JP2003505678A - Method for measuring coagulation factor activity in whole blood - Google Patents

Abstract

  (57) [Summary] The present invention relates to a method for rapidly assessing the overall clotting properties of a patient's blood sample by measuring and comparing blood clotting times with and without the addition of a procoagulant or anticoagulant inhibitor. . When the sample is whole blood, the resulting clotting time represents the overall clotting activity of the plasma and blood cell components, and indicates existing or impending pathologies arising from abnormal clotting capacity. The present invention also provides a method of measuring a patient's risk for a thrombotic event by determining a functional current level of one or more procoagulants or anticoagulants in whole blood. Further, a method for measuring the effectiveness of anticoagulant treatment is provided. Kits for performing the method of the invention are also provided.

Description

Detailed Description of the Invention

This invention was made with government support under grant number HL48872, which was sponsored by the National Institutes of Health. The US Government has certain rights in this invention.

FIELD OF THE INVENTION The present invention relates generally to the field of medical diagnostics and disease prevention. More particularly, the present invention provides for the determination of blood coagulation rates using whole blood samples in the presence or absence of at least one inhibitor of procoagulant or anticoagulant. The present invention relates to a diagnostic method and a test kit for rapidly evaluating blood coagulation activity. The clotting activity of individual blood samples and the difference in activity between samples is an indication of the presence or potential development of a particular medical condition.

[0003] tendency BACKGROUND blood invention solidifies rapidly too, the development of more serious medical conditions, it is important sign of progression and recovery. These conditions result directly from or are modulated by the blood coagulation process. Examples of such symptoms include, among others:
Includes heart attack, stroke, coronary artery disease, deep vein thrombosis, and pulmonary embolism.
Of these diseases, coronary artery disease is the leading cause of death in the United States.

Furthermore, certain clinical conditions such as vascular disease, surgery, trauma, malignant tumors, prosthetic vascular devices, general anesthesia, pregnancy, oral contraceptives, systemic lupus erythematosus, and infectious diseases can result in blood coagulation that is harmful to the individual. Predisposes to the event. Often, patients with acute symptoms that appear to result from abnormal blood coagulation appear in the emergency room. A method for rapidly detecting a patient's current risk for clot formation in a whole blood sample will help determine whether to apply or exclude thrombotic events and coagulopathy. This method will also improve the performance of emergency health care for patients in need while early identifying patients who may develop potentially fatal coagulation pathologies.

Blood may also clot too slowly or not at all, which may cause bleeding or other blood coagulation disorders. Hemophilia is an example of an inherited bleeding disorder. In addition, diseases affecting the liver such as alcoholic cirrhosis and acute and chronic hepatitis,
Since the liver is the organ that synthesizes many coagulation factors, it is associated with multiple coagulation abnormalities.

The most well known hereditary coagulation disorders are hemophilia A and B, which are associated with decreased activity of Factor VIII and Factor IX, respectively. The severity of the disorder depends on the degree of deficiency of each blood coagulation factor. Severe cases develop at an early age, and children with hemophilia show easy bleeding in large joints such as the knee and a marked loss of clot formation. In its milder form, hemophilia does not appear until older.

Treatment of hemophilia generally consists of infusion of a blood product concentrate in which large amounts of blood coagulation factors, Factor VIII or Factor IX, are present. Many hemophiliacs can lead a relatively normal life, but require special attention when participating in sports and during surgery or dental care. Unfortunately, 10% of hemophiliacs present antibodies against factor VIII and are difficult to treat.

A condition in which blood coagulates too quickly (ie, hypercoagulability).
ability)) is also a pathological symptom. Diffuse intravascular coagulation (DIC) is an example of an acquired blood coagulation disorder characterized by pathologically rapid blood coagulation.

Blood coagulation is a complex process involving multiple initiators, activator cascades, enzymes, and modulators that ultimately forms fibrin, which polymerizes into insoluble clots. The intrinsic and extrinsic blood coagulation pathways are described, for example, in Davie et al., “The Coagulation Cascade: Initiation, Maintenan,”
ce, and Regulation) ", Biochemistry, vol.30 (43): 10363-70 (1991).

[0010] Classically, the tendency of blood to clot is determined manually or automatically by measuring the time taken for a sample of plasma or blood to form insoluble fibrin chains or clots. For example, clot formation may be performed visually by observing the formation of fibrin chains, or by an automated method by measuring mechanical or electrical impedance to detect viscosity changes or by photo-optical detection. It may be detected.

The blood coagulation time can be measured directly on freshly drawn blood without the addition of anticoagulants. Alternatively, blood containing an anticoagulant that binds calcium ions such as citric acid may be used. In this case, blood coagulation time measurements are initiated by adding calcium salts to reverse the effect of the anticoagulant. This latter measurement is called the recalcification time. Prothrombin time (PT
) Measurement, activated partial thromboplastin time (APTT) measurement, thrombin time measurement, and fibrinogen level test. The detection of thrombotic events
It can be carried out by measuring the level of soluble fibrin or fibrin degradation products in the circulation.

[0012] Determining clotting time is determined by the unusually long clotting time of hemophilia, Von, which is typically diagnostic.
Most commonly used to diagnose diseases such as Willebrand disease, Christmas disease and certain liver diseases. Despite the many serious conditions associated with abnormally fast blood clotting, current assays are diagnostic in identifying almost all but these most rapid pathological clotting pathologies. It does not have the sensitivity that is well worth it.

The PT and APTT tests are not useful for detecting clinically pathological hypercoagulable states. Generally, these tests are used to detect symptoms of prolonged blood clotting time, ie, hypocoagulability. Usually these tests are performed on plasma, but both plasmas do not contain activated platelets and monocytes, which can significantly contribute to modification of the coagulation state. In addition, these tests used reagents, which are themselves procoagulants, in addition to the samples, with plasma clotting times of approximately 6 minutes to values of approximately 10-13 seconds and 25-39 seconds for PT and APTT, respectively. Shorten to. "Laboratory Test Handbook", 4th Edition, Lex
See i-Comp Inc., 1996, pp. 227 (APTT) and 262 (PT). These common plasma-based blood coagulation time measurements are the best predictors of thrombotic events modulated by cellular components of blood by eliminating the effects of cellular components of whole blood such as monocytes. And does not give a complete diagnostic value.

Further, monitoring anticoagulant therapy, such as heparin and warfarin,
It would be improved by measuring the clotting capacity of whole blood rather than plasma alone. The presence of these therapeutically administered anticoagulants modulates the ability of blood coagulation by cells as well as soluble (plasma) blood components.

