greilich PE Carr ME, Zekert SL, Dent RM: Quantitative Assessment of Platelet Function and Clot Structure in Patients with Severe Coronary Artery Disease. Am JMedSci 307:15-20,1994.

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EP00957363A 1999-08-13 2000-08-11 Verwendung der zusammenziehbaren kraft von blutplättchen und des elastischen modulus von blutgerinnseln als klinische marker Withdrawn EP1210101A4 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US14859599P 1999-08-13 1999-08-13
US148595P 1999-08-13
PCT/US2000/021848 WO2001012211A1 (en) 1999-08-13 2000-08-11 Method of using platelet contractile force and whole blood clot elastic modulus as clinical markers

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EP1210101A1 true EP1210101A1 (de) 2002-06-05
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EP (1) EP1210101A4 (de)
JP (1) JP2003507693A (de)
KR (1) KR20020043556A (de)
CN (1) CN1370075A (de)
AU (1) AU778160B2 (de)
CA (1) CA2380972A1 (de)
WO (1) WO2001012211A1 (de)

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US6573104B2 (en) * 2001-05-10 2003-06-03 Hemodyne, Incorporated Disposable cup and cone used in blood analysis instrumentation

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US5293772A (en) * 1992-01-17 1994-03-15 Center For Innovative Technology Instrumentation and method for evaluating platelet performance during clotting and dissolution of blood clots and for evaluating erythrocyte flexibility
US5205159A (en) * 1992-01-17 1993-04-27 Virginia Commonwealth University Apparatus and method for measuring clot elastic modulus and force development on the same blood sample
US5691160A (en) * 1992-08-14 1997-11-25 Biogen, Inc. Effects of actin filaments of fibrin clot structure and lysis

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CN1370075A (zh) 2002-09-18
AU778160B2 (en) 2004-11-18
EP1210101A4 (de) 2005-10-12
WO2001012211A1 (en) 2001-02-22
CA2380972A1 (en) 2001-02-22
KR20020043556A (ko) 2002-06-10
JP2003507693A (ja) 2003-02-25

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