1. Field of the Invention
The present invention relates to a method for testing liquid samples, wherein the liquid samples are tested within disposable cuvettes. More particularly, the invention relates to blood clotting assays performed using fresh, non-citrated blood and specific reagents placed in a disposable cuvette.
2. Description of Related Art
Blood clotting is a complex process involving three interacting components: blood vessels, blood coagulation factors, and blood platelets. Blood coagulation factors are proteins or glycoproteins which freely circulate within the body. The blood coagulation factors interact in a mechanism commonly referred to as the coagulation cascade. In this proteolytic activation process, inactive coagulation factors are chemically converted to active enzymes. The active enzyme subsequently converts an inactive enzyme precursor or zymogen to an active state resulting in the conversion of prothrombin to thrombin and fibrinogen to fibrin. The activation of zymogens is crucial to the process.
The final step of clot formation is the conversion of plasma soluble fibrinogen to insoluble fibrin as a result of the cleavage of peptide bonds. Cleavage occurs as the result of the proteolytic enzyme thrombin, which is produced from prothrombin. Conversion of prothrombin to thrombin requires a number of proteins called clotting factors, in addition to calcium. The fibrin clot is a crosslinked matrix which entraps the formed elements of the blood thereby sealing off the site of bleeding. Formed elements consist of platelets, white blood cells, and red blood cells.
Platelets are cell fragments having multiple roles in the clotting process. By attaching to the exposed collagen matrix of broken blood vessels and to each other, a primary platelet plug seals off the bleeding site. During the aggregation of platelets, chemical components are released into the plasma which are important in the clotting process. One component is platelet factor 3, which is a phospholipid serving as a necessary cofactor in the coagulation cascade.
There are significant differences in the clotting system of the body, i.e., blood solids including red blood cells, white blood cells, and platelets, and the circulatory coagulation factors proteins associated with the plasma. It is the production and metabolism of the coagulation factors which vary within fairly wide ranges among individuals. Whole blood clotting and plasma clotting have different mechanisms and substances. Plasma lacks many elements present in whole blood, namely, platelets, red blood cells, and white blood cells.
Specific blood clotting assays enable the clinician to determine the integrity of the blood coagulation cascade and the efficacy of therapy. An example of a clotting assay is the Partial Thromboplastin Time (PTT) test. This test was first described in an article entitled xe2x80x9cNewer Approaches To The Study Of Hemophilia and Hemiphiloidal Statesxe2x80x9d by K. M. Brinkhous et al., JAMA 154:481-486, 1954, and an article entitled xe2x80x9cEffect of Antihemophilic Factor On One Stage Clotting Testsxe2x80x9d by R. D. Langdell et al., J. Lab. Clin. Med. 41:637-647, 1953. The principle of the PTT is that citrated platelet poor, i.e, depleted, plasma (PPP) is added to a mixture containing platelet factor 3 (PF3) substitute and calcium. The substitute is a phospholipid derived from brain and lung tissue, and is described in an article entitled xe2x80x9cA Brain Extract As A Substitute In The Thromboplastin Generation Testxe2x80x9d by W. H. Bell and H. G. Alton, Nature 174:880-881, 1954. The PF3 substitute takes the place of platelets in the clotting process. Calcium is required as a necessary component for clotting since the calcium normally present in circulating blood is rendered unusable by the addition of citrate.
The PTT assay is a valuable clinical test, but a relative lack of reproducibility among different individuals makes it difficult to establish a range of normal values. Much of the variability has been attributed to the processes involved in the initial activation of the coagulation cascade. This xe2x80x9ccontact activationxe2x80x9d sequence was found to be highly variable among individuals. By standardizing the rate at which the activation occurs, the reliability of the PTT assay was greatly improved.
Standardization is accomplished by adding an activator to the PTT mixture. Conducting the PTT in the presence of an activator was first described by Proctor and Rapaport in an article entitled xe2x80x9cThe Partial Thromboplastin Time With Kaolinxe2x80x9d by R. P. Proctor et al., Am J. Clin. Path. 36:212-219, 1961. Known as the Activated Partial Thromboplastin Time (APTT) test, the particulate activator kaolin was first used, and later studies demonstrated the use of a soluble plasma activator, ellagic acid. See the article entitled xe2x80x9cActivation of Hageman Factor by Solutions of Ellagic Acidxe2x80x9d by O. D. Ratnoff and J. D. Crum, J.Lab.Clin.Med. 63:359-377, 1964. The APTT assays presently used in the clinical laboratory are minor modifications of the earlier tests. See the chapter, xe2x80x9cRecalcification Time Test And Its Modification (Partial Thromboplastin Time, Activated Partial Thromboplastin Time And Expanded Partial Thromboplastin Time)xe2x80x9d by C. Hougie, in the text: Hematology, 3rd edition, ed. Williams, McGraw Hill Book Co., N.Y., ppg. 1662-1664, 1983.
