AU783760B2

AU783760B2 - Method for detecting, treating, and monitoring conditions associated with activation of the coagulation response

Info

Publication number: AU783760B2
Application number: AU67690/00A
Authority: AU
Inventors: David E. Berg; Lois Hill Berg; Harold H. Harrison
Original assignee: Individual
Current assignee: Individual
Priority date: 1999-08-13
Filing date: 2000-08-11
Publication date: 2005-12-01
Anticipated expiration: 2020-08-11
Also published as: GB2368642A; CA2385502C; GB2368642B; CA2385502A1; WO2001012839A1; AU6769000A; GB0203838D0

Description

WO 01/12839 PCT/US00/22177 METHOD FOR DETECTING, TREATING, AND MONITORING CONDITIONS ASSOCIATED WITH ACTIVATION OF THE COAGULATION RESPONSE FIELD OF THE INVENTION This Non-Provisional Application claims the benefit of U.S. Provisional Application Serial No. 60/148,799 filed on August 13, 1999. This invention relates to methods for detecting, treating, and monitoring conditions associated with activation of the coagulation response which respond to anti-coagulant therapy.

BACKGROUND OF THE INVENTION Chronic fatigue syndrome (CFS) and fibromyalgia (FM) have been considered diagnoses of exclusion where no other diagnosis fit well. CFS has been defined by specific requirements of fatigue, its duration, associated symptoms, and initial clinical and laboratory evaluation. There has existed no reliable laboratory means for determining whether an individual was suffering from CFS, FM, or some other disease. Accordingly, a felt need for a method of testing for CFS and FM existed.

SUMMARY OF THE INVENTION This invention relates to a method for using a novel combination of assays to detect minimal activation of the coagulation response for determining whether a patient whose initial clinical evaluation indicates chronic fatigue syndrome, fibromyalgia, and related conditions can be treated using anticoagulant therapies. If activation of the coagulation response is detected, the present invention further includes treatment of CFS, FM or related condition using anticoagulant therapies.

We have discovered that we may reliably diagnose a patient suffering from CFS or FM by evaluating the status of the coagulation response in that patient by using a novel 1 SUBSTITUTE SHEET (RULE 26) combination of tests which can detect minimal activation of the coagulation response in a patient. This novel combination preferably includes tests for determining levels of fibrinogen, prothrombin fragmentl 2, thrombin/antithrombin complexes, soluble fibrin monomer, and platelet activation by flow cytometry. These assays are preferred because they are highly sensitive to minimal deviation from normal. However, in some circumstances, other assays may be suitable. Deviation from the normal values in any two of the five assays permits diagnosis of CFS, FM, or other disease associated with activation of the coagulation response.

Accordingly, in one aspect the invention provides an ex vivo method for diagnosing a blood sample comprising steps of: identifying conditions that each cause a low level activation of the coagulation response in blood; providing a blood sample; providing different quantitative blood tests that are each for identifying low level activation of the coagulation response in blood; I>performing each of the different blood tests on the blood sample; and if at least two of the different blood tests identify low level activation of the coagulation response in the blood sample are abnormal, using the at least two of the blood tests to assist in diagnosing the blood sample with one of 20 the conditions.

The method may further comprise the steps of providing a population of blood samples, and performing the method for each of the blood samples of the population.

The different blood tests may comprise tests for at least two of fibrinogen, prothrombin fragment 1+2, thrombin/antithrombin complexes, and soluble fibrin monomer. Further, the different blood tests may comprise tests for at least three of fibrinogen, prothrombin fragment 1+2, thrombin/antithrombin complexes, and soluble fibrin monomer The different blood tests may comprise tests for at least two of fibrinogen, prothrombin fragment 1+2, thrombin/antithrombin complexes, soluble fibrin monomer, and platelet activation. Further, the different blood tests may comprise tests for at least three of fibrinogen, prothrombin fragment 1+2, thrombin/antithrombin complexes, soluble fibrin monomer, and platelet activation.