There are several important initiators and modulators of the blood coagulation process in whole blood. One such molecule is a procoagulant protein, also known as blood coagulation factor III and called tissue factor, which is a transmembrane glycoprotein present on the surface of circulating cells known as monocytes. Tissue factor is also found in phospholipid vesicles in plasma. Elevated circulating tissue factor levels are associated with many thrombotic disorders and conditions. For example, tissue factor has been found in circulating cells and plasma vesicles from patients with cancer, infections, and thrombotic disorders such as heart attack and stroke. The level of tissue factor activity in whole blood is a diagnostically useful parameter for identifying patients at risk for thrombotic events.

Tissue factor (TF) must form an active complex with plasma coagulation factor Factor VII or its activated form Factor VIIa. The TF: Factor VIIa complex then contains the zymogens Factor IX and Factor X, respectively, in the enzymatically active form of Factor I.
Activates factor Xa and factor Xa. Factor Xa binds to Factor Va to form a prothrombinase complex (active coagulant), which then cleaves prothrombin to thrombin. Thrombin, in turn, cleaves fibrinogen to produce fibrin, which forms a clot.

Methods for the direct measurement of tissue factor have been described. In addition to the immunoassay method described in U.S. Pat.No. 5,403,716, U.S. Pat.No. 4,814,247 and Spillert and L.
Exposure of whole blood to endotoxin as described in azaro, 1993, J. Nat. Med. Assoc. 85: 611-616 allows assessment of TF levels within hours. The method using endotoxin measures the modified remineralization time of a blood sample. This assay represents tissue factor expression that is present when endotoxin or other immunomodulatory agent creates a condition that mimics a disease or trauma, thereby predisposing a patient to blood clotting when experiencing such a condition. taking measurement. The present invention provides another important method for assessing tissue factor, which is a simple method for determining the actual value of tissue factor activity in circulating whole blood, thereby providing the patient's current clot. It is a method of measuring the formation risk.

For example, a sonoclott blood coagulation and platelet function analyzer (Sonocl) that measures the viscous resistance of fibrin chains using a disposable vibration probe immersed in whole blood.
ot Coagulation and Platelet Function Analyzer) (Sienco, Wheat Ridge, CO
) Can be used to measure fibrin formation. Instead, a "HEMOCHRON ^™ " system (International Technidyne Corp.) that uses magnets that are precisely aligned in the test tube and a magnetic detector that is located in the instrument that detects clot formation.
May be used.

Currently, there are no standard clinical assays to measure tissue factor (TF) or tissue factor pathway inhibitor (TFPI) functional activity. However, in the research setting, there are assays that measure the activity of certain procoagulant or anticoagulant substances. For example, the percentage of Factor XII activity present in plasma can be determined by the degree of correction obtained when the plasma is added to severely Factor XII deficient plasma.
This assay is a modification of the APTT test, which measures the ability of patient plasma to "correct" APTT in plasma containing less than 1% Factor XII. The amount of correction achieved by diluting the patient's plasma is compared to the correction obtained with known concentrations of Factor XII. Normal plasma is considered to exhibit 100% correction.

The percentage of thrombin (factor II) activity present in plasma may also be determined by the degree of correction obtained when the plasma is added to severely factor II deficient plasma. . This assay is a modification of the prothrombin time test, with a first
The ability of the patient's plasma to "correct" the PT of plasma containing Factor II is measured. The amount of correction achieved by dilution of the patient's plasma is compared to the correction obtained with known concentrations of Factor II. Normal plasma is considered to exhibit 100% correction. However, current blood clotting factor assays of the type discussed may only be useful in a clinical setting to detect symptoms of hypocoagulability (ie, pathologically slow blood coagulation).

F 1.2 prothrombin fragment, D-dimer, soluble fibrin and thrombin /
Immunoassays exist for multiple markers of blood coagulation cascade activation, such as the antithrombin III complex. In general, these blood coagulation immunoassays have limited acceptance outside the research setting, as these kits are slow and relatively laborious ELISA procedures.

Therefore, a rapid and simple in vitro method for the evaluation of overall blood coagulation ability, which correlates with the in vivo blood coagulation risk and the influence of the specific influence of coagulation-promoting substances or anticoagulants on blood coagulation. It is desirable to provide. This assessment method provides healthcare professionals with (1) assessment of patient symptoms; (2) selection of appropriate treatment courses; (3) diagnostic and monitoring for monitoring the speed and effectiveness of surgical and non-surgical treatments. It will provide clinically useful data. No rapid method of assessing global blood coagulation was available to date to identify and evaluate the contribution of tissue factor and other procoagulant and anticoagulant substances. Detection of elevated levels of procoagulants and anticoagulants allows for earlier treatment and thereby improved prognosis. Currently, there is no whole blood coagulation assay that accurately evaluates this hypercoagulable or hypocoagulable state.
The method of the invention comprises comparing a patient's whole blood clotting time in the presence or absence of at least one inhibitor of procoagulant or anticoagulant activity to determine a hypercoagulable or hypocoagulable state. To measure.

SUMMARY OF THE INVENTION The present invention provides for global coagulation of a blood sample of a patient by measuring and comparing blood coagulation times with or without the addition of procoagulant or anticoagulant inhibitors. Provide a way to quickly assess a property. If the sample is whole blood, the resulting blood clotting time will be indicative of the overall clotting activity of the blood's plasma and cellular components, indicating existing or future pathology resulting from abnormal blood clotting ability.

One object of the present invention is a method of measuring a patient's risk for a thrombotic event by functionally determining the current level of one or more procoagulant or anticoagulant in whole blood. Is to provide.

A further object of the present invention is to provide a method for measuring the efficacy of anticoagulant drug treatments such as warfarin or low molecular weight heparin, which method first exposes a whole blood sample to an inhibitor and then It is characterized in that the coagulation activity of the whole blood sample is measured by measuring the coagulation time of the blood sample by a standard method. Coagulation time values, or the difference between control and inhibitor treated sample values, is useful for monitoring anticoagulant therapy.

A further object of the present invention is to provide a method of monitoring recovery of a patient from adverse blood coagulation related conditions by monitoring blood coagulation according to the methods described herein.

Yet another object of the present invention is to provide a diagnostic kit for measuring the clotting time of whole blood and plasma in the presence and absence of at least one inhibitor of procoagulant or anticoagulant. is there.

These and other aspects of the invention will be better understood by reference to the following detailed description.

The cause disease detailed description abnormalities range of blood coagulation of the invention. In particular, factors that increase the coagulability or prothrombin potential of blood are highly undesirable in most cases and can lead to serious medical conditions such as heart attack, stroke, coronary artery disease, deep vein thrombosis, and pulmonary embolism. In addition, certain clinical conditions such as vascular disease, surgery, trauma, malignancy, the presence of prosthetic vascular devices, general anesthesia, pregnancy, use of oral contraceptives, systemic lupus erythematosus, and infections can cause patients to have adverse blood effects. It can be a predisposition to suffer the coagulation phenomenon. These conditions modify the blood coagulation state, predominantly and potentiate the prothrombin pathway compared to the protective anticoagulant pathway.

