Chemistry: analytical and immunological testing – Clotting or clotting factor level tests
Reexamination Certificate
1999-04-14
2002-09-17
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Clotting or clotting factor level tests
C436S164000, C436S165000, C422S051000, C422S073000, C356S246000, C435S013000, C073S064410, C073S064430, C600S369000
Reexamination Certificate
active
06451610
ABSTRACT:
BACKGROUND OF THE INVENTION
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 “Newer Approaches To The Study Of Hemophilia & Hemiphiloidal States” by K. M. Brinkhous et al., JAMA 154:481-486, 1954, and an article entitled “Effect of Antihemophilic Factor On One Stage Clotting Tests” 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 “A Brain Extract As A Substitute In The Thromboplastin Generation Test” 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 “contact activation” 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 “The Partial Thromboplastin Time With Kaolin” 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 “Activation of Hageman Factor by Solutions of Ellagic Acid” 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, “Recalcification Time Test And Its Modification (Partial Thromboplastin Time, Activated Partial Thromboplastin Time And Expanded Partial Thromboplastin Time)” 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 37° 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 w
Gorman Anne Jessica
Gorman John
Samo Robert
Duane Morris LLP
International Technidyne Corporation
Plevy Arthur L.
Wallenhorst Maureen M.
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