Whole blood control sample system and method for preparing and using the above.

Abstract

Disclosed is the preparation and use of a system that provides a surrogate whole blood control that uses coagulation modifiers to simulate human whole blood in coagulation assays.

Description

【Technical Field】 【0001】 Cross - reference to related applications 【0001】This application claims the benefit of U.S. Provisional Patent Application No. 63 / 014,852, filed Apr. 24, 2020, which is hereby incorporated by reference in its entirety. 【0002】 【0002】The present invention is directed to the preparation and use of a system that provides an alternative whole blood control for simulating human whole blood in a coagulation assay. 【Background Art】 【0003】 【0003】In the United States, the number of patients requiring anticoagulation reaches millions annually, with over 600,000 patients having thromboembolic events or pulmonary embolisms (CDC Fast Stats 2012) and over 1 million joint replacements (knee and hip) (CDC Statistics 2010) requiring prophylactic postoperative anticoagulation, and the majority of 2.66 million patients with atrial fibrillation being prophylactically treated with anticoagulants (CDC Atrial Fibrillation Fact Sheet). 【0004】 【0004】Anticoagulation is time - consuming and burdensome for both physicians and patients. Traditional anticoagulants (heparin and warfarin) have a significant bleeding risk while having a long history of success in clinical use over the years. (Palareti G 2011, FDA Safety Alert 2012) Due to the incidence of major bleeding associated with traditional anticoagulants, the demand for new therapeutic anticoagulants has been met by the introduction of low - molecular - weight heparin (LMWH) products and selective oral inhibitors of coagulation factors IIa and Xa. Non - vitamin K oral anticoagulants (NOACs) have several advantages including rapid therapeutic effect, ease of dosing, and the need for no monitoring, but each is associated with a risk of major bleeding (ISMP 2012). 【0005】

[0005] In response to this, point-of-care (POC) coagulators have been developed, such as those described in U.S. Patents 9,910,053 and 10,534,006, whose disclosures are incorporated herein by reference in their entirety. For example, Perosphere Technologies Inc. (Danbury, CT) has developed a POC coagulator, a portable, battery-powered device that uses a disposable silicon and glass microfluidic cuvette for measuring coagulation time. Coagulation is initiated by contact between the blood sample and the glass surface inside the cuvette. The coagulator utilizes continuous near-infrared (IR) (1,300 nm) spectroscopy to determine the time elapsed between the addition of the blood sample to the test strip and fibrin association, the final step in the coagulation cascade. The addition of blood to the test strip causes a decrease in IR transmission due to the absorption of light by the blood, initiating automatic measurement of coagulation time. Initially, the IR transmission signal rises over time as red blood cells settle and aggregate, making it less likely to block the light path, i.e., reducing the opacity between the IR emitter and detector. Activation within the test strip occurs upon exposure of blood to the glass surface, activating coagulation via an intrinsic pathway beginning with factor XII. As the coagulation cascade progresses to the point of fibrinogen conversion to fibrin mediated by factor IIa, fibrin begins to aggregate. The aggregated fibrin network blocks the IR light path, i.e., increasing opacity and resulting in decreased IR transmission. In a time-course plot of IR transmission, the point at which fibrin aggregates and registers as a maximum or peak is reported as the coagulation time. Because activation occurs at the beginning of the intrinsic pathway and blood clot detection occurs at the end of the common pathway, the POC coagulator showed sensitivity to a wide range of anticoagulants, including edoxaban, rivaroxaban, apixaban, dabigatran, unfractionated heparin, and low molecular weight heparin (LMWH). [Overview of the Initiative] [Problems that the invention aims to solve] 【0006】

[0006] As a point-of-care diagnostic device, Perosphere Technologies' PoC coagulators require freshly collected human whole blood for measuring coagulation time. However, fresh human whole blood is unstable and therefore cannot be aliquoted, stored, or transported. Thus, there is a need to prepare alternative whole blood controls for evaluating the accuracy of POC coagulators, such as those described in U.S. Patents 9,910,053 and 10,534,006, particularly those developed by Perosphere Technologies Inc., and for routine verification of their performance. The object of the present invention is to prepare an alternative whole blood control sample system that, when used, can mimic the coagulation properties of fresh human whole blood, mediate coagulation times similar to those of human whole blood treated with different anticoagulants, and have excellent stability for storage and transport. 【0007】

