Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving blood clotting factor
Reexamination Certificate
1993-12-17
2001-06-19
Saucher, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving blood clotting factor
Reexamination Certificate
active
06248547
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates, in general, to the production of serum, and, more particularly to a reagent preparation for the rapid production of a high quality serum specimen upon which diagnostic blood chemical analyses can be performed.
BACKGROUND OF THE INVENTION
Serum represents the liquid portion of the blood which remains following the clotting of the formed blood elements and blood coagulation factors. The serum contains various blood proteins, sugars, fats, enzymes and charged particles (i.e., electrolytes) which are necessary for normal metabolic processes. Devoid from the serum are the elements consumed during the clotting process namely red blood cells, white blood cells, platelets and the blood coagulation factors. Serum, obtained by centrifugation of clotted blood, is used to conduct chemical analyses which are valuable tests used to diagnose and monitor muscle and organ function, metabolic balances, and basic physiologic functions. By removing the substances consumed during clotting, a better substrate upon which to perform chemical analyses is produced. Thus, serum is the preferential test substrate on which to perform analytical tests such as enzyme, electrolyte, protein and glucose assays since the interference of unwanted substances has been removed. These analytical tests are routinely performed using automated chemistry analyzers such as the Hitachi 717, Technicon SMAC, and Ciba-Corning 5500 Express.
In the past, the production of serum from whole blood has been a passive process in which freshly collected blood is added to a glass test tube and allowed to clot. Blood, once removed from the body, has a natural tendency to clot and its exposure to a surface such as glass promotes clotting in a more efficient manner. Contact with a glass surface causes the activation of coagulation factors which interact in a mechanism commonly referred to as the coagulation cascade. In this process, an inactive coagulation factor is chemically converted to an active enzyme which subsequently converts yet another inactive precursor. The coagulation factors are identified by Roman Numerals; Factors I-XIII. The end result of the coagulation cascade is a conversion of the soluble plasma protein fibrinogen, to an insoluble protein, fibrin, whereby the fibrin clot entraps the white cells, red cells, and platelets forming a solid gelatinous mass. Substances not consumed in the process remain free of the gelatinous mass and are found in the liquid matrix. It is this liquid portion which serves as the best test substrate upon which to perform serum chemistry analyses.
The passive clotting process described above has several inadequacies. Blood from normal healthy individuals will routinely clot in approximately 6 to 10 minutes in a glass test tube. However, blood from sick individuals who may have deficiencies of coagulation proteins or from patients who are receiving anticoagulation therapy (i.e., oral anticoagulants or heparin) may require extensive time to clot (i.e., 2-8 hours). Typically highly heparinized blood is between 2-5 units per milliliter and possibly higher. Consequently, in the past, there has been a delay associated with the obtaining of blood specimens and the performance of the analytical tests, thereby affecting the ability of the clinician to quickly provide optimal patient care. In addition, the blood from individuals with deficiencies of coagulation proteins or patients receiving anticoagulation therapy may never form a complete and adequate fibrin mass. Incomplete clotting in heparinized blood specimens results in a poor quality serum substrate upon which to perform the chemical test. Furthermore, heparin anticoagulated blood which may not clot initially may actually begin to clot once placed in the chemistry analyzer, thereby clogging the system and causing an instrument shutdown.
In order to improve the clot forming process, laboratorians have routinely added the clot promoting agent thrombin to the blood specimen. Thrombin actively converts fibrinogen to fibrin, thereby forming the clot more efficiently then the slower glass-activated clotting process. Although thrombin may improve clotting in normal blood, it's effects on heparinized blood are minimal since heparin acts as an inhibitor of thrombin. As a result, collection of heparinized blood in these thrombin containing serum test tubes provides little improvement of the serum specimens, both in quality as well as in the time required to prepare the specimens, as compared to the plain glass test tube. Thrombin containing test tubes are available from Becton-Dickinson and Company, VACUTAINER Systems, of Rutherford, N.J. 07070. Though it is possible to overcome the anticoagulant effect of heparin with very large amounts of thrombin, the fibrin mass so formed is often an incomplete one. Consequently, following the centrifugation of an apparently clotted specimen, delayed clotting occurs in the serum supernatant rendering it of little use in analytical tests. Moreover, such an excessive thrombin quantity will interfere with the proper performance of the chemical analyses, thereby altering the test results. Furthermore, excessive procoagulants such as thrombin can cause some red blood cell hemolysis, whereby the red blood cell bursts open contaminating the serum specimen with intracellular particles. The above stated problems relating to the rapid production of a high quality serum specimen from heparinized patients on which to perform chemical analyses are significant since many patients receiving heparin anticoagulation therapy require repetitive serum chemistry analyses.
SUMMARY OF THE INVENTION
The problems and disadvantages associated with the prior art methods of obtaining serum discussed above are overcome in accordance with the present invention by providing a cocktail preparation to enable relatively rapid clotting of highly heparinized blood when the preparation is mixed with a given volume of blood. The preparation contains thrombin, a heparin neutralizing substance, and a snake venom which is capable of converting fibrinogen to fibrin and is unaffected by the presence of heparin. The thrombin, heparin neutralizing substance, and snake venom are present in the preparation in sufficient amounts to rapidly clot the highly heparinized blood sample to enable the serum to be separated from the clotted matter. The serum is then chemically tested is to provide clinically accurate blood chemistry results.
By providing such a clot promoting cocktail, it is now possible to prepare serum specimens and obtain accurate blood chemistry analyses of highly heparinized patients in a rapid fashion. Clinicians who rely upon results of such tests to monitor and adjust patient therapies now have the means to more closely identify patient deficiencies and provide the appropriate treatment. Thus, the patient benefits from a more closely monitored therapeutic regimen.
DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS
The present invention describes a unique combination of clot promoting reagents which when combined with fresh normal blood, fresh blood from coagulation deficient patients, or fresh blood from heparinized patients results in rapid clotting of the blood. By applying this reagent combination in very low concentration it is possible to clot non anticoagulated blood from normal individuals within two minutes and blood from patients receiving heparin therapy within five minutes. Prior to the development of this clot promoting preparation, non anticoagulated blood required three to five times as long to clot (i.e., 6-10 minutes) and heparinized blood would not clot for several hours. Some heparinized blood samples actually remained non-coagulated after 6-8 hours.
The reagents used to formulate the cocktail include two clot promoting enzymes, thrombin and snake venom, and a heparin neutralizing reagent, protamine sulfate. Thrombin may be obtained from the plasma of several animal species including, but not limited to, cows (i.e., bovine), sheep, horse, pig, rat, mouse, and humans. In t
Duane Morris & Heckscher LLP
International Technidyne Corp.
Plevy, Esq. Arthur L.
Saucher Sandra E.
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