In the method, the different blood tests may comprise tests for fibrinogen, prothrombin fragment 1+2, thrombin/antithrombin complexes, soluble fibrin monomer, and platelet activation.

In another aspect, the invention provides an ex vivo method for diagnosing a blood sample comprising steps of: identifying a condition that causes low level activation of the coagulation response in blood; 15 providing a blood sample; providing different blood tests that are each for identifying low level activation of the coagulation response in blood; performing the different quantitative blood tests on the blood sample; and if at least two of the different blood tests identify low level activation of 20 the coagulation response in the blood sample, using the at least two of the blood tests to assist in diagnosing the blood sample with the condition.

The method may further comprise the steps of providing a population of blood samples and performing the method for each of the blood samples of the population.

In yet another aspect, the invention provides an ex vivo method for diagnosing a blood sample comprising steps of: identifying conditions that o 1 each cause a low level activation of the coagulation response in blood; providing a blood sample; providing different blood tests that are each for identifying low level activation of the coagulation response in blood, S: the blood tests comprising tests for at least two of fibrinogen, prothrombin fragment 1+2, thrombin/antithrombin complexes, soluble fibrin 20 monomer, and platelet activation; oo: obtaining a result for each of the blood tests; observing the results; and if at least two of the results are abnormal, using the abnormal results to assist in diagnosing the blood sample with one of the conditions.

In yet another aspect, the invention provides an ex vivo method for diagnosing a blood sample comprising steps of: identifying a condition that causes a low level activation of the coagulation response in blood; providing a blood sample; providing different blood tests that are each for identifying low level activation of the coagulation response in blood; the blood tests comprising tests for fibrinogen, prothrombin fragment 1+2, thrombin/antithrombin complexes, soluble fibrin monomer, and platelet activation; and if at least two of the results are abnormal, using the abnormal results to assist in diagnosing the condition.

Once a condition associated with activation of the coagulation response has been diagnosed, the patient is treated with anticoagulant therapy, such as heparin followed by warfarin or warfarin alone. Coumarins or coumarin derivatives may also be used. Heparin can be defined as heparin (porcine or bovine) or any of its derivatives, such as low molecular weight heparin (LMWH), oral heparin, heparinoids, or any other designer heparin-like drugs.

It is presently preferred that a low dose anticoagulant therapy be used. Patient progression and recovery is then monitored using the novel combination of assays.

We postulate that a majority of individuals diagnosed as CFS and/or FM on clinical criteria may be defined as having antiphospholipid antibody syndrome (APS) 25 with endothelial cells as a major disease target with or without platelet activation.

Laboratory findings in patients suffering with APS include anticardiolipin antibodies, lupus anticoagulants, antiphosphatidylserine antibodies, anti-B2GPI antibodies.

Clinical findings include thrombocytopenia, neurological complications, venous thrombosis, arterial thrombosis, and/or recurrent fetal loss. Patients with primary APS (PAPS) presently have no clinical or laboratory evidence of another definable autoimmune disease. Antiphospholipid antibodies WO 01/12839 PCTUS00/22177 have long been associated with a hypercoagulable state, involving both procoagulant activity as well as inhibition of anticoagulant and fibrinolytic activity. In CFS and/or FM patients, the principal antibodies found to date are the anti-B2GPI antibodies. This precedes the generation of a hypercoagulable state based on our proposed model.

Endothelial cells are protected in the microvascular circulation by B 2 GPI and Annexin V proteins. This protective layer helps endothelial cells maintain an anticoagulant environment. Exposure to pathogens, such as herpes viruses (HV, HHV6, EBV), cytomegalovirus (CMV), mycoplasma, chlamydia pneumonia, or some vaccines can result in both active persistent infection and latent infection in mononuclear and endothelial cells.

Some pathogens like CMV and HV constitutively express phosphatidylserine-like procoagulant activity, capable of binding Xa and Va to form the prothrombinase complex.