[0007] The preparation and use of alternative whole blood control systems for providing whole blood control samples are described herein. A typical alternative whole blood control system comprises three components: a lyophilized product prepared from animal red blood cells and plasma, a diluent solution, and an activating solution, and the three components combined provide a whole blood control sample. In particular, the whole blood control sample system of the present invention comprises (a) at least one lyophilized product containing (i) fixed red blood cells from one or more mammalian species, and (ii) plasma from one or more mammalian species, (b) at least one diluent, and (c) at least one activating solution, wherein at least one of the lyophilized product, diluent, or activating solution contains a coagulation regulatory factor. The advantageous whole blood control system of the present invention differs from a typical whole blood control system which comprises only two components, namely a lyophilized product prepared from mammalian red blood cells and plasma, and a diluent solution containing an activator for rehydrating the blood mixture and restarting the coagulation cascade. Significantly, the whole blood control system of the present invention separates the diluent and activator, most preferably the lyophilized product and diluent being substantially free of coagulation activators. This provides a more accurate and consistent whole blood control system because the lyophilized product is more completely rehydrated with the diluent compared to prior art systems. [Means for solving the problem] 【0008】

[0008] In one embodiment, red blood cells from a single mammalian species are fixed with a crosslinking reagent containing an aliphatic aldehyde. Fixation of red blood cells helps to increase the stability of the red blood cells during lyophilization and long-term storage. The fixed red blood cells are mixed with mammalian plasma from a single species, or a mixture of plasmas from different species with different clotting times. The red blood cell / plasma mixture is then lyophilized to obtain a lyophilized product. In some embodiments, when a mixture of plasmas from different species is used, the mixing ratio of the different plasmas may be modified to achieve different clotting times for whole blood controls. In some embodiments, mammalian fibrinogen may be added to the red blood cell / plasma mixture to modify the clotting time of alternative whole blood controls. In some embodiments, activators containing thromboplastin, cephalin, or kaolin are added to the red blood cell / plasma mixture to promote clotting activation. In other embodiments, the activation solution contains calcium chloride, as well as clotting regulators such as serine protease inhibitors, including, for example, trypsin inhibitors such as rabbit brain cephalin and / or benzamidine. By changing the concentration of calcium chloride or a protease inhibitor, which is a coagulation regulator, the clotting time of the surrogate whole blood control may be adjusted to provide a range of values ​​that represent those obtained from whole blood samples taken from patients who are not receiving anticoagulants (untreated) and that represent the range of clotting times observed in patients receiving anticoagulants at different therapeutic levels. In certain embodiments, the surrogate whole blood control prepared as described herein may be used as a control for other whole blood coagulation assays, including whole blood PT, ACT, or PTT assays. 【0009】

[0009] Yet another aspect of the present invention relates to a method for preparing a whole blood control sample system, comprising the steps of (a) providing at least one lyophilized product comprising (i) fixed red blood cells derived from one or more mammalian species and (ii) plasma derived from one or more species; (b) providing at least one diluent; (c) providing at least one activating solution, wherein at least one of the lyophilized product, diluent or activating solution contains a coagulation modifier; and (d) adjusting the concentration of the coagulation modifier to provide a whole blood control system in which a measured aliquot of the lyophilized product, diluent and activating solution is combined to form a whole blood control sample having a defined coagulation time. Most preferably, the method comprises the step of providing the coagulation modifier only in the activating solution. 【0010】

[0010] In yet another embodiment, the present invention relates to a whole blood control sample obtained by a combination of aliquots of a lyophilized material, an aliquot of a diluent, and an aliquot of an activating solution. Advantageously, the whole blood control system may comprise different concentrations of coagulation modifiers in the activating solution, most preferably, so that each obtained whole blood control sample prepared in the system has a different coagulation time. Preferably, when using the whole blood control system of the present invention, the lyophilized material is first mixed with the diluent for a period of time that allows for substantially sufficient rehydration of the lyophilized material, and then the activating solution is added. 【0011】

[0011] The present invention will be better understood by referring to the following drawings, which are for illustrative purposes only. [Brief explanation of the drawing] 【0012】 [Figure 1]

[0012] This graph shows that the coagulation time of a surrogate whole blood control shows a linear response to the concentration of benzamidine in the lyophilized product. [Figure 2] 【0013】 This graph shows that the coagulation time of a surrogate whole blood control exhibits a linear response to the concentration of benzamidine in the activation solution. [Modes for carrying out the invention]

[0013] I. Definition 【0014】 As used herein, “fixative” refers to a chemical substance that fixes red blood cells to the surface of red blood cells by cross-linking proteins or other molecules.

[0014] 【0015】 As used herein, "activator" refers to a reagent capable of accelerating coagulation in an alternative whole blood control. 【0016】 As used herein, "serine protease inhibitor" refers to a family of proteins and small molecules that antagonize the activity of serine proteases.