The overall coagulability of blood is controlled by both factors contributed by the soluble (plasma) part of the blood as well as by the cellular part. Traditional clotting or blood coagulation capacity measures, such as prothrombin time (PT) and active partial thromboplastin time (APTT), among others, commonly use plasma to measure blood coagulation capacity. However, these plasma-based methods eliminate the contribution to blood coagulation capacity provided by the cell components. One example is the contribution of tissue factor to blood coagulation. As mentioned above, tissue factor is an initiator and modulator of blood coagulation and may be present in blood. High levels are associated with pathological conditions. In addition to tissue factor, other components present in or on the cellular components of blood may modulate blood coagulation capacity and may also contribute to the blood's tendency to coagulate in vivo.

In certain embodiments, the methods of the invention involve measuring whole blood coagulation with or without inhibitors of procoagulants or anticoagulants. The magnitude of the difference in clotting time with or without inhibitor is proportional to the amount of procoagulant or anticoagulant contained in the sample, ie, the greater the difference, the more factor measured. Absolute coagulation time is important in addition to the difference in coagulation time, as patients may have increased coagulation capacity due to abnormalities other than high levels of procoagulant.

To describe performing the assay of the present invention with an inhibitor of tissue factor, the modified remineralization time test described by Spillert and Lazaro, ie endotoxin incubated with a whole blood sample, is associated with tissue factor In contrast to the method that induces the synthesis of A. pneumoniae, which in turn affects the clotting properties of blood samples, the method of the present invention does not measure the effect of tissue factor synthesis on blood clotting ability. Instead, the method of the invention measures the effect of tissue factor extant in a whole blood sample on blood coagulability. See, for example, Santucci et al., “Measurement of Tissue Factor Activity in Whole B”, incorporated herein by reference.
Lood) ”, Thromb. Haemost., vol. 83 (3): 445-54 (2000).

The method of the present invention may be carried out with fresh whole blood, to which an inhibitor is added, and then the coagulability of the blood sample and the inhibitor-free sample is determined by standard methods. Alternatively, blood samples may be citrated, oxalate, ED
It may be collected in the presence of certain anticoagulants such as TA. The anticoagulant is free of anticoagulants that block the intrinsic pathway of clot formation, ie the anticoagulant may block extrinsic or common pathways. Then add the inhibitor,
Thereafter, the coagulability of blood is determined by standard methods. Any known method of measuring blood coagulation capacity may be used in the method of the present invention.

When blood is collected with an anticoagulant, the effect of the anticoagulant in the blood sample must be counteracted when measuring blood coagulation capacity or clotting time. This nullification is achieved, for example, by adding a calcium salt such as calcium chloride. The coagulation time measurement of an anticoagulated blood sample by reactivating the coagulation process by the addition of calcium salts is called remineralization time.

Any inhibitor of a procoagulant or anticoagulant is suitable for use in the methods of the invention, as long as it is specific for the particular procoagulant or anticoagulant. Suitable inhibitors include, inter alia, antibodies or substrate analogs to procoagulants or anticoagulants. In certain preferred embodiments, the analog is a peptide. Suitable inhibitors are well known to those skilled in the art and are usually commercially available.

In a preferred embodiment, inhibitory antibodies or combinations of antibodies that exhibit sufficient affinity for tissue factor may be used as inhibitors of the TF: Factor VIIa complex. In certain embodiments, two antibodies designated VD10 and VIC12 are used in combination. The concentration of the antibody or antibody combination in the reagent is provided in a form that can be easily added to the blood sample so as to give a final concentration suitable for carrying out the method of the invention. In another embodiment, the inhibitor is Factor VIIai, which is a catalytically inactive species of Factor VIIa.

Measurement of clotting time by the method of the invention also provides certain useful information of diagnostic and clinical utility in identifying and monitoring certain disease states associated with thrombosis. It may be carried out for the presence of additional compounds. Compounds such as homocysteine, tissue factor, Russell's viper snake venom and other procoagulant venoms are contemplated. Other modulators of the blood coagulation process contemplated for use in the present invention include thrombin, platelet activating factor, fibrinogen, kaolin, celite, adenosine diphosphate, arachidonic acid, collagen, and procoagulants such as ristocetin. . Factors having anticoagulant activity useful as modulators of the blood coagulation process of the present invention include protein C, protein S, antithrombin III, thrombomodulin, tissue plasminogen activator, urokinase, streptokinase, tissue factor pathway inhibitor and phon.・ Billbrand factor is mentioned. A therapeutic agent capable of controlling blood coagulation activity can be added and used as the modulator of the present invention. further,
Cancer cell extracts and amniotic fluid can act as modulators.

The present invention is not limited to any particular method of measuring blood coagulation. Any of a number of methods available for measuring blood coagulation may be utilized in the present invention, including manual, semi-automated, and automated methods, and their corresponding equipment or instruments. Suitable instruments for this purpose include, for example, all instruments that measure the mechanical impedance caused by the initiation of clot formation. Reagents that initiate blood coagulation or affect blood coagulation time are provided in a variety of forms, including, but not limited to, solution, lyophilized or air dried forms, or dried carded forms.

For example, the SONOCLOT ^™ coagulation analyzer sold by Sienco, Inc. measures viscoelasticity as a function of mechanical impedance of the test sample. Such an assay is very sensitive to fibrin formation, resulting in improved sensitivity and reproducibility of results.

Another device, thromboelastography (TEG), may be utilized to measure viscoelasticity. An example of this type of instrument is the Computerized Thromboelastography (CTEG) supplied by Haemoscope Corp. SONOCLOT ^™ and CTEG can record changes in the blood coagulation process by measuring changes in blood viscosity or elasticity, respectively. You get a complete graph of the whole process.

Other instruments, such as HEMOCHRON ^™ , measure blood coagulation time, but do not give a graph of changes in blood coagulation parameters as a function of time. Hemocron (
The HEMOCHRON ^™ system (International Technidyne Corp.) detects clot formation using a magnet that is precisely aligned in a test tube and a magnetic detector located in the instrument.

