JPH0511264B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to stabilized control products useful in the clinical field, and in particular to stabilized coagulation control reagents and plasma-renin control reagents. This invention is related to a related application entitled "Stabilized Multiparameter Control Products (ORD-50)" filed concurrently by the inventors of the present invention. Hemostasis is a complex life-saving process involving blood vessels, plasma, platelets, vessel walls, nerves and humoral activity, all aimed at stopping bleeding. Although much research has been carried out, particularly in recent decades, on the process of coagulation in particular and on hemostasis in general, the details of the interaction of the contributing mechanisms in particular are not completely understood. For example, more than 35 compounds are known to be involved in the production of hard blood clots. Thus, on the surface, blood clotting is one of the complex chemical processes that occur within the body. fact,
Because there are so many separated components in the coagulation process, the International Committeeon organized by the International Hematology Congress
Nomenclature fer Blood Coagulalion adopted a series of names that have now become generally accepted. The most common of these names (commonly used interchangeably) and designations of factors include: factor fibrinogen, factor prothrombin, factor thromboplastin, factor calcium, factor proaccelelin, Stable factor, factor (currently unused), factor serum prothrombin conversion accelerator (SPCA), stable factor, factor antihemophilic factor (AHF), factor Christmas factor, plasma thromboplastin component (PTC), Factor Stuart factor, Stuart-Prower factor, Factor XI plasma thromboplastin precursor, Factor XII Hageman factor, Factor fibrin stabilizing factor, profibrinolysin plasminogen, fibrinolysin plasmin. Diagnostic determination of the presence and quantity of the various factors mentioned above is important because many clinically relevant diseases are associated with abnormal coagulation. For example, the absence of factor factor is the most common and important cause of inherited clotting defects such as hemophilia A, which has been known for over 2000 years. Deficiencies in plasma concentrations of factor factor are commonly associated with genetically determined deficiencies that predispose to type B hemophilia. Bleeding results from a lack of factors;
On the other hand, lack of factor causes long clotting times, but substantial clotting occurs. In addition, hepatopathy or vitamin K deficiency is accompanied by abnormal values of coagulation factors and coagulation factors, because these factors are mainly produced in the liver. Other causes of abnormal clotting are related to decreased platelet counts, thrombocytopenia;
It is often accompanied by abnormal levels of coagulation and coagulation factors, because these are produced in the liver. Other causes of abnormal coagulation are related to decreased platelet counts, thrombocytopenia, often accompanied by increased peripheral destruction by pernicious anemia, certain drug treatments, phototherapy or antibodies. The clinical value of clotting time tests is not limited to the detection of genetic or pathological conditions, but is also useful in adjusting anticoagulant therapy. Anticoagulants typically inhibit coagulation mechanisms, such as by heparin-mediated inhibition of factor by antithrombin. Deficiencies in the clotting mechanism due to congenital defects, pathological conditions or anticoagulant therapy are commonly caused by Robert D. Langdell, In
Clinical Diagnosis, Davidson and Henry, 1979
``Coagulation and Hemostasis'', Chapter 7. As commonly understood, coagulation occurs by two routes, the so-called internal route and the extrinsic route. The former is generally initiated by the presence of phospholipids and calcium on the surface (considered a primary activator) through a number of processes that substantially stimulate the production of a stabilized fibrin clot. be done. The partial thromboplastin (PTT) test typically measures the internal pathway by which most congenital deficiencies occur. This type of test therefore serves as an excellent preliminary surgical coagulation screening test;
The PTT test does not measure platelet activity because the reagent used in this test is typically a platelet substitute. The extrinsic route is generally initiated by injury to the tissue, and the resulting exudation of tissue thromboplastin acts on the enzymes and factors, followed by a series of processes that result in fibrin coagulation. This extrinsic pathway is commonly tested by the so-called one-stage prothrombin time test, which detects most factors inhibited by oral anticoagulants versus anticoagulant therapy controls. Thrombin time tests typically measure the amount and reactivity of fibrinogen. This test is a rapid quantitative test and is therefore the test of choice for intravascular coagulation and fibrinolysis analysis. The above is summarized in the following table and
Ortho Diagnostics.Inc., Raritan, Newjersey
Published “Concentric Concepts of Coagulation”
1971, 1975”.

[Table] →→Thron→→Fibrin→→Stabilized fiber
bottle coagulation evelyn coagulation
protro
Nbin
(thrombin time test)
Roman numerals represent the number of factors.