[0015] 【0017】 As used herein, "aliquot" refers to the measured volume of lyophilized material, diluent, and activating solution combined to provide a whole blood control system. 【0018】 As used herein, “coagulation modifiers” refer to one or more serine protease inhibitors and / or rabbit brain cephalins that are effective in modulating coagulation time by different concentrations of the modifier in a whole blood control. While calcium chloride, required in the activation solution, can be used to adjust coagulation time by changing its concentration, calcium chloride is not considered a coagulation modifier as used herein; that is, the whole blood control system of the present invention must contain both calcium chloride and coagulation modifiers. II. Preparation A. Fixed red blood cells 【0019】 To prepare fixed red blood cells, mammalian red blood cells can be collected by centrifugation and washed with saline to remove residual plasma proteins. The separated red blood cells are fixed with a fixative to prevent lysis of the red blood cells during lyophilization. Glutaraldehyde is a preferred fixative. The fixation time and concentration of glutaraldehyde may be modified to optimize the stability of the red blood cells. After fixation, the fixation solution is removed, and the fixed red blood cells are washed several times with saline to remove any residual fixative. Fixed red blood cells are typically stable when stored at 4°C. Fixed red blood cells are readily available for purchase. B. Plasma base for resuspending fixed red blood cells 【0020】 To provide the coagulation factors and fibrinogen required for the coagulation of an alternative whole blood control, mammalian plasma from a single species may be used to resuspend fixed red blood cells. Freshly prepared plasma from mammalian whole blood is used immediately to resuspend fixed red blood cells. Alternatively, freshly prepared mammalian plasma may be frozen immediately after preparation for storage and transport. The frozen plasma is thawed in a 37°C water bath before use to preserve the activity of the coagulation factors. In some embodiments, the plasma base is prepared by mixing plasma from different mammalian species in different mixing ratios to adjust the coagulation time of the whole blood control. This is because plasma from different species has different coagulation times. In certain embodiments, activators such as cephalin, thromboplastin, or kaolin may be added to the plasma base. These activators accelerate the coagulation initiation process, thereby reducing the coagulation time of the alternative whole blood control. In other embodiments, coagulation modifiers such as serine protease inhibitors (e.g., benzamidine) are added to the activation solution at different concentrations. Benzamidine inhibits the activity of coagulation factors such as factors Xa and IIa, thereby prolonging the clotting time of surrogate whole blood controls. Significantly, it has been found that by changing the concentration of benzamidine, the resulting clotting time of surrogate whole blood controls can be adjusted to represent the range of clotting times observed in patients receiving different anticoagulants at different doses. If desired, the coagulation modifiers may be contained in lyophilized products or even diluents. However, most preferably, the coagulation modifiers are contained in the activation solution.

[0016] 【0021】 It should be understood that the whole blood and plasma discussed above are collected in containers containing an anticoagulant such as sodium citrate or citric acid in the form of acid-citric acid-dextrose, or EDTA, which chelates calcium, disrupts the coagulation cascade, and prevents coagulation, thus enabling storage and processing. C. Activation solution 【0022】The activation solution contains calcium chloride, which neutralizes sodium citrate in the plasma base and leads to activation of coagulation in the surrogate whole blood control. In one embodiment, the concentration of calcium chloride is modified to adjust the coagulation time of the surrogate whole blood control. In certain embodiments, a serine protease inhibitor (e.g., benzamidine) is added to the activation solution at different concentrations. By changing the concentration of the serine protease inhibitor, the coagulation time of the surrogate whole blood control can be adjusted to represent a range of coagulation times observed in patients receiving different anticoagulants at different doses.

[0017] 【0023】 When preparing the whole blood control system of the present invention, the ratio of fixed red blood cells to plasma in the lyophilized material is generally in the range of about 0.2:0.8 to about 0.5:0.5 by weight, preferably about 0.25:0.75 to about 0.45:0.55, and most preferably about 0.3:0.7 to about 0.4:0.6. The diluent is generally water, preferably water for injection (WFI). The activation solution generally contains calcium chloride at a concentration of about 14 mM to about 40 mM, preferably about 18 mM to about 36 mM, and more preferably about 20 mM to about 30 mM. When the coagulation regulatory factor is a serine protease inhibitor such as benzamidine and may be present in a lyophilized product, diluent, or activated solution, more preferably in a lyophilized product or activated solution, most preferably in an activated solution, the serine protease inhibitor such as benzamidine is generally present in a range of about 0.11 mM to about 11 mM, preferably about 0.22 mM to about 8.8 mM, more preferably in a range of about 0.33 mM to about 6.6 mM. When the coagulation regulatory factor is rabbit brain cephalin and may be present in a lyophilized product, diluent, or activated solution, more preferably in a lyophilized product or activated solution, most preferably in a lyophilized product, the rabbit brain cephalin is generally present in a range of about 0.048 mg / mL to about 0.96 mg / mL, preferably about 0.096 mg / mL to 0.48 mg / mL, more preferably in a range of about 0.1 mg / mL to 0.36 mg / mL.