In certain embodiments of the invention, no use of anticoagulants is required in the event of an emergency, for example, in an emergency room when assaying a patient suspected of an acute thrombotic event such as heart attack or stroke. Alternatively, the assay may be performed directly with a fresh blood sample. The blood coagulation analyzer may be pre-filled with a necessary reagent such as an antibody and the blood coagulation time may be measured together with a control sample to which no antibody is added. Alternatively, blood is first collected using an anticoagulant that binds calcium ions, such as citric acid, oxalic acid, EDTA, and then blood coagulation time is determined under conventional laboratory conditions. In samples containing one or more of these anticoagulants, calcium salts must be added to initiate blood clotting. The time required to form the fibrin polymer is referred to in this case as the remineralization time. In another embodiment of the present invention, when blood is drawn in the presence of a specific inhibitor of the endogenous contact activated blood coagulation pathway, such as maize trypsin inhibitor, a procoagulant or anticoagulant of blood coagulation. Improved sensitivity and specificity in the detection of substances is obtained.

As an example of performing an assay for anticoagulated blood, 1 milliliter of citrated blood is combined with an inhibitory monoclonal anti-tissue factor antibody (final concentration
10 microgram / ml). Another aliquot of 1 ml citrated blood was prepared with control antibody or protein control. After mixing the samples, they were placed in a 37 ° C incubator. After a given incubation time, 300 microliters of each sample was mixed with 40 microliters of 0.1 M calcium chloride and the remineralization time (the time required to form fibrin) was measured using an automated instrument. The difference in remineralization time between control and inhibitor (antibody) -containing samples can be used to diagnose whether a patient has abnormal blood coagulation due to increased tissue factor and needs therapeutic intervention. Indicated.

In a further embodiment of the present invention, there is provided a test kit for measuring blood coagulation ability, which provides an appropriate concentration of an inhibitor for measuring blood coagulation time or remineralization time according to the method of the present invention. It

Tissue factor (TF), also known as factor III, results in the initiation of the extrinsic blood coagulation pathway. Tissue factor is mainly present in circulating monocytes. Certain disease states may predispose an individual's monocytes to primary immunization with tissue factor, which may exhibit an abnormally fast tendency of whole blood clotting time. Such patients may be at risk for thrombosis or other events.

At present, there is no method for accurately evaluating this hypercoagulable state. Utilizing the present invention, hypercoagulability can be assessed by detecting circulating TF levels through comparison of unstimulated blood coagulation times in the presence and absence of anti-TF antibody. In another aspect of the invention, a patient in the presence and absence of anti-TF antibody
By comparing the LPS-stimulated whole blood coagulation times, it is possible to measure the physiological possibility of hypercoagulability.

Example 1 The tissue factor whole blood assay described in this example involves a test method performed on a Hemochron instrument. The assay utilizes an anti-TF antibody inhibition test to assess endogenous circulating tissue factor levels in whole blood.

Materials and Reagents Required to Evaluate TF in Blood Circulation Hemochron P213 Sample Tube 0.01 M Calcium Chloride Stock Solution Control Reagent Vials Containing Non-Inhibitory Antibody Reagent Containing Dry Anti-Tissue Factor Antibody Vial Assay Quality Control Reagent Hemoliance RecombiPlasTin Stock Solution (lipidated recombinant tissue factor) TF Dilution Solution (20 mM HEPES 150 mM Sodium Chloride, pH 7.4 and 0.10 mg / mL Bovine Serum Albumin) Liquid Citrate Anticoagulant and Corn Trypsin Inhibitor (CTI )
Blood collection tube containing

Preparation of Hemochron P213 Tubes Hemochron P213 tubes are pre-filled with 50 μL of 0.10 M calcium chloride solution prior to performing the unstimulated blood coagulation time test. Store the tube at room temperature with the stopper closed.

Quality Control Sample Preparation for Unstimulated Whole Blood Clotting Time Negative Control = TF Dilution Solution High Positive TF Control: Make a 1: 100 dilution of lipidated recombinant TF stock solution. That is, lipidated recombination
Add 10 μL of TF stock solution to 0.99 mL of TF diluted solution. Low positive TF control: Make a 1: 7 dilution of the high positive TF control solution. That is, 100 μL of high positive TF control is added to 0.60 mL of TF diluted solution.

[0051] After discarding the blood of the blood collecting first few mL, collecting blood in 5mL citrate / CTI blood collection tubes. Blood must be analyzed within 4 hours of collection.

Unstimulated Blood Coagulation Time Test for Circulating TF (10 min Incubation) 1 Citric Acid / CT per 8 Test Vials for Unstimulated Blood Coagulation Time Test
I Provide a blood collection tube. Place 10 μl of either negative or positive TF control reagent in the four test vials listed below. : Control vial + Negative TF control Anti-TF antibody vial + Negative TF control Control vial + Positive TF control Anti-TF vial + Positive TF control Transfer 0.50 ml of citrate / CTI anticoagulated blood to each test vial. Mix several times by gentle tube inversion. Place in 37 ° C water bath for 10 minutes. After the 10 minute incubation is complete, 0.40 ml of blood is transferred from each test vial to a Hemochron P213 sample tube containing 50 μl calcium chloride.

On Hemochron 8000, test = "ACT" and tube = "P214 /
Select 5 ". Press" Start "to start the clot timer. Gently swirl the Hemochron tube to mix the blood with the calcium chloride solution. Tube Hemochron sample Place in tube Rotate tube in sample well until green light illuminates.

Typical unstimulated blood clotting time Control vial + negative TF control:> 850 seconds TF antibody vial + negative TF control:> 850 seconds Control vial + high positive TF control: 250-350 seconds Control vial + low positive control : 650-750 seconds TF antibody vial + low or high positive control:> 850 seconds To illustrate the use of the invention in circulating TF detection to assess hypercoagulability, Figure 2 shows the presence of anti-TF antibody. And detection of TF in exogenously added whole blood through comparison of blood clotting times after incubation for 10 minutes at 37 ° C in the absence and presence.

Example 2 This example describes a lipopolysaccharide stimulation test procedure on a Hemochron instrument that assesses TF production in whole blood in response to endotoxin (lipopolysaccharide) stimulation.

Materials and Reagents Required to Evaluate TF Production in Whole Blood in Response to LPS Stimulation Hemochron P213 Sample Tube 0.10 M Calcium Chloride Stock Solution Reagent vial containing dried LPS Dry LPS and dried anti-tissue Reagent Vials Containing Factor Antibody Liquid Citrate Anticoagulant and Corn Trypsin Inhibitor (CTI)
Blood collection tube containing

Preparation of Hemochron P213 Tubes Hemochron (Hemochro) prior to performing the blood coagulation time test of stimulated tissue factor.
n) Prefill the P213 tube with 50 μL of 0.10 M calcium chloride solution. Store the tube at room temperature with the stopper closed.

[0058] After discarding the blood of the blood collecting first few mL, collecting blood, in 5ml citrate / CTI blood collection tube contained in the kit. Blood must be analyzed within 4 hours of collection.