By eliminating the variable nature of activation, the APTT test is more reliable than PTT. It is particularly useful in identification of clotting factor deficiencies, of which the most common are the Hemophilias: Hemophilia A (Factor VIII deficiency), Hemophilia B (Factor XI deficiency), and Hemophilia C (Factor XII deficiency). It is also valuable as a means to monitor the effect of clot inhibiting agents such as heparin, an animal derived substance which interferes with the formation of the fibrin clot.
The APTT test is generally performed with citrated platelet poor plasma, and employs two active ingredients, a contact activator consisting of kaolin or ellagic acid, and a source of phospholipids. Other contact activators include celite, glass beads, and colloidal silica. Sources of phospholipids include, for example, phosphatidyl choline, phosphatidyl serine, ethanolamine, and other neutral lipids. The blood sample is obtained by hospital personnel and transported to a central laboratory for testing. Plasma is obtained from citrated whole blood. The plasma is placed in a reaction vessel to which the APTT reagents and calcium are added. The sample is incubated for three to five minutes at 37xc2x0 C. with an equal volume of liquid APTT reagent to allow full activation of Factors XI and XII. The phospholipid allows the remaining factors in the intrinsic pathway (prothrombin complex factors) to activate in the presence of calcium. In the absence of calcium, the pathway is blocked and the activated Factors XI and XII accumulate. Thereafter, the mixture is recalcified to activate the prothrombin complex factors and to determine the clotting time. Under these reaction conditions, a normal APTT clotting time would be in the range of twenty-five to forty seconds, depending upon the reagent, the instrument, and the patient being tested. The two step process is necessary as the APTT test requires a period of contact activation, which generally ranges from about two to five minutes.
This type of processing has proven economical and practical in screening large numbers of patient plasmas. Although practical in the previous example, the present APTT test is unacceptable in many clinical settings where delay in time between drawing the blood sample and obtaining the APTT result is critical. In response to this problem, a more practical test used is the Activated Clotting Time Test (ACT) (Hattersley, JAMA 196:150-154, 1966). Unlike the APTT, the ACT is performed at the bedside of the patient using non-anticoagulated (non-citrated) blood.
The difference between the ACT and the APTT test is that clot formation proceeds in the ACT test in whole blood in the presence of an activator. Scientists have advocated that such an analysis using whole blood is more indicative of the true coagulation state than inducing clot formation in plasma. Consequently, the APTT test using whole blood has been proposed as a more accurate measure of the coagulant state than the ACT test (P. Hattersley, Heparin Anticoagulation, in the test Laboratory Hemotology, ed. by J. Koepke, Churchill, Livingston, p. 789-818, 1984).
Many devices have been made that are capable of performing a coagulation time test, especially in a laboratory environment. Few of the devices are portable and simple enough to operate by a patient in the home. One of the most difficult aspects of using these devices is the taking of a blood sample from the patient, and the administration of the blood sample to the testing equipment before the natural blood clotting mechanism begins.
Many of the devices and methods for measuring the coagulation time of blood samples are assigned to the assignee herein. For example, U.S. Pat. No. 5,372,946 to Cusak et al., entitled BLOOD COAGULATION TIME TEST APPARATUS AND METHOD, issued on Dec. 13, 1994; relates to a disposable cuvette for retaining a sample of blood and an automated test apparatus and method for analyzing the sample of blood within the cuvette to determine a coagulation time for the sample of blood. The blood is deposited in a fluid reservoir of the cuvette. Within the cuvette is a capillary conduit with at least one restricted region. The blood is caused to traverse the restricted region and a testing machine measures the coagulation time.
Methods of performing blood coagulation tests can be implemented using the device described in the following U.S. patents to Gavin et al. and assigned to the assignee herein: U.S. Pat. No. 5,731,212 entitled TEST APPARATUS AND METHOD FOR TESTING CUVETTE ACCOMMODATED SAMPLES, issued on Mar. 24, 1998; U.S. Pat. No. 5,591,403, entitled PORTABLE PROTHROMBIN TIME TEST APPARATUS AND ASSOCIATED METHOD OF PERFORMING A PROTHROMBIN TIME TEST, issued on Jan. 7, 1997; which is a division of U.S. Pat. No. 5,534,226, entitled PORTABLE TEST APPARATUS AND ASSOCIATED METHOD OF PERFORMING A BLOOD COAGULATION TEST, issued on Jul. 9, 1996; which is a division of U.S. Pat. No. 5,504,011, entitled PORTABLE TEST APPARATUS AND ASSOCIATED METHOD OF PERFORMING A BLOOD COAGULATION TEST, issued on Apr. 2, 1996; the disclosures of which are incorporated herein by reference. The portable test apparatus includes a disposable cuvette with multiple conduits. Each of the conduits contains a dried or lyophilized activation reagent. The blood in each of the conduits is reciprocally moved across a restricted region until a predetermined degree of coagulation occurs.