[0018] 【0024】In one aspect, the whole blood control sample system includes an aliquot of the lyophilizate, an aliquot of the diluent, and an aliquot of the activation solution, which are mixed together before introduction into the coagulometer. Preferably, the lyophilizate is first added to the diluent, and then, after the lyophilizate is substantially rehydrated by the diluent, the activation solution is added to the mixture. Typically, the ratio of the lyophilizate to the diluent to the activation solution is in the range of about 1:0.2:0.8 to about 1:0.8:0.2 by volume, preferably about 1:0.3:0.7 to about 1:0.7:0.3, and most preferably 1:0.4:0.6 to about 1:0.6:0.4. It should be understood that each aliquot of the lyophilizate, diluent, and activation solution of the whole blood control sample system may be supplied in a separate container, meaning that they are mixed at the time of use to provide the whole blood control system. It is also possible to provide a single container having three separate compartments with means for mixing all three compartments. In one aspect of the present invention, the whole blood control sample system contains two aliquots of the lyophilizate, two aliquots of the diluent, and two aliquots of the activation solution, and the concentration of the coagulation regulator in either the lyophilizate, the diluent, or the activation solution is different. Thus, when the aliquots of the lyophilizate, diluent, and activation solution are combined, the resulting coagulation time of the whole blood control system obtained from one combination is different from that of other combinations. In another aspect, the whole blood control sample system may include three or four or five or more aliquots of each of the lyophilizate, diluent, and activation solution, and each combination has different concentrations of the coagulation regulator. Preferably, the whole blood control system having multiple aliquots of the lyophilizate, diluent, and activation solution has a coagulation regulator that is present in the activation solution but not in the lyophilizate and the diluent. Such a whole blood control system is preferably provided as a kit including instructions for mixing the lyophilizate, diluent, and activation solution. When the coagulation regulator is present in the activation solution, it is preferred to first mix the lyophilizate and the diluent, and then introduce the activation solution. 【0019】 【0025】Typically, the difference in the amount of a coagulation regulatory factor such as benzamidine in each whole blood control system having more than one aliquot of each of the lyophilizate, diluent, and activation solution is from about 0.55 mM to about 4.4 mM, preferably from about 1.1 mM to about 3.3 mM, and most preferably from about 1.1 mM to about 2.2 mM. 【0020】 【0026】 In a particularly preferred embodiment, having a plurality of activation solutions with different concentrations of coagulation regulatory factors allows each system to utilize a single lyophilizate and a single diluent, i.e., each aliquot of the lyophilizate in the whole blood control system is the same, and each aliquot of the diluent in the whole blood control system is the same. The plurality of activation solutions in the system advantageously allows for the range of coagulation times obtained. Further, when the coagulation activator in the activation solution is separated from the diluent, more time is ensured after the addition of the diluent to the lyophilizate, resulting in more complete and consistent rehydration of the lyophilizate. This provides a significant advantage compared to existing coagulation controls that have a single diluent with the activator in the diluent and thus must be introduced into the measuring device immediately after reconstitution. In particular, the above invention in which the whole blood control system is provided as three separate components, namely a lyophilizate, a diluent, and an activation solution, provides a whole blood control system with improved accuracy and precision over prior art systems by the ability to achieve more complete and consistent rehydration of the lyophilizate. 【0021】 【0027】 In yet another aspect of the invention, the whole blood control sample system of the invention consists essentially of (a) a single lyophilizate containing (i) fixed red blood cells from one or more mammalian species and (ii) plasma from one or more mammalian species, (b) a single diluent, and (c) at least one activation solution, wherein the lyophilizate and the diluent are substantially free of a coagulation activator. Preferably, the activation solution further contains a coagulation regulatory factor. The fact that the lyophilizate and the diluent are free of a coagulation activator allows the addition of the activation solution after the lyophilizate has been more consistently and completely rehydrated by the diluent when preparing the whole blood control sample. 【Example】 【0022】 Example 1 By using plasma bases prepared with different mixing ratios using plasma from different mammalian species, the clotting time of alternative whole blood controls can be adjusted. 【0028】 Plasma was prepared by mixing sheep plasma and horse plasma in different mixing ratios of 9:1, 7:3, 3:7, and 1:9. Plasma bases were further prepared by mixing 70% (w / v) plasma, 18% (w / v) sheep fibrinogen solution (45.72 mg / mL in 20 mM sodium citrate-HCl, pH 7.4), and 12% (w / v) physiological saline. Whole blood controls containing 35% (w / v) glutaraldehyde-fixed horse erythrocytes and 65% (w / v) plasma base were recalcified with 0.2 M calcium chloride in a volume ratio of 17:1, and the samples were immediately tested using a Perosphere Technologies PoC coagulator. As shown in Table 1, a higher proportion of horse plasma was associated with longer coagulation times.