HHV6 is found in about 70% of all CFS patients. In several studies, this same 70% infection rate is seen in multiple sclerosis patients with HHV6. HHV6 is also implicated in chronic myelopathy. Endothelial cells serve as a reservoir for harboring HHV6. Infected endothelial cells lose the ability to synthesize prostacyclin with associated incapacity to deter platelet adhesion. In addition, CMV and HV express tissue factor antigen on each virus surface. HV can induce a prothrombotic phenotype in vascular endothelial cells. This phenotype markedly reduces heparin sulfate protcoglycan synthesis and surface expression by endothelial cells.

Thrombomodulin expression is also reduced in infected endothelium. Due to fibrin deposition, fibrinolysis activation may also be diminished. Activation of endothelial cells is seen by surface expression of P-selectin and von Willebrand Factor (vWF). Thrombin generated after the assembly of the prothrombinase complex on the virus-infected 3 SUBSTITUTE SHEET (RULE 26) WO 01/12839 PCTIUS00/22177 endothelium mobilizes vWF from the Weibel-Palade body to the endothelial cell surface, where it acts as a platelet receptor. Cell-independent thrombin generation may be the earliest event in vascular pathology mediated by HV.

Because exposure and expression of phosphatidylserine (PS) is part of the infectious process, those exposed phospholipids activate the immune system to form antiphospholipid antibodies. The primary targets of these immunoglobulin (Ig)G, IgM and IgA antibodies are the protective proteins for endothelial cells, specifically B2GPI and Annexin V. As with other APS diseases, there is an increased incidence of thrombocytopenia in HHV6 patients.

With the loss of this protective layer due to APL antibodies, coagulation proteins can bind, react and form thrombin If this process is not property inhibited (thrombin-antithrombin complexes), then excess thrombin can convert fibrinogen to soluble fibrin monomer (SFM). SFM is a sticky protein that increases blood viscosity and can coat endothelial cells surfaces as fibrin or fibrinoid material. Patients with CFS and FM symptoms typically have a hypercoagulable state demonstrated by increased markers of coagulation activation and increased blood viscosity due to the generation of soluble fibrin monomer (SFM). Once CFS and FM is diagnosed using our combination of assays, CFS/FM patients may be treated with anticoagulant therapies, and their treatment and recovery monitored using our combination of tests.

Patients with immune mediated chronic inflammatory disorders of many types can have low level activation of the coagulation response. Therefore, patients with a spectrum of chronic inflammatory processes may have low level activation of coagulation as part of their pathophysiology. We postulate that our tests for activation of the coagulation and platelet 4 SUBSTITUTE SHEET (RULE 26) WO 01/12839 PCT/US00/22177 systems also have application to other conditions which stem from activation of the coagulation response. This has been validated by preliminary studies of patients suffering with multiple sclerosis, breast implant sickness syndrome, fetal wastage syndrome, gulf war illness, inflammatory bowel disease, autism. As with CFS and FM, once diagnosed using our combination of assays, these patients may be treated with anticoagulant therapies, and their treatment and recovery monitored using our combination of tests.

We postulate that our combination of tests for detecting minimal activation of coagulation response also has application to detecting and treating, Sjogrens syndrome, late Lyme disease (also called chronic Lyme disease), transient ischemic attack, attention deficit disorder, Alzheimer's disease, Parkinson's disease, as well as some cardiovascular diseases.

Once diagnosed using our combination of assays, these patients should also benefit from treatment with anticoagulant therapies, and their treatment and recovery monitored using our combination of tests.

The Presently Preferred Assays Used To Determine Minimal Activation of Coagulation Response Although thrombin generation is among the first steps in the coagulation response, thrombin itself is difficult to quantify because it lasts only 30 seconds in the blood circulation.

Consequently, other measurable components of the coagulation response must be considered.