In giving such attention to the coagulation capacity and the presence or absence of special factors in a particular patient, appropriate measures should be taken to confirm the proper operation of manual and automatic methods designed to measure the amount of these factors and coagulation in general. It is obvious that appropriate controls must be obtained. Traditionally, such controls that are available typically have relatively short lifetimes in both liquid and dry states, depending in part on the unstable nature of the ingredients, or are susceptible to drying processes such as freeze-drying. It was characterized by being intolerable. It is an object of the present invention to provide plasma renin and coagulation control reagents that have suitable amounts of certain factors present in a stable state and can be lyophilized. It is another object of the present invention to provide a method by which coagulation control and plasma renin control reagents can be stabilized. It is another object of the present invention to provide a stabilized state reagent useful for manual and automated renal hypertension and coagulation testing. In accordance with the objects and principles of the present invention, there are provided coagulation control reagents and plasma renin control reagents that are substantially stabilized by the addition of plexiform stabilizing means. For coagulation control reagents,
adding an anticoagulant medium to the substantially defibrinated plasma;
This keeps the coagulation components intact and suppresses residual fibrin formation. In addition, about 3 to 10% by volume of a plexiform stabilizing medium selected from the group consisting of sucrose, reducing monosaccharide sugars, and reducing disaccharide sugars.
of the final product. Suitable plexiform stabilizing media are selected from the group consisting of maltose, mannitol, cellobiose, glucose and lactose, the last being most preferred. Additionally, depending on the desired type of coagulation control reagent, certain coagulation factors or factors, such as the first and/or the third, can be removed. The plasma renin control reagent of the present invention is prepared from selected plasma to which an anticoagulant medium in which the coagulation components remain substantially intact, and a plexiform stabilizing medium similar to that described for the coagulation control reagent. Become. Ideally, the anticoagulant medium is selected from the group consisting of citrate, oxalate, EDTA (ethylenediaminetetraacetic acid), heparin, and any combination thereof. In the most preferred embodiment, substantially all of the water is removed by known methods such as freeze drying. The reagent products of the invention are useful for use in manual as well as automated methods in chemical analysis;
and is particularly useful for tests involving coagulation and plasma renin activity. These tests include, for example, renal hypertension tests, actual coagulation tests, tests for angiotensin and/or
as well as renin activity tests, which are typically tested only on fresh human plasma. As expected, especially with respect to the last test, it is difficult to obtain suitable control reagents, especially in a suitably stabilized state. Such stabilized conditions are particularly desirable in the clinical field to minimize costs and increase the comparability of data on a daily testing basis. Ideally, the control reagent material should resemble the patient sample as closely as possible, except that the control may be artificially altered to some extent to provide a factor-absent control to the degree normally expected in a particular disease state. . For example, a patient who has suffered a heart infarction may be expected to have increased amounts of renin. It is known that renin acts on plasma proteins, thereby producing angiotensin, which is responsible for the increase in blood pressure caused by narrowed blood vessels. Renin, a proteolytic enzyme that appears to occur in efferent golmerular arterioles, reacts with hypertensinogen to produce angiotensin. This last one is the decapeptide hormone, which, in addition to affecting vascular narrowing, also alters aldosterone secretion by the adrenal cortex. Angiotensin is also known as hypertensin. Aldosterone is a steroid hormone that primarily has the effect of regulating sodium and potassium metabolism, thereby exerting an important effect on cardiac function. Therefore, to confirm an accurate test method, a plasma renin control reagent is substantially necessary and particularly desirable when stable. According to the purposes of the present invention, such control reagents are now possible. This ideally consists of plasma from humans, preferably selected for its high renin content. In addition, an anticoagulant may be added, which may keep the clotting components intact and inhibit fibrin formation. Such anticoagulants may be selected from the group consisting of citrate, oxalate, EDTA, heparin or combinations thereof. The desired stability is achieved by adding a plexus stabilizing medium selected from the group consisting of reducing monosaccharide sugars and reducing disaccharide sugars. Preferred embodiments use maltose, mannitol, cellobiose, lactose or glucose. The plexiform stabilizing medium is added so that the final % concentration is preferably in the range of about 3% to 10%. This is the related application shown above.
As described in more detail in ORD-50, plexiform stabilizing media are believed to provide a three-dimensional "crystalline" structure, which allows proteins, analytes,
and the components of the control reagent are held in stabilized proximity. The inventors herein further believe that the plexiform stabilizing medium serves to hold the individual components in a substantially rigid state, thereby eliminating any degradation that may accompany or result from the physical movement of the analyte. or the degree of instability is reduced, although it is hoped that such considerations do not limit the invention. Additionally, the plexiform stabilizing medium acts to assist in sublimation of water during water removal processes such as freeze drying. Although the actual mechanism by which this is achieved is unknown, it is particularly important for ORD-
50 patent applications were found regarding clinical chemistry control reagents. These advantages can also be expected in the subsequent lyophilization of the control reagent of the invention. Similarly, a coagulation control reagent is ideally produced from plasma plus anticoagulants such as those described above, so that clotting components remain intact and residual fibrin formation is substantially suppressed. be done. In addition to the plexiform stabilizing media described above, the coagulation control reagent can be tailored to remove one or more of the specific coagulation factors according to the type of coagulation control reagent desired. For example, reagents that are substantially devoid of factor (fibrinogen) or factor (antihemophilic factor) are desirable, and these components can then be removed, for example by ethanol extraction or cooling, or combined with other factors. can be removed by other methods known in the art. Together with the plasma renin control reagent, the plexiform stabilizing medium provides a three-dimensional structure that improves stability, speed of regeneration following lyophilization, and superior reagent bottling. - Greatly increases the reproducibility of reagent bottles. Furthermore, as more fully explained in the ORD-50 patent application, the rate at which lyophilization is achieved can actually be increased despite the lowered freezing point. This is thought to be primarily due to the removal of the eutectic plateau.In addition, for reasons that cannot be fully explained, the regenerated material typically exhibits greater optical clarity. , at least in part due to reduced protein degradation, particularly problems during freeze-drying.Other advantages are also obtained with the present invention, including that defibrinated plasma is transferred to a plexiform stabilizing medium. appears to be more completely defibrinated compared to less accelerated defibrinated plasma; thus, the harder gel-like clot seen here. is a natural progression from this, and is also shown to be able to enhance the overall stability of other unique clotting factors present in plasma. Substantially all of the water has been removed by known methods, including without limitation lyophilization, and serves to increase shelf life.