[0023] [Table 1]

[0024] Example 2 Adjust the clotting time of alternative whole blood controls by changing the concentration of benzamidine in the lyophilized material. 【0029】Plasma was prepared by mixing sheep plasma and horse plasma in a 1:9 ratio. Sheep fibrinogen solution (45.72 mg / mL in 20 mM sodium citrate-HCl, pH 7.4) was diluted to 40 mg / mL with physiological saline. A plasma base was further prepared by mixing 77% (w / v) plasma and 23% (w / v) sheep fibrinogen solution (40 mg / mL). A whole blood control was prepared by resuspending 35% (w / v) 2% glutaraldehyde-fixed horse erythrocytes in a 65% (w / v) plasma base and adding rabbit brain cephalin to a final concentration of 0.144 mg / mL. Different concentrations of benzamidine were added to the preparations. The whole blood control was then lyophilized in glass vials. To test the effect of lyophilized benzamidine on clotting time, a lyophilized alternative whole blood control was reconstituted in a diluent solution to half its original volume, incubated at room temperature for 6 minutes, and mixed by gently rotating the glass vial for a further 1 minute. The control was then activated by adding 22 mM calcium chloride in a 1:1 volume ratio, followed by 6 rapid inversions. The samples were tested immediately after activation using a Perosphere Technologies PoC coagulator. As shown in Table 2, clotting time increased with increasing benzamidine concentration, and the dose-response curve was linear (Figure 1).

[0025] [Table 2]

[0026] 【0030】 As illustrated in Figure 1, the coagulation time of the surrogate whole blood control showed a linear response to the concentration of benzamidine in the lyophilized material. Example 3 The clotting time of whole blood controls is adjusted by changing the concentration of calcium chloride in the activation solution. 【0031】Plasma was prepared by mixing sheep plasma and horse plasma in a 1:9 ratio. Sheep fibrinogen solution (45.72 mg / mL in 20 mM sodium citrate-HCl, pH 7.4) was diluted to 40 mg / mL with physiological saline. A plasma base was further prepared by mixing 77% (w / v) plasma and 23% (w / v) sheep fibrinogen solution (40 mg / mL). A whole blood control was prepared by resuspending 35% (w / v) 2% glutaraldehyde-fixed horse erythrocytes in a 65% (w / v) plasma base and adding rabbit brain cephalin to a final concentration of 0.144 mg / mL. The whole blood control was then freeze-thawed and lyophilized in a glass vial. To test the effect of calcium chloride concentration in the activation solution on coagulation time, lyophilized alternative whole blood controls were reconstituted in a diluent solution to half their original volume, incubated at room temperature for 6 minutes, and mixed by gently rotating the glass vial for an additional minute. The controls were then activated by adding calcium chloride solutions of different concentrations in a 1:1 volume ratio, followed by 6 rapid inversions. The samples were tested immediately after activation using a Perosphere Technologies PoC coagulator. As shown in Table 3, coagulation time could be adjusted by changing the concentration of calcium chloride in the activation solution.

[0027] [Table 3]

[0028] Example 4 Adjust the clotting time of alternative whole blood controls by changing the concentration of benzamidine in the activation solution. 【0032】Plasma was prepared by mixing sheep plasma and horse plasma in a 1:9 ratio. Sheep fibrinogen solution (45.72 mg / mL in 20 mM sodium citrate-HCl, pH 7.4) was diluted to 40 mg / mL with physiological saline. A plasma base was further prepared by mixing 77% (w / v) plasma and 23% (w / v) sheep fibrinogen solution (40 mg / mL). A whole blood control was prepared by resuspending 35% (w / v) 3% glutaraldehyde-fixed horse erythrocytes in a 65% (w / v) plasma base and adding rabbit brain cephalin to a final concentration of 0.144 mg / mL. The whole blood control was then freeze-thawed and lyophilized in a glass vial. To test the effect of benzamidine concentration in the activation solution on coagulation time, a lyophilized alternative whole blood control was prepared in a diluent solution to half its original volume, incubated at room temperature for 6 minutes, and mixed by gently rotating the glass vial for a further 1 minute. The control was then activated by adding an activation solution containing 22 mM CaCl2 and different concentrations of benzamidine in a 1:1 volume ratio, followed by 6 rapid inversions. The samples were tested immediately after activation using a Perosphere Technologies PoC coagulator. As shown in Table 4, coagulation time showed a linear response with respect to the concentration of benzamidine in the activation solution.