LPS Stimulated Blood Coagulation Time Test (2 hour Incubation) Transfer 0.50 ml of citrate / CTI anticoagulated sample blood into each test vial listed below. : Vial containing dry LPS and control reagent Vial containing both dry LPS and dry anti-TF antibody Mix several times by gentle inversion of the test vial. 2 in 37 ℃ water bath
Put time

After the 2 hour incubation period is complete, mix the test vials by gentle tube inversion. 0.40 ml of blood, calcium chloride 50 from each test vial
Transfer to a Hemochron P213 sample tube containing μl.

On the Hemochron 8000, test = "ACT" and tube = "P214 /
5 "Select. Press" Start "to start the clotting timer. Gently swirl the Hemochron tube containing the remineralized blood. Place it on the Hemochron. Turn the tube until a green light is illuminated.

[0062]   Typical coagulation time 2 hours after LPS stimulation   LPS vial: 250-350 seconds   LPS and anti-TF antibody vials:> 850 seconds

Example 3 The tissue factor whole blood assay described in this example includes a test method performed on a Sonoclot ^™ instrument. This assay uses an anti-TF antibody inhibition test to test LPS
Assess tissue factor levels in stimulated whole blood.

Sample Requirements Using a 19 mm gauge needle, for example, “Collection, Transport and Processi of Blood Samples for Blood Coagulation Tests, and Performance of Coagulation Assays.
ng of Blood Specimens for Coagulation Testing and Performance of Coagula
(Analysis Assays) "(National Committee for C
linical Laboratory Standards) Document # H21-A-2, Volume XI, No. 23). Blood was collected first in a waste tube (blue vacutainer at the tip) and then in a plastic vacutainer tube containing 50 μg / ml of corn trypsin inhibitor (CTI) and 0.5 ml of 3.2% sodium citrate. Use as test sample. At least 4 for the vacutainer tube.
You have to put 5 ml of blood. If not, the test will not continue. If good blood flow is not obtained at the time of sample collection, the sample is discarded and another venipuncture attempt is made on the patient's other arm. After blood collection, gently invert the vacutainer 5-7 times. Note: After collection, place the sample at room temperature.

Reagent Preparation and Storage 1. Vial with cap (2.0-ml polypropylene) and lyophilized reagent from Sienco. Vials are color coded on the cap: clear cap (for control mAb), white insert (for TF mAb cocktail), yellow insert (
No reagent), or blue insert (containing 20 microliters of 0.5 mg / ml lyophilized Difco endotoxin). Each patient sample is for each color
You will need a total of 4 tubes, one for each. Store at 2-8 ° C.

2. Calcium chloride 0.1M (Analytical Control Systems, Inc., Fishers, Ind.). 2
Store at ~ 8 ° C.

1. Tris buffered saline containing anti-osteocalcin monoclonal antibody (pH 7.4
) 1 mg / ml BSA. Store at 2-8 ° C.

2. Tris buffered saline containing tissue factor monoclonal antibody (pH 7.4) 1 mg / ml BS
A. Store at 2-8 ° C.

Required Equipment 1. Sonoclot ^™ analyzer 2. Handling instructions 3. Cuvettes supplied by Sienco. Five cuvettes are required for each patient sample, four to hold the blood sample and one to warm the calcium chloride. Four. Magnetic stirring bar supplied by Sienco 5. Probe supplied by Sienco 6. Probe extractor supplied by Sienco 7. Heating block or water bath set to 37 degrees Celsius 8. Timer 9. Eppendorf pipettes and pipette tips (40 microliters, 300 microliters, and 1000 microliters) 10.3 x 100 mm Haematologic Technologies Inc. vacutainer containing 0.5 ml of 3.2% buffered citric acid and 50 μg / ml corn trypsin inhibitor 11 ．19mm gauge needle

Operation 1. Sample preparation: Place 1 clear vial, 1 white vial, 1 yellow vial, and 1 blue vial in a test tube rack. Manually mix the vacuum vessel 6-10 times to ensure homogeneity. Open the vacuum vessel under the hood or after the splash guard.

2. Pipette 1.0 ml of blood into each clear and white vial. Add 5 microliters of control antibody to a clear vial. Add 5 microliters of TF mAb cocktail to a white vial. Cap each vial, gently invert 6-10 times and place the vial in a water bath. Set the timer to 10 minutes.

3. Pipette 1.0 ml of blood into each yellow and blue vial. Do not add any reagents to these vials. Cap each vial and gently 6-10
Turn over and place vial in water bath. Set the timer to 2 hours.

4. Sample test: a) Pipette 300 microliters of calcium chloride into a cuvette located in one of the side wells of a Sonoclot analyzer and warm to 37 ° C.

B) Place the probe into the appropriate position on the Sonoclot ™ with slight twisting. While twisting the cuvette slightly, place it firmly in the lower part of the receiver. c) Insert one magnetic stir bar into the cuvette. d) Add 40 μl of 0.1 M calcium chloride to the cuvette. Put on the lid. e) Remove vials of appropriate color from the water bath after the correct incubation time and gently invert 6-10 times. Remove the cap and pipette 300 μl of sample into the cuvette of the Sonoclot Analyzer. Avoid foam formation.

F) The same sample is gently re-aspirated once (to avoid platelet activation) and mixed well with the calcium chloride already in the cuvette. Once the sample has been delivered into the cuvette and mixed with calcium chloride, press the metal toggle switch on the Sonoclot analyzer to the "start" (down) position and activate the magnetic stirrer. Wait for the voice and instructions on the screen and close the lid. Close the port head. This operation introduces the probe into the sample and begins the test. g) Sound will sound when the test is complete. Read the values on the Sonoclot Analyzer data panel and chart record. The Sonoclot Analyzer automatically calculates the clotting time. Even if the voice sounds earlier, the test should continue for at least 20 minutes to obtain additional raw data.

H) Promptly record whole blood coagulation time and at the same time vial type (clear, white, yellow,
Or blue), patient identification number and date and time of the study. i) Dispose of the cuvette, probe and probe extractor. j) For each sample vial, after incubating for an appropriate time,
Repeat step.

3. Safety precautions: Technicians must take general precautions to eliminate the possibility of contracting diseases through pathogens contained in blood. As a minimum, precautions must include goggles, gloves, and suitable work clothing. Appropriate disposal techniques for pipettes, tips, vials, and sample containers should be utilized.

Comments on Procedures 1. Check the date of the reagent. Reagents should not have reached their expiration date. 2. Keep the incubation time and temperature accurate. These steps are extremely important. 3. Handle patient samples with care. Four. Use plastic material for all pipette tips. Keep the glass out of contact with blood in any environment. Five. Remove the blood spatter using a suitable disinfection method. 6. Store test kit material at 2-8 ° C. Do not use after the expiration date. This test is for in vitro diagnostic studies only.