Ideally, the resulting dry product is stored at about 2-8°C and regenerated immediately after use. As used herein, stability means that an activity of at least 9% of the initially measured activity is subsequently measured. As those skilled in the art will readily appreciate, numerous non-substantive modifications or substitutions of the above-described components can be made without departing from the spirit or scope of the invention.

Claims (1)

Claims: 1 a plasma; b an anticoagulant medium that retains clotting components intact and suppresses fibrin formation; c present in a final volume percent range of about 3% to 10%;
a plexiform stabilizing medium selected from the group consisting of sucrose, reducing monosaccharide sugars and reducing disaccharide sugars which increases the integrity of the coagulation component; and d. A stabilized coagulation control reagent that does not require the presence of a protective gas, characterized in that the plasma can be handled manually in order to adjust one or more of the coagulation factors. 2. The reagent according to claim 1, wherein the plexiform stabilizing medium is selected from the group consisting of sucrose, lactose, mannitol, cellobiose and maltose. 3 Claim 2 in which the plasma is human plasma
Reagents listed in section. 4 Anticoagulants include citrate, oxalate,
4. A reagent according to claim 3 selected from the group consisting of EDTA, heparin and combinations thereof. 5. The reagent according to claim 4, wherein the coagulation factor to be removed is selected from the group consisting of factor 1, factor 3, and combinations thereof. 6. The reagent according to claim 4, wherein the plexiform stabilizing medium is lactose. 7. The reagent according to claim 4, wherein the plexiform stabilizing medium is sucrose. 8. The reagent according to claim 4, wherein the plexiform stabilizing medium is cellobiose. 9. The reagent according to claim 4, wherein substantially all of the water has been removed. 10. The reagent of claim 6, wherein substantially all of the water has been removed. 11. The reagent according to claim 7, wherein substantially all of the water has been removed. 12. The reagent of claim 8, wherein substantially all of the water has been removed. 13 a. preparing whole blood; b. removing substantially all cells from the whole blood to obtain plasma; c. treating the whole blood with an anti-inflammatory agent that retains clotting components intact and inhibits fibrin formation. add a coagulation medium; d adjust one or more of the specific coagulation factors according to the type of coagulation control reagent desired; and e. the presence of a protective gas characterized in that it includes the steps (a) to (b) above, adding a plexus stabilizing medium selected from the group consisting of sugars, reducing monosaccharide sugars and reducing disaccharide sugars; 14. Method of making a stabilized coagulation control reagent that does not require 14. The method of claim 13, wherein the plexiform stabilizing medium is selected from the group consisting of sucrose, lactose, mannitol, cellobiose and maltose. 15. The method of claim 13, wherein the plexiform stabilizing medium is lactose. 16 The anticoagulant medium is citrate, oxalate,
15. The method of claim 14 selected from the group consisting of EDTA, heparin and combinations thereof. 17 The anticoagulant medium is citrate, siolate,
16. The method of claim 15 selected from the group consisting of EDTA, heparin and combinations thereof. 18. The method of claim 16, wherein the factor removal step includes a step selected from the group consisting of factor removal by cooling, factor removal by ethanol extraction, and combinations thereof. 19. The method of claim 17, wherein the removing step includes a step selected from the group consisting of factor factor removal by cooling, factor factor removal by ethanol extraction, and combinations thereof. 20. The method of claim 13, further comprising the step of removing substantially all of the water. 21. The method of claim 18, further comprising the step of removing substantially all of the water. 22. The method of claim 19, further comprising the step of removing substantially all of the water. 23. The method according to claim 20, wherein the water removal step is performed by freeze drying. 24. The method according to claim 21, wherein the water removal step is performed by freeze drying. 25. The method according to claim 22, wherein the water removal step is performed by freeze drying.