Prothrombin fragment 1+2, which is released when prothrombin is converted into thrombin, can be used to indicate activation of the coagulation response. Increased levels ofthrombin/antithrombin complexes (TATs) also indicate thrombin generation and an attempt to remove excess thrombin. Thus, an increased level of TATs also indicates activation of the coagulation response. When excess thrombin is generated by the SUBSTITUTE SHEET (RULE 26) WO 01/12839 PCT/US00/22177 coagulation response, fibrinogen is cleaved to soluble fibrin monomer (SFM). Accordingly, increased levels of SFM also indicate activation of the coagulation response. With the consumption offibrinogen, the body may compensate by increasing fibrinogen levels slightly above the normal range; therefore, increased fibrinogen levels are also an indicator of activation of the coagulation response. Finally, platelet activation may also be used as an indicator of coagulation response activation.

The tests discussed above are the assays presently preferred, it being understood that other tests sensitive to minimal activation of the coagulation response may be substituted for the assays discussed.

DETAILED DESCRIPTION OF THE INVENTION For the purposes of describing the invention, we discuss the form of the method of the present invention which is presently preferred; it being understood, however, that this invention is not limited to the precise arrangements, instrumentalities, and assays discussed.

Our new method for diagnosing CFS, FM, and related conditions uses a novel combination of assays for detecting minimal activation of the coagulation response. Highly sensitive tests are used which are capable of detecting minimal deviations from normal levels offibrinogen, prothrombin fragment 1 2, thrombin/antithrombin complexes, soluble fibrin monomer, and platelet activation by flow cytometry, platelet aggregation, or related techniques. According to our invention, if a patient receives an initial clinical evaluation indicating CFS and/or FM and if 2 or more of the above assays show abnormal results, that patient can be reliably diagnosed with CFS and/or FM. Once a condition has been diagnosed that is responsive to anti-coagulant therapy, the patient is treated with low dose anticoagulant 6 SUBSTITUTE SHEET (RULE 26) WO 01/12839 PCT/US00/22177 therapy, such as heparin followed by warfarin, or pharmacologically and conceptually similar anticoagulant regimens. Patient progression and recovery is then monitored using the novel combination of assays.

In order to test our method, we have conducted a blinded study of 54 CFS and/or FM patients and 23 controls, for a total of 77 individuals. According to our method, blood drawn from all individuals was subjected to each of the 5 tests. Using our presently preferred combination of 5 assays, we determined that the CFS and FM patients could be reliably distinguished from the controls. Individuals whose blood tested outside of the normal range for 2 or more of the 5 tests were diagnosed as suffering from CFS and/or FM. 22 of the 23 controls were correctly identified. One control was positive in two assays for a false positivity rate of Of the 54 patients with CFS or FM, 4 had normal values, for a false negative rate of This shows that 92+% of CFS and/or FM patients had a demonstrable hypercoagulable state.

The following table sets out the mean values for each group tested.

7 SUBSTITUTE SHEET (RULE 26) WO 01/12839 PCT/US00/22177 TESTS: FIB mg/dl F1+2 nM T/AT ug/1 SFM nmol/i PA (Platelet CD62P% Activation) Reference Normal <310 <1.1 1.0-4.1 <20 Normal <26 Range Controls 23 280 1.0. 1.6 10 0% Positive 17.5 #Abnormal 2/23 3/23 4/23 3/23 0/23 5/23 /Normal Patients 54 367 1.2 1.6 22 42% Positive 22 #Abnormal 45/54 26/54 25/54 32/54 22/54 21/52 /Normal P Value <0.001 <0.005 <0.025 <0.001 <0.001 <0.10 As stated above, in CFS and FM patients, the principal antibodies found to date are the anti-B2GPI antibodies. Anti-B2GPI antibodies can precede the generation of a hypercoagulable state. In a normal capillary endothelial cell, phosphatidylserine is on the inside of the cell membrane. When phosphatidylserine is transported to the surface of the endothelial cell, it is isolated from blood flowing through the vessels by a layer of B2GPI and Annexin V proteins, which cover the phosphatidylserine. When the immune system is activated, anti-B2GPI and anti-annexin V antibodies, collectively antiphospholipid antibodies, are generated. These antibodies are IgG, IgA, and IgM, the majority of which bind to the B2GPI and annexin V proteins dislodging them from the phosphatidylserine. When the phosphatidylserine on the vessel walls is exposed, coagulation factor tenase and prothrombinase complexes bind to the surfaces of the endothelial cells which coat the vessel walls. When prothrombin complexes bind to the cell walls, prothrombin is converted to thrombin, which releases the activation peptide prothrombin fragment 1 2.