Quality control 1. The viscous oil control provided with the Sonoclot Analyzer must be performed as described in the operating instructions. 2. The technician must perform regular duplicate readings of the sample (at least once for each shift or every 25 readings, whichever is more frequent). In doing so, take two 300 μl samples from the same incubation vial and place them in two instruments and record whole blood clotting time readings simultaneously. Use a new pipette tip for each sample taken from the vial. The value measured twice must be within 10% of the average value. 3. The instrument shall be evaluated daily for quality control in accordance with the manufacturer's specifications in Chapter 4 of the Sonoclot Operating Instructions.

Sources of Error Errors that may occur when performing an assay include technician error, instrument or supply problems,
And due to recording error. The main sources of error for each group are: 1. Technician error-insufficient or traumatic venipuncture, poorly sampled,
Sample not properly mixed and inverted, sample or calcium of inadequate volume, incubation time error, instrumentation error, data transfer error, or sample confusion. 2. Instrument errors-Errors in following the instructions, errors in performing the instrument manufacturer's quality control procedures, errors in performing the blood coagulation quality control procedures, temperature errors, and persistent discrepancies between duplicate samples. 3. Recording Errors-Errors in saving accurate and timely notes, data transfer errors, errors in recording vial lot numbers.

Subject Population Blood donor subjects were selected from a population of healthy individuals at the General Clinical Research Center (GCRC) of the Scripps Institute (La Jolla, California). Subjects were not chronically treated and did not use non-steroidal anti-inflammatory drugs (NSAIDS) for 20 days prior to blood donation. The use of human blood samples was approved and controlled by the Human Subjects Research Committee.

Determination of Blood Coagulation Time Whole blood coagulation time was determined using a Sonoclot blood coagulation and platelet function analyzer (Sienco, Inc., Wheat Ridge, CO) as described above, which was 300 μl. The viscous drag (viscous drag) of the fibrin chain is measured using a disposable vibration probe immersed in whole blood (1, 2). The blood coagulation time is the number of seconds until the impedance of the remineralized sample rises by 6 units above the baseline, and the software (Sile) is modified to use a custom onset algorithm.
nco) to calculate (Coagulation Diagnostics Inc., Be
thesda, MD). Tested whole blood samples were collected as described in 5 ml vacuum vessel glass test tubes containing 0.5 ml of 3.2% sodium citrate (Becton Dickinson, Franklin Lakes, NJ) without injury. The time from blood collection to the assay was less than 4 hours. The blood-containing tube was inverted several times to remix the whole blood before aliquoting the incubation tube. Blood (1 ml) was placed in a plastic tube at 37 ° C for 10 minutes or 2 and 4 hours, and bacterial lipopolysaccharide (LPS) (10 µg / ml) (E. coli 055: B5 Westphal, Difco; Detroit, MI) was added. Incubation was performed with and without addition. No agitation was performed during the incubation at 37 ° C. After the incubation, the blood was remixed and 300 μl was taken as aliquots in warmed cuvettes pre-filled with 40 microliters of 0.1 M CaCl ₂ and a magnetic stir bar. Then, after stirring for 10 seconds, the blood coagulation time was determined.

Effect of TF activity inhibition on blood coagulation time The effect of the inhibitory anti-TF antibody on blood coagulation time was determined by using the cocktail of mouse IgG ₁ MAbs (3) (9C3, 5G9, 6B4) inhibitory against human tissue factor at the final concentration. It was determined by adding 10 g / ml. Non-inhibitory mouse IgG ₁ antibody (10H10) was used as a control.

To determine the contribution of TF to the clotting time of whole blood, we examined the effect of a cocktail of inhibitory anti-TF antibodies on clotting time. The inhibitory anti-human TF monoclonal antibody significantly prolonged the coagulation time of LPS-stimulated blood from healthy volunteers but not unstimulated blood. Control non-inhibitory antibody had no effect on stimulated or unstimulated blood (Figure 3). Further studies conducted on 19 healthy individuals showed that inhibitory anti-TF antibodies did not affect mean baseline coagulation time (10 min) (478 ± 78 with antibody vs. no antibody). 437
± 113, average ± SD). These data show that TF in whole blood stimulated with the assay was TF
It was shown that dependent fibrin chain formation was measured. Recombinant lipidated TF added to whole blood shortened the coagulation time of remineralized whole blood in a dose-dependent manner over the range 0-80 pg / mL (FIG. 4).

Example 4 Role of Contact Activation Pathway on Coagulation Time of Unstimulated Blood In order to evaluate the contribution of the contact activation pathway to coagulation of whole blood, the present inventors have conducted three research methods. Used: i) Factor XII, Factor XI,
Or coagulated blood reconstituted with factor VII deficient plasma; ii) using a maize trypsin inhibitor that inhibits factor XIIa; iii) using an inhibitory anti-factor XIa antibody that inhibits factor XIa activity I was there. To determine the effect of deficient plasma, cells were separated from plasma by centrifugation at 850 xg for 10 minutes, washed, and resuspended in the following plasma: autologous, normal pool, Factor XI deficiency, Factor XII deficiency and Factor VII deficiency (Sigma). For experiments analyzing Factor XIIa inhibition, we used the maize trypsin inhibitor (32 g / ml) (Haematologic Tech.
nology, Essex Junction, VT) was used. For experiments analyzing factor XIa inhibition, goat anti-human factor Xia antibody (10 g / ml) (provided by Dr. K. Mann) or control non-immune goat antibody was added to whole blood prior to incubation at 37 ° C. added. In all cases, blood was incubated for 10 minutes at 37 ° C. before measuring whole blood clotting time.

Prior to measuring clotting time, cells isolated from whole blood were reconstituted with various plasmas. Cells reconstituted with autologous plasma showed a coagulation time that was slightly faster than that of naive blood (Fig. 5A), which may be a reflection of partial activation of monocytes and platelets during cell isolation. . Cells reconstituted with normal or factor VII-deficient plasma showed clotting times similar to those observed for autologous plasma, a result consistent with studies of anti-TF antibodies that did not show TF activity in unstimulated blood. did. Autologous plasma and normal or first
The small difference between Factor VII-deficient plasma is believed to be due to the difference between fresh plasma vs. frozen / lyophilized plasma. In contrast to the results for Factor VII-deficient plasma, cells were
The coagulation time was significantly prolonged when reconstituted with factor I and factor XII deficient plasma, suggesting that the contact activation pathway contributes to the coagulation time of unstimulated blood. Similarly, the inhibitory anti-factor XIa antibody significantly prolonged the clotting time of unstimulated whole blood (Fig. 5B).
.