[0029] [Table 4]

[0030] 【0033】 Figure 2 illustrates that the coagulation time of a surrogate whole blood control shows a linear response to the concentration of benzamidine in the activation solution. Example 5 The clotting time of surrogate whole blood controls is adjusted by adding rabbit brain cephalin. 【0034】Plasma was prepared by mixing sheep plasma and horse plasma in a 1:9 ratio. Sheep fibrinogen solution (45.72 mg / mL in 20 mM sodium citrate-HCl, pH 7.4) was diluted to 40 mg / mL with physiological saline. A plasma base was further prepared by mixing 77% (w / v) plasma and 23% (w / v) sheep fibrinogen solution (40 mg / mL). Whole blood controls containing 35% (w / v) glutaraldehyde-fixed horse erythrocytes and 65% (w / v) plasma base, with or without rabbit brain cephalin added to a final concentration of 0.144 mg / mL, were recalcified with 0.2 M calcium chloride in a 17:1 volume ratio, and the samples were immediately tested using a Perosphere Technologies PoC coagulation analyzer. As shown in Table 5, the addition of rabbit brain cephalin significantly reduced the coagulation time.

[0031] [Table 5]

[0032] Example 6 Prepare activator solutions containing different concentrations of benzamidine to meet the clotting time range of a whole blood control. 【0035】 Four activator solutions were prepared, each containing the following components: 1) 0 mM benzamidine and 22 mM CaCl2 in physiological saline; 2) 2.5 mM benzamidine and 22 mM CaCl2 in physiological saline; 3) 5 mM benzamidine and 22 mM CaCl2 in physiological saline; and 4) 10 mM benzamidine and 22 mM CaCl2 in physiological saline.

[0033] 【0036】DI water was used as the diluent solution. Lipoderized whole blood controls were reconstituted by adding 0.5 mL of the diluent to each control and incubating the controls at room temperature for 6 minutes. Each vial was rotated for 1 minute to ensure reconstitution, and then 0.5 mL of the above activator solution was individually added to separate vials of the reconstituted whole blood controls. After closing the vials and shaking, each sample was loaded into three Perosphere Technologies PoC coagulation meters. The average coagulation time for each of the four activators described above was calculated, and the average coagulation time was plotted against the concentration of benzamidine in each activator. Using the equations defined in the resulting plots, the benzamidine concentrations for the four coagulation time ranges described below were calculated.

[0034] 【0037】 The ranges of solidification times used were as follows: Level 1: 150–250 seconds; Level 2: 220–320 seconds; Level 3: 290–390 seconds; and Level 4: 370–470 seconds. Four activator solutions were then prepared by weighing appropriate amounts of benzamidine based on the equations calculated above, so that each individual solution provided each desired level of solidification time. One liter each of the activator solutions (Levels 1, 2, 3, and 4) was prepared, each containing the calculated amount of benzamidine and 22 mM CaCl2. References