The human body responds to the generation of excess or inappropriate thrombin levels by synthesizing the inhibitor antithrombin. Antithrombin combines with thrombin to form thrombin/antithrombin complexes, which are then removed from the blood.

8 SUBSTITUTE SHEET (RULE 26) WO 01/12839 PCT/US00/22177 Antithrombin is a slow inhibitor of thrombin, but in the presence of heparin, antithrombin reacts with thrombin at a greatly increased rate. Activated protein C/antitrypsin complex is a secondary inhibitor of thrombin generation.

When more thrombin is generated than can be removed by the thrombin inhibitors, thrombin reacts with fibrinogen to create an intermediate protein called soluble fibrin monomer. Soluble fibrin monomer is a sticky protein which increases blood viscosity and forms deposits on capillary wall endothelial cells. The soluble fibrin monomer which is deposited on the capillary walls, a phenomenon called fibrin deposition, may block the passage of nutrients through the capillary walls to the surrounding tissues, whether it is another capillary, muscle or organ tissue. We postulate that the increase in blood viscosity in combination with associated blockage in nutrient transfer results in the fatigue of CFS and the muscle pain of FM. This could also be part of the explanation (pathology) of fetal demise and spontaneous abortions seen in recurrent miscarriages (fetal wastage syndrome).

In addition to the creation of soluble fibrin monomer, thrombin also activates factor XIII to XIIla in the blood, causing it to react with the soluble fibrin monomer to create insoluble fibrin strands, which may in turn generate a clot or fibrin deposition on vascular walls, further inhibiting blood flow and nutrient transfer. Once fibrin deposition or blood clot formation occurs, it becomes necessary to dissolve the fibrin. Protein C, which is activated by the excess thrombin production, helps to activate the fibrinolytic system when tissue plasminogen activator converts the protein plasminogen into plasmin. Plasmin is a very potent enzyme for fibrin depositions or clots and keeps the circulatory system of blood vessels open and clean. Tissue plasminogen activator is conventionally injected into patients 9 SUBSTITUTE SHEET (RULE 26) WO 01/12839 PCT/US00/22177 to dissolve blood clots, such as heart attacks, myocardial infarctions, strokes, blood clots in the eye, etc.

Platelet activation within the blood is also a contributing factor in thrombin formation.

Platelet activation reinforces the generation of thrombin by providing a major surface for thrombin formation. Platelets may be directly activated by the immune system when immunoglobulins activate glycoprotein receptors and cause platelets to become "sticky".

This "sticky state" can result in fibrin formation, vascular and endothelial cell changes, and, at times, platelet clumping. Platelet activation can be measured by a special laboratory procedure called flow cytometry, platelet aggregation or related techniques.

Differences in genetic makeup are the most likely reason that all people exposed to an immune system activating pathogen do not develop diseases associated with a hypercoagulable state, such as chronic fatigue syndrome and fibromyalgia. Patients who have a hereditary deficiency for thrombophilia or hypofibrinolysis may be unable to properly control thrombin generation. Genetic defects in the proteins which regulate the coagulation process, are most likely the cause of long term activation of the immune system which results in a persistent hypercoagulable state. Thrombophilia may be caused by one or more genetic abnormalities in proteins which presently include protein C, protein S, antithrombin, factor V leiden, factor II gene mutation, thrombomodulin, heparin cofactor II, or factors II, VII, IX, X, XI, XII, and others which may be found for related genes. Hypofibrinolysis may be caused by one or more genetic abnormalities factors which presently include plasminogen, tPA, urokinase, PA-1, lipoprotein(a), homocysteine, factor V leiden, factor XI, and TAFI, and others which may be found for related genes.