An APTT test is described in U.S. Pat. No. 5,091,304 to La Duca, entitled WHOLE BLOOD ACTIVATED PARTIAL THROMBOPLASTIN TIME TEST AND ASSOCIATED APPARATUS, issued on Feb. 25, 1992; assigned to the assignee herein, where the citrate anticoagulant step is simultaneously combined with the contact activation step to allow one to employ fresh, whole blood specimens. The specific combination in a test tube of reagents which are lyophilized or in liquid form include citrate, activator, and phospholipid. Freshly drawn blood is added to the test tube, which is agitated and heated for three minutes. Calcium is then added to initiate clotting. The blood is inspected every 3-5 seconds until clot formation is observed. Upon formation of a sufficient clot, the blood fails to move freely within the test tube upon tilting.
The present methods of determining clotting time provide clotting times of 70 to 90 seconds, depending upon the patient being tested, and only loosely correlate to any given laboratory test employing plasma in an APTT test. The inability to duplicate the hemostatic status of the patient is a problem when employing whole blood in these laboratory assays.
There remains a need for a method of performing a coagulation-based assay employing whole blood which does not need to be citrated prior to performing the assay which avoids inconvenience to the patient while providing accurate results when screening for clotting abnormalities. The APTT times obtained from the present method are substantially close to those obtained in a clinical laboratory which employs citrated whole blood to obtain normal donor clotting times of 25-35 seconds.
Briefly described, the invention comprises a method of performing a coagulation-based assay on a fresh whole blood sample. A cuvette having at least one conduit has at least one restriction disposed between the first and second ends of the conduit. After drawing a sample of blood from a patient, a predetermined volume of the blood sample is introduced into the first end of the conduit. At a first position in the conduit is an anticoagulating agent capable of mixing with the blood sample. At a second position in the conduit is a coagulation reagent capable of mixing with the blood sample. At a third position located between said at least one restriction and the second end of the conduit is a recalcification agent.
The whole blood sample is mixed by pneumatic pumping at the first position for a predetermined period of time after which the blood sample is moved to the second position, where it incubates with the coagulation reagent, or APTT reagent for a predetermined period of time at a predetermined temperature. After mixing the sample with the coagulation reagent for approximately three to five minutes, the sample is moved to the third position in the conduit. At the third position, the sample is recalcified. The time until clot formation is measured by visual detection after pulling the sample through the restriction to the third position where it contacts a calcium agent.
Suitable anticoagulating reagents or calcium chelating agents which chelate the free calcium ions present in the blood include citrates, aminocarboxylates such as EDTA, oxalates, and heparin, and the like, as discussed in e.g. xe2x80x9cDiagnostic Hematologyxe2x80x9d by Lawrence Powers, Chapter 25, pages 417 to 420. In a preferred embodiment, a citrate salt is used. Suitable APTT reagents include diatomaceous earth, sodium citrate, phospholipid, and a barbital buffered saline, comprising sodium barbital, sodium chloride, sodium azide, and bovine albumen, and the like, as described in U.S. Pat. No. 5,091,304 to La Duca, the disclosure of which is incorporated herein by reference. Other APTT reagents are known, and any suitable coagulation reagent may be employed. Suitable coagulation reagents include APTT, ACT, prothrombin time, or snake venom reagents (e.g. Russel""s viper venom), and the like, depending upon the assay to be performed. The recalcification reagent includes calcium in the form of a salt, such as calcium chloride, calcium gluconate, calcium glycine, and calcium imidodiacetate, and the like. Suitable stabilizers, surfactants, fillers and buffers can optionally be added to the recalcification reagent. Suitable surfactants include Pluronic L18, F68, and the like. Suitable fillers include Trehalose, M700, and the like. Suitable buffers include Hepes buffer having a pH of about 7.4 and a concentration of about 0.02M.
The method of the present invention is used to monitor the integrity of the intrinsic pathway of blood coagulation. In the present invention, calcium is always present, that is, there is no need to citrate or chelate the calcium present in whole blood prior to placing it in the cuvette.