[0035] [Table 6] The following is a description of the claims as they were at the time of filing the application. [Claim 1] A whole blood control sample system, ai Fixed red blood cells derived from one or more mammalian species, and ii. Plasma derived from one or more mammalian species A freeze-dried product containing at least one type of freeze-dried product, b. At least one diluent, c. At least one type of activating solution and A whole blood control sample system comprising, and wherein at least one of a lyophilized product, diluent, or activating solution contains a coagulation regulatory factor. [Claim 2] The whole blood control sample system according to claim 1, wherein the coagulation regulatory factor is a serine protease inhibitor and / or rabbit brain cephalin. [Claim 3] The whole blood control sample system according to claim 2, wherein the coagulation regulatory factor is a serine protease inhibitor. [Claim 4] The whole blood control sample system according to claim 3, wherein the serine protease inhibitor is benzamidine. [Claim 5] The whole blood control sample system according to any one of claims 1 to 4, wherein the coagulation regulatory factor is present in at least one activated solution but not in at least one diluent and at least one lyophilized product. [Claim 6] The whole blood control sample system according to claim 5, wherein the system comprises two or more activating solutions, each activating solution containing different concentrations of coagulation regulators, a single lyophilized product, and a single diluent. [Claim 7] The whole blood control sample system according to claim 6, wherein the system is provided as a kit having two or more aliquots of an activating solution, two or more aliquots of a lyophilized product, and two or more aliquots of a diluent. [Claim 8] The whole blood control sample system according to claim 6, wherein the system comprises two activation solutions, each activation solution having different concentrations of coagulation regulatory factors. [Claim 9] The whole blood control sample system according to claim 8, wherein the system is provided as a kit having two aliquots of an activating solution, two aliquots of a lyophilized product, and two aliquots of a diluent. [Claim 10] The whole blood control sample system according to any one of claims 1 to 4, wherein the coagulation regulatory factor is present in at least one lyophilized product and not in at least one diluent and at least one activating solution. [Claim 11] The whole blood control sample system according to claim 10, wherein the system comprises two or more lyophilized products, each lyophilized product containing different concentrations of coagulation regulators, a single diluent, and a single activating solution. [Claim 12] The whole blood control sample system according to claim 11, wherein the system is provided as a kit having two or more aliquots of an activating solution, two or more aliquots of a lyophilized product, and two or more aliquots of a diluent. [Claim 13] The whole blood control sample system according to claim 11, wherein the system comprises two lyophilized products, each lyophilized product having different concentrations of coagulation regulatory factors. [Claim 14] The whole blood control sample system according to claim 13, wherein the system is provided as a kit having two aliquots of an activating solution, two aliquots of a lyophilized product, and two aliquots of a diluent. [Claim 15] The whole blood control sample system according to any one of claims 1 to 4, wherein the activation solution contains calcium chloride. [Claim 16] The whole blood control sample system according to claim 3 or 4, wherein at least one lyophilized product or at least one activated solution contains an effective amount of rabbit brain cephalin to further modulate the coagulation time of the whole blood control sample system. [Claim 17] A method for preparing a whole blood control sample system, ai Fixed red blood cells derived from one or more mammalian species, and ii. Plasma derived from one or more species A step of providing at least one freeze-dried product, b. A step of providing at least one diluent, c. A step of providing at least one type of activation solution. Herein, at least one of the freeze-dried product, diluent, or activating solution contains a coagulation regulatory factor. Furthermore, d. A step of adjusting the concentration of coagulation regulatory factors to provide a whole blood control sample having a defined coagulation time. Methods that include... [Claim 18] The method according to claim 17, wherein the coagulation regulatory factor is a serine protease inhibitor and / or rabbit brain cephalin. [Claim 19] The method according to claim 18, wherein the coagulation regulatory factor is a serine protease inhibitor. [Claim 20] The method according to claim 19, wherein the serine protease inhibitor is benzamidine. [Claim 21] The method according to any one of claims 17 to 20, wherein the coagulation regulatory factor is present in at least one activated solution. [Claim 22] The method according to claim 21, wherein at least two aliquots of the same lyophilized material are provided, and at least two activation solutions are provided, each activation solution containing different amounts of a coagulation modifier to provide different coagulation times when the aliquots of the lyophilized material, diluent and activation solution are combined. [Claim 23] The method according to claim 22, wherein the system comprises aliquots of two identical lyophilized products, aliquots of two identical diluents, and two aliquots of an activating solution, each of the two aliquots of the activating solution having different concentrations of a coagulation modifier. [Claim 24] The method according to any one of claims 17 to 20, wherein the coagulation regulatory factor is present in at least one freeze-dried product. [Claim 25] The method according to claim 24, wherein at least two identical aliquots of activating solutions are provided, and at least two aliquots of lyophilized products are provided, each aliquot of a lyophilized product containing different amounts of a coagulation modifier to provide different coagulation times when the aliquots of the lyophilized product, diluent and activating solution are combined. [Claim 26] The method according to claim 25, wherein the system comprises two identical aliquots of an activator solution, two identical aliquots of a diluent, and two aliquots of a lyophilized product, each of the two aliquots of the lyophilized product containing different concentrations of a coagulation modifier. [Claim 27] The method according to any one of claims 17 to 20, further comprising the step of mixing an aliquot of at least one lyophilized product, an aliquot of at least one diluent, and an aliquot of at least one activator solution to provide a whole blood control sample.