SUBSTITUTE SHEET (RULE 26) WO 01/12839 PCT/US00/22177 Patients with immune mediated chronic inflammatory disorders of many types can have low level activation of the coagulation response. Therefore, patients with a spectrum of chronic inflammatory processes may have low level activation of coagulation as part of their pathophysiology. We postulate that our tests for activation of the coagulation and platelet systems also have application to other conditions which stem from activation of the coagulation response. This has been validated by preliminary studies of patients suffering with multiple sclerosis, breast implant sickness syndrome, gulf war illness, inflammatory bowel disease, autism, and fetal wastage syndrome. As with CFS and FM, once diagnosed using our combination of assays, these patients may be treated with anticoagulant therapies, and their treatment and recovery monitored using our combination of tests. We propose that a common feature of these conditions is the chronic inflammatory process that stimulates low level activation of coagulation.

The following table sets out our preliminary findings for autism, attention deficit disorder, multiple sclerosis, Parkinson's disease, Sjogrens syndrome, and gulf war illness.

Individuals who were diagnosed as suffering from one of these chronic inflammatory disorders tested outside of the normal range for 2 or more of the 5 tests in our method.

11 SUBSTITUTE SHEET (RULE 26) WO 01/12839 PTUOI2 PCT/USOO/22177 Disease TESTS Number of FEB mgtdl FI+2 T/AT SFM% PA Individuials tiM ugA nmo/1 (Platelet Tested Autism 3 #Abnormal 1/3 3/3 0/3 3/3 0/3 Attention 2 Deficit #Abnormal V21/2 1/2 V20/2 Disorder Multiple 3 Sclerosis #IAbnormal 1/3 1/3 2/3 2/3 0/3 Normal Parkinson's 2 Disease #Abnormal /21/2 V/2 0/2 0/2 Sjogrens I Disease #Abnormal 1/1 0/1 0/1 1/1 0/1 Nrml Gulf War 33 Illness #Abnormal 17/33 11/33 6/33 17/33 15/33 /Normal We postulate that our combination of tests for detecting minimal activation of coagulation response also has application to detecting and treating other immune mediated chronic inflammatory disorders such as Sjogrens syndrome, late Lyme disease (also called chronic Lyme disease), transient ischem-:ic attack, attention deficit disorder, Alzheimer's disease, Parkinson's disease, as well as some cardiovascular diseases. Once diagnosed using our combination of assays, these patients should also benefit from treatment with anticoagulant therapies, and their treatment and recovery monitored using our combination of tests.

12 SUBSTITUTE SHEET (RULE 26) It is to be understood that the words "comprise", "comprises", "comprising", "comprised" or the like, when used in this specification, are to be given a nonexhaustive meaning.

o* o o oo

Claims

1. An ex vivo method for diagnosing a blood sample comprising steps of: identifying conditions that each cause a low level activation of the coagulation response in blood; providing a blood sample; providing different quantitative blood tests that are each for identifying low level activation of the coagulation response in blood; performing each of the different blood tests on the blood sample; and if at least two of the different blood tests identify low level activation of the coagulation response in the blood sample are abnormal, using the at o least two of the blood tests to assist in diagnosing the blood sample with one of the conditions.

2. The method of claim 1, further comprising the steps of providing a population of blood samples, and performing the method of claim 1 for each of the blood samples of the population.

G* The method of claim 1, wherein the different blood tests comprise tests for at least two of fibrinogen, prothrombin fragment 1+2, *thrombin/antithrombin complexes, and soluble fibrin monomer.