We used a trypsin cone inhibitor to block factor XIIa activity (4). Again, we observed a consistent prolongation of blood coagulation time in the presence of corn trypsin inhibitor (Fig. 5C). The mean difference in coagulation time between with and without corn trypsin inhibitor was 141 ± 88 seconds (mean ± SD
, N = 28). Importantly, corn trypsin inhibitors did not block the coagulation time of LPS-stimulated blood, which we have shown to be TF-dependent (see Figure 3) (Figure 5C). Taken together, these studies show that the contact activation pathway contributes to the clotting time of unstimulated blood, but does not significantly affect the shortening of clotting time in LPS-stimulated blood.

Example 5 Effect of inhibitory anticoagulant on whole blood coagulation time Unfractionated heparin, low molecular weight heparin (LMWH) on coagulation time of LPS-stimulated blood
And the effect of hirudin anticoagulant was tested. The results are shown in FIGS. 6A to 6C, respectively. Unfractionated heparin and LMWH inhibit both thrombin and factor Xa. However, unfractionated heparin exhibits higher antithrombin activity compared to its antifactor Xa activity (5). In contrast, LMWHs have low antithrombin activity compared to their antifactor Xa activity (5). Hirudin selectively inhibits thrombin (6
). Administration of clinically relevant doses of these three anticoagulants to LPS-stimulated blood prolonged blood clotting time in a dose-dependent manner. These data indicate that this blood coagulation time assay can be used to monitor anticoagulant therapy in a clinical setting.

Example 6 Clotting Time of Blood from Patients with Unstable Angina In order to study unstable angina, we present an emergency treatment at The John Hopkins Hospital, Baltimore. Blood from patients (n = 8) diagnosed with unstable angina who were admitted to the chamber was analyzed. Patients taking anticoagulants were excluded. Healthy volunteers from the same place (
The group of n = 37) was used as a control group. The use of human blood samples was approved and controlled by the Human Subjects Research Committee. Elevated TF protein levels in blood from patients with unstable angina have been reported in the literature (7,8,9,10).
). We tested the clotting time of unstimulated blood from patients admitted to the emergency room with unstable angina. The clotting time of unstimulated blood from unstable angina patients was significantly faster than that from a group of healthy volunteers (Figure 7). These results indicate that patients with unstable angina have elevated levels of circulating TF activity.

While the above specification teaches the principles of the invention using the examples provided for purposes of illustration, one of ordinary skill in the art will appreciate upon reading this disclosure that various modifications in form and detail are made by the invention. It will be understood that it can be done without departing from the true scope of. The cited patents and publications are incorporated herein by reference in their entirety.

References 1. Chandler WL, Schmer G. "Evaluation of a new dynamic viscometer for measuring the visco.
sity of whole blood and plasma) Clin Chem 1986; (32): 505-507. 2. Hett DA, Walker SN, Pilkington SN, Smith DC.
"Clot analysis)" Br J Anaesth 1995; 75: 771-776. 3. Morrissey JH, Fair DS, Edgington TS. "Monoclonal antibody analysis of purified and cell-
Associated tissue factor) "Thromb Res 1988; 52: 247-261. 4. Rand MD, Lock JB, van't Veer C, Gaffney DP, Mann KG." Blood clotting in minimally modified whole blood " minimally altered whole blood) "Bl
ood 1996; 88: 3432-3445. 5. Canton, MM in “Clinical Hematology and F
undamental of Hemostasis) '', 2nd edition, Harmening DM, edited; 1992, FA Davis
Company, Philadelphia, pp.510-512. 6. Bates SM, Weitz JI. “Direct thrombin inhibitors for the treatment of arterial thrombosis:
Direct thrombin inhibitors for tre
atment of arterial thrombosis: potential differences between bivalirudin
and Hirudin) Am. J. Cardiol. 1998; 82: 12P-8P. 7. Leatham E, Bath P, Tooze J, Camm A. "Increased monocyte tissue factor expression in coronary vascular disease. in coronary di
sease) '' Br Heart J 1995; 73: 10-13. 8. Suefuji H, Ogawa H, Yasue H, Kaikita K, Soejima H, Motoyama T, Mizuno
Y, Oshima S, Saito T, Tsuji I, Kumeda K, Kamikubo Y, Nakamura S. “Increased plasma tissue factor level in acute myocardial infarction
levels in acute myocardial infarction) '' Am Heart J 1997; 134: 253-259. 9. Misumi K, Ogawa H, Yasue H, Soejima H, Suefuji H, Nishiyama K, Takazo
e K, Kugiyama K, Tsuji I, Kumeda K, Nakamura S. "Comparison of plasma tissue factor lev
els in unstable and stable angina pectoris) '' Am J Cardiol 1998; 81 (1):
22-26. 10. Agraou B, Corseaux D, McFadden EP, Bauters A, Cosson A, Jude B. “Effects of coronary angioplasty on monocyte tissue factor response in patients with stable and unstable angina. coronary angioplasty on monocyte tissue factor resp
onse in patients with stable or unstable angina) '' Thromb Res 1997; 88 (
2): 237-243.

[Brief description of drawings]

FIG. 1 shows the central role of monocyte TF at the initiation of fibrin clot formation in whole blood. The TF: VIIa complex activates Factor X to Factor Xa and Factor IX to Factor IXa. Thrombin (Factor II) activates platelets, which form the thrombin generating surface for the prothrombinase complex (Xa: Va). Fibrinogen is cleaved to give fibrin clots. This figure shows Kjalke et al., "Active site inactivated Factors VIIa, Xa, and IXa inhibit individual steps in a cell-based model of tissue factor-initiated blood coagulation (Active site-inactivated.
Factors VIIa, Xa, and IXa inhibit individual steps in a cell-based model
of tissue factor-initiated coagulation), Thromb. Haemost., 80: 578-8
4 (1998).

FIG. 2 shows the results of detecting exogenously added TF in whole blood through comparison of blood coagulation time after incubation for 10 minutes at 37 ° C. in the presence and absence of anti-TF antibody. Indicates.

FIG. 3 shows the effect of anti-TF antibody on remineralized whole blood coagulation time. LPS (10 μg / mL)
Incubation of whole blood with C. for 2 hours at 37 ° C shortened the blood coagulation time induced by TF exposure. Addition of anti-TF antibody blocked LPS-mediated reduction of remineralized whole blood coagulation time. In contrast, the addition of non-inhibitory control antibody did not affect LPS blood coagulation time.