Claims

[Claim 1] A whole blood control sample system, a. i. Fixed red blood cells derived from one or more mammalian species, ii. Plasma derived from one or more mammalian species, and iii. An anticoagulant selected from the group consisting of sodium citrate, acid-citric acid-dextrose, and EDTA. A freeze-dried product containing at least one type of freeze-dried product, b. At least one diluent, c. At least one activation solution containing a coagulation activator, which is calcium chloride. The above includes, and at least one of the freeze-dried product, diluent, or activating solution contains a coagulation modifier, The freeze-dried product and the diluent are substantially free of the coagulation activator, and the coagulation regulatory factor is a serine protease inhibitor and / or rabbit brain cephalin. The whole blood control sample system mentioned above. [Claim 2] The whole blood control sample system according to claim 1, wherein the coagulation regulatory factor is a serine protease inhibitor. [Claim 3] The whole blood control sample system according to claim 2, wherein the serine protease inhibitor is benzamidine. [Claim 4] The whole blood control sample system according to any one of claims 1 to 3, wherein the coagulation regulatory factor is present in at least one of the activation solutions and is not present in at least one of the diluents and at least one of the freeze-dried products. [Claim 5] The whole blood control sample system according to claim 4, wherein the system comprises two or more activating solutions, each activating solution containing different concentrations of coagulation regulators, a single lyophilized product, and a single diluent. [Claim 6] The whole blood control sample system according to claim 5, wherein the system is provided as a kit having two or more aliquots of an activation solution, two or more aliquots of a lyophilized product, and two or more aliquots of a diluent. [Claim 7] The whole blood control sample system according to claim 5, wherein the system comprises two types of activation solutions, each activation solution having different concentrations of the coagulation regulatory factor. [Claim 8] The whole blood control sample system according to claim 7, wherein the system is provided as a kit having two aliquots of an activating solution, two aliquots of a lyophilized product, and two aliquots of a diluent. [Claim 9] The whole blood control sample system according to any one of claims 1 to 3, wherein the coagulation regulatory factor is present in at least one of the freeze-dried products and is not present in at least one of the diluents and at least one of the activating solutions. [Claim 10] The whole blood control sample system according to claim 9, wherein the system comprises two or more lyophilized products, each lyophilized product containing different concentrations of a coagulation regulatory factor, a single diluent, and a single activating solution. [Claim 11] The whole blood control sample system according to claim 10, wherein the system is provided as a kit having two or more aliquots of an activation solution, two or more aliquots of a lyophilized product, and two or more aliquots of a diluent. [Claim 12] The whole blood control sample system according to claim 10, wherein the system comprises two types of freeze-dried products, each freeze-dried product having different concentrations of coagulation regulatory factors. [Claim 13] The whole blood control sample system according to claim 12, wherein the system is provided as a kit having two aliquots of an activating solution, two aliquots of a lyophilized product, and two aliquots of a diluent. [Claim 14] The whole blood control sample system according to claim 2 or 3, wherein at least one of the lyophilized products or at least one of the activating solutions contains an effective amount of rabbit brain cephalin to further adjust the coagulation time of the whole blood control sample system. [Claim 15] A method for preparing a whole blood control sample system, a. i. Fixed red blood cells derived from one or more mammalian species, ii. Plasma derived from one or more mammalian species; and iii. An anticoagulant selected from the group consisting of sodium citrate, acid-citric acid-dextrose, and EDTA. A step of providing at least one freeze-dried product, b. Providing at least one diluent, c. Providing at least one activation solution containing a coagulation activator that is calcium chloride, Herein, at least one of the lyophilized product, diluent, or activating solution contains a coagulation modifier, the lyophilized product and the diluent are substantially free of the coagulation activator, and the coagulation modifier is a serine protease inhibitor and / or rabbit brain cephalin. d. The step of adjusting the concentration of coagulation regulatory factors to provide a whole blood control sample having a defined coagulation time, Methods that include... [Claim 16] The method according to claim 15, wherein the coagulation regulatory factor is a serine protease inhibitor. [Claim 17] The method according to claim 16, wherein the serine protease inhibitor is benzamidine. [Claim 18] The method according to any one of claims 15 to 17, wherein the coagulation regulatory factor is present in at least one of the activation solutions. [Claim 19] The method according to claim 18, wherein at least two aliquots of the same lyophilized material are provided, and at least two activation solutions are provided, each activation solution containing different amounts of a coagulation modifier to provide different coagulation times when the aliquots of the lyophilized material, diluent and activation solution are combined. [Claim 20] The method according to claim 19, wherein the system comprises aliquots of two identical freeze-dried products, aliquots of two identical diluents, and two aliquots of an activating solution, each of the two aliquots of the activating solution having different concentrations of a coagulation modifier. [Claim 21] The method according to any one of claims 15 to 17, wherein the coagulation regulatory factor is present in at least one of the freeze-dried products. [Claim 22] The method according to claim 21, wherein at least two identical aliquots of the activating solution are provided, and at least two aliquots of the lyophilized product are provided, each aliquot of the lyophilized product containing different amounts of a coagulation modifier to provide different coagulation times when the aliquots of the lyophilized product, diluent and activating solution are combined. [Claim 23] The method according to claim 22, wherein the system comprises two identical aliquots of an activating solution, two identical aliquots of a diluent, and two aliquots of a lyophilized product, each of the two aliquots of the lyophilized product containing different concentrations of a coagulation modifier. [Claim 24] The method according to any one of claims 15 to 17, further comprising the step of mixing an aliquot of at least one of the freeze-dried products, an aliquot of at least one of the diluents, and an aliquot of at least one of the activating solutions to provide a whole blood control sample.