4. The method of claim 1, wherein the different blood tests comprise tests for at least three of fibrinogen, prothrombin fragment 1+2, thrombin/antithrombin complexes, and soluble fibrin monomer.

The method of claim 1, wherein the different blood tests comprise tests for at least two of fibrinogen, prothrombin fragment 1+2, thrombin/antithrombin complexes, soluble fibrin monomer, and platelet activation.

6. The method of claim 1, wherein the different blood tests comprise tests for at least three of fibrinogen, prothrombin fragment 1+2, thrombin/antithrombin complexes, soluble fibrin monomer, and platelet activation.

7. The method of claim 1, wherein the different blood tests comprise tests for fibrinogen, prothrombin fragment 1+2, thrombin/antithrombin complexes, soluble fibrin monomer, and platelet activation.

8. An ex vivo method for diagnosing a blood sample comprising steps of: identifying a condition that causes low level activation of the o coagulation response in blood; providing a blood sample; providing different blood tests that are each for identifying low level activation of the coagulation response in blood; performing the different quantitative blood tests on the blood sample; and if at least two of the different blood tests identify low level activation of the coagulation response in the blood sample, using the at least two of the blood tests to assist in diagnosing the blood sample with the condition.

9. The method of claim 8 further comprising the steps of providing a population of blood samples and performing the method of claim 8 for each of the blood samples of the population.

The method of claim 8, wherein the different blood tests comprise tests for at least two of fibrinogen, prothrombin fragment 1+2, thrombin/antithrombin complexes, and soluble fibrin monomer.

11. The method of claim 8, wherein the different blood tests comprise tests for at least three of fibrinogen, prothrombin fragment 1+2, thrombin/antithrombin complexes, and soluble fibrin monomer.

12. The method of claim 8, wherein the different blood tests comprise tests for at least two of fibrinogen, prothrombin fragment 1+2, thrombin/antithrombin complexes, soluble fibrin monomer, and platelet activation.

13. The method of claim 8, wherein the different blood tests comprise tests for at least three of fibrinogen, prothrombin fragment 1+2, thrombin/antithrombin complexes, soluble fibrin monomer, and platelet activation.

14. The method of claim 8, wherein the different blood tests comprise tests for fibrinogen, prothrombin fragment 1+2, thrombin/antithrombin complexes, soluble fibrin monomer, and platelet activation.

15. An ex vivo method for diagnosing a blood sample comprising steps of: identifying conditions that each cause a low level activation of the 1 coagulation response in blood; providing a blood sample; providing different blood tests that are each for identifying low level activation of the coagulation response in blood, the blood tests comprising tests for at least two of fibrinogen, prothrombin fragment 1+2, thrombin/antithrombin complexes, soluble fibrin monomer, and platelet activation; obtaining a result for each of the blood tests; observing the results; and if at least two of the results are abnormal, using the abnormal results to assist in diagnosing the blood sample with one of the conditions.

16. The method of claim 15 further comprising the steps of providing a population of blood samples, and performing the method of claim 15 for each of the blood samples of the population.

17. An ex vivo method for diagnosing a blood sample comprising steps of: identifying a condition that causes a low level activation of the coagulation response in blood; providing a blood sample; providing different blood tests that are each for identifying low level activation of the coagulation response in blood; the blood tests comprising tests for fibrinogen, prothrombin fragment 1+2, thrombin/antithrombin complexes, soluble fibrin monomer, and platelet activation; and if at least two of the results are abnormal, using the abnormal results to assist in diagnosing the condition. S

18. The method of claim 17 further comprising the steps of providing a ,o 9population of blood samples, and performing the method of claim 17 for each of the blood samples of the population. *SS•

19. A method as hereinbefore described, with reference to the examples. Dated this 2 0 th day of April 2005 David E. Berg, Lois Hill Berg and Harold H. Harrison by their Patent Attorneys Pipers Mills Oakley