FIG. 4 shows the effect of recombinant TF on remineralized whole blood clotting time. Recombinant lipidated TF added to whole blood shortened remineralized whole blood clotting time over a range of 0-80 pg / mL (mean ± 95% confidence interval of the mean, at each point). n = 11 repeated test value)
.

FIG. 5a shows clotting times for cells isolated from blood and mixed with various plasmas. Figure 5b shows inhibitory anti-XI-XI added to blood before incubation at 37 ° C for 10 minutes.
The effect of factor antibody (100 μg / mL) is shown (mean ± standard deviation, n = 3). Addition of anti-factor XIa antibody prolonged blood coagulation time. In contrast, the addition of the corresponding amount of control antibody did not affect blood coagulation time. FIG. 5c shows the effect of corn trypsin inhibitor (CTI) (32 μg / mL) added to blood before incubation for 2 hours at 37 ° C. with or without LPS stimulation (mean ± standard deviation, n =). 3).

FIG. 6a shows the effect of unfractionated heparin (0-0.1 U / ml) on the clotting time of LPS-stimulated blood. Figure 6b shows low molecular weight heparin (LMWH; 0-0.25U /
(ml) effect. FIG. 6c shows the effect of hirudin (0-0.1 U / ml) on the clotting time of LPS-stimulated blood.

FIG. 7 compares remineralized whole blood coagulation times in patients with unstable angina (n = 8) and healthy controls (n = 37). Open circles represent individuals outside the 5th and 95th percentiles of clotting time.

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor McMan, Nigel             United States 92122 California             State, San Diego, Stress             Treat 5832 (72) Inventor McDonnell, John, Jay.             20895 Maryland,             Kensington, Evert Street             4008 F term (reference) 2G045 AA10 BB41 BB46 BB50 BB51                       BB53 CA25

Claims (29)

[Claims] 1. A method for determining the presence and function of the presence of procoagulants in whole blood, comprising: (a) collecting a whole blood sample; (b) dividing the whole blood sample into at least two aliquots. (C) adding at least one inhibitor of a procoagulant to one of the aliquots; (d) incubating the aliquots; (e) measuring the clotting time for each aliquot; and (f) the aliquot of each aliquot. A method as described above comprising comparing clotting times. 2. A coagulation promoting substance is α ₂ antiplasmin, fibrinogen,
High molecular weight kininogen, kallikrein, prekallikrein, tissue factor, factor II, factor IIa, factor Va, factor VIIa, factor VIIIa, factor IXa, factor Xa, factor X
2. The method of claim 1, selected from the group consisting of Factor Ia, Factor XIIa, and Factor XIIIa. 3. The method of claim 1, further comprising adding a contact activation inhibitor to the whole blood sample or collecting the sample or whole blood in the presence of the contact activation inhibitor. 4. The method of claim 3, wherein the contact activation inhibitor is a maize trypsin inhibitor. 5. The method of claim 1, wherein the inhibitor of a procoagulant is an antibody or analog of a procoagulant substrate. 6. The method of claim 5, wherein the inhibitor is an antibody. 7. The method of claim 5, wherein the analog is a peptide. 8. The method of claim 6, further comprising adding a control antibody to the aliquot that does not contain the antibody inhibitor of the procoagulant, in an amount substantially equivalent to the antibody inhibitor of the procoagulant. Method of adding control antibody. 9. The method of claim 1, wherein the procoagulant is not tissue factor. 10. A method of determining the presence and function of the presence of anticoagulants in whole blood, comprising: (a) collecting a whole blood sample; (b) dividing the whole blood sample into at least two aliquots. (C) adding an inhibitor of an anticoagulant to one of the aliquots; (d) incubating the aliquots; (e) measuring the clotting time for each aliquot; and (f) each aliquot. A method as described above comprising comparing the clotting times of the. 11. The anticoagulant is α ₁ antitrypsin, activated protein C,
Antithrombin III, C1 esterase inhibitor, heparin, protein C,
11. The method of claim 10, selected from the group consisting of protein S, thrombomodulin, and tissue factor pathway inhibitor. 12. The method of claim 10, further comprising adding a contact activation inhibitor to a sample of whole blood or collecting the sample or whole blood in the presence of the contact activation inhibitor. 13. The method of claim 12, wherein the contact activation inhibitor is a corn trypsin inhibitor. 14. The method of claim 10, wherein the inhibitor of anticoagulant is an antibody. 15. The method of claim 14, further comprising adding a control antibody to the aliquot that does not contain the antibody inhibitor of anticoagulant, in an amount substantially equivalent to the antibody inhibitor of anticoagulant. A method as described above, characterized in that a control antibody is added. 16. The method of claim 1, wherein the whole blood sample is collected in a solution containing citric acid and the blood is remineralized prior to step (e). 17. The method of claim 10, wherein the whole blood sample is collected in a solution containing citric acid and the blood is remineralized prior to step (e). 18. The method of claim 16, wherein a sample of whole blood is collected in a solution containing citric acid and the blood is remineralized prior to step (e). 19. A kit for determining the presence and function of a procoagulant in whole blood, comprising: (a) a vial containing an inhibitor of the procoagulant; (b) a non-inhibitory control reagent. A kit containing a vial containing; and (c) a vial containing a liquid citric acid anticoagulant. 20. The kit of claim 19, wherein the vial containing the liquid citrate anticoagulant further comprises corn trypsin inhibitor. 21. The kit according to claim 20, wherein the coagulation promoting substance is tissue factor. 22. The method of claim 21, wherein the inhibitor is at least one antibody.
The kit described in. 23. A kit for determining the presence and function of the presence of anticoagulants in whole blood, comprising: (a) a vial containing an inhibitor of an anticoagulant; (b) a non-inhibitory control reagent. A kit containing a vial containing; and (c) a vial containing a liquid citric acid anticoagulant. 24. The kit of claim 23, wherein the vial containing the liquid citrate anticoagulant further comprises corn trypsin inhibitor. 25. The inhibitor of claim 24, wherein the inhibitor is at least one antibody.
The kit described in. 26. A method of determining the presence and function of the presence of procoagulants in whole blood, comprising: (a) collecting a whole blood sample; (b) dividing the whole blood sample into at least four aliquots. (C) adding an immunomodulator to two of the aliquots; (d) at least one inhibitor of a procoagulant is added to one of the aliquots not containing the immunomodulator and the aliquot containing the immunomodulator. A method as described above comprising adding to one; (e) incubating aliquots; (f) measuring the clotting time for each aliquot; and (g) comparing the clotting time of each aliquot. 27. The method of claim 26, wherein the whole blood sample further comprises an anticoagulant and a corn trypsin inhibitor. 28. The method of claim 27, wherein the immune modulator is endotoxin. 29. The method of claim 27, wherein the inhibitor is an antibody.