WHOLE BLOOD CONTROL SAMPLE SYSTEMS AND METHOD OF PREPARING AND USING THE SAME

Abstract

The preparation and use of systems to provide surrogate whole blood controls using coagulation modifiers to simulate human whole blood in coagulation assays are disclosed.

Claims

1. A whole blood control sample system comprising: a. at least one lyophilizate comprising: i. fixed red blood cells sourced from one or more mammalian species; and ii. plasma sourced from one or more mammalian species; b. at least one diluent; and c. at least one activation solution, wherein at least one of the lyophilizate, diluent or activation solution contains a coagulation modulator.

2. The whole blood control sample system of claim 1, wherein the coagulation modulator is a serine protease inhibitor and/or rabbit brain cephalin.

3. The whole blood control sample system of claim 1, wherein the coagulation modulator is a serine protease inhibitor.

4. The whole blood control sample system of claim 3, wherein the serine protease inhibitor is benzamidine.

5. The whole blood control sample system of claim 1, wherein the coagulation modulator is present in the at least one activation solution and is not present in the at least one diluent and the at least one lyophilizate.

6. The whole blood control sample system of claim 5, wherein the system comprises two or more activation solutions, with each activation solution containing a different concentration of coagulation modulator, a single lyophilizate and a single diluent.

7. The whole blood control sample system of claim 6, wherein the system is provided as a kit having two or more aliquots of activation solution, two or more aliquots of lyophilizate and two or more aliquots of diluent.

8. The whole blood control sample system of claim 6, wherein the system comprises two activation solutions with each having different concentrations of the coagulation modulator.

9. The whole blood control sample system of claim 8, wherein the system is provided as a kit having two aliquots of activation solution, two aliquots of lyophilizate and two aliquots of diluent.

10. The whole blood control sample system of claim 1, wherein the coagulation modulator is present in the at least one lyophilizate and is not present in the at least one diluent and the at least one activation solution.

11. The whole blood control sample system of claim 10, wherein the system comprises two or more lyophilizate, with each lyophilizate containing a different concentration of coagulation modulator, a single diluent and a single activation solution.

12. The whole blood control sample system of claim 11, wherein the system is provided as a kit having two or more aliquots of activation solution, two or more aliquots of lyophilizate and two or more aliquots of diluent.

13. The whole blood control sample system of claim 11, wherein the system comprises two lyophilizates with each having different concentrations of coagulation modulator.

14. The whole blood control sample system of claim 13, wherein the system is provided as a kit having two aliquots of activation solution, two aliquots of lyophilizate and two aliquots of diluent.

15. The whole blood control sample system of claim 1, wherein the at least one activation solution comprises calcium chloride.

16. The whole blood control sample system of claim 2, wherein either the at least one lyophilizate or the at least one activation solution includes an effective amount of the rabbit brain cephalin to further modulate the clotting time of the whole blood control sample system.

17. A method of preparing a whole blood control sample system comprising steps of: a. providing at least one lyophilizate comprising: i. fixed red blood cells sourced from one or more mammalian species; and ii. plasma sourced from one or more species; b. providing at least one diluent; c. providing at least one activation solution; wherein the at least one of the lyophilizate, the diluent or activation solution contains a coagulation modulator; and d. adjusting the concentration of the coagulation modulator to provide a whole blood control sample having a defined clotting time.

18. The method of claim 17, wherein the coagulation modulator is a serine protease inhibitor and/or rabbit brain cephalin.

19. The method of claim 17, wherein the coagulation modulator is a serine protease inhibitor.

20. The method of claim 19, wherein the serine protease inhibitor is benzamidine.

21. The method of claim 17, wherein the coagulation modulator is present in the at least one activation solution.

22. The method of claim 21, wherein at least two identical lyophilizates aliquots are provided and at least two activation solutions are provided, wherein each activation solution contains different amounts of coagulation modulator so as to provide a different clotting time, when an aliquot of lyophilizate, diluent and activation solution are combined.

23. The method of claim 22, wherein the system contains two identical lyophilizates aliquots, two identical diluents aliquots and two aliquots of activation solution each of the two aliquots of activation solution having a different concentrations of coagulation modulator.

24. The method of claim 17, wherein the coagulation modulator is present in the at least one lyophilizate.

25. The method of claim 24, wherein at least two identical activation solution aliquots are provided and at least two lyophilizate aliquots provided, wherein each aliquot of lyophilizate contains different amounts of coagulation modulator so as to provide a different clotting time, when an aliquot of lyophilizate, diluent and activator solution are combined.

26. The method of claim 25, wherein the system contains two identical aliquots of activator solution, two identical diluent aliquots and two lyophilizate aliquots, each of the two lyophilizate aliquots having different concentrations of coagulation modulator.

27. The method of claim 17, further comprising a step of mixing an aliquot of the at least one lyophilizate, an aliquot of the at least one diluent and an aliquot of the at least one activator solution to provide a whole blood control sample.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The present invention will be more fully understood by reference to the following drawings, which are for illustrative purposes only.

[0012] FIG. 1 illustrates that clotting time of the surrogate whole blood control shows linear response to benzamidine concentration in lyophilizate.

[0013] FIG. 2 illustrates that clotting time of surrogate whole blood control shows linear response to benzamidine concentration in activation solution.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0014] As used herein, “fixative” refers to a chemical that fix red blood cells by crosslinking proteins or other molecules on the surface of red blood cells.

[0015] As used herein, “activators” refer to reagents that can accelerate the clotting of surrogate whole blood controls.

[0016] As used herein, “serine protease inhibitors” refer to family of proteins and small molecules that antagonize the activity of serine proteases.

[0017] As used herein, “aliquots” refer to measured amounts of lyophilizate, diluent and activation solution that are combined to provide a whole blood control system.

[0018] As used herein, “coagulation modulator” refers to one or more serine protease inhibitors and/or rabbit brain cephalin that are effective to adjust clotting time by varying concentration of the modulator in the whole blood control. Although calcium chloride, which is required in the activation solution, can be varied in concentration to adjust clotting time, calcium chloride is not considered a coagulation modulator as used herein, i.e., the whole blood control system of the present invention must include both calcium chloride and a coagulation modulator.

Preparation

[0019] A. Fixed Red Blood Cells

[0020] To prepare fixed red blood cells, mammalian red blood cells may be collected by centrifugation and washed by saline to remove residual plasma proteins. The separated red blood cells are fixed with fixatives to prevent lysis of red blood cells upon lyophilization. Glutaraldehyde is a preferred fixative. The fixation time and concentration of glutaraldehyde can be varied to optimize the stability of red blood cells. After fixation, the fixature solution is removed and the fixed red blood cells are washed with saline several times to remove residual fixative. The fixed red blood cells are typically stable upon storage at 4° C. Fixed red blood cells are readily available for purchase.

[0021] B. Plasma Base for the Resuspension of Fixed Red Blood Cells

[0022] To provide the coagulation factors and fibrinogen needed for the coagulation of surrogate whole blood controls, mammalian plasma from a single species may be used to resuspend fixed red blood cells. Plasma freshly prepared from mammalian whole blood is immediately used to resuspend the fixed red blood cells. Alternatively, freshly prepared mammalian plasma can be frozen immediately after preparation for storage and transportation. Before use, frozen plasma is thawed in a water bath at 37° C. to preserve the activity of coagulation factors. In some embodiments, the plasma base is prepared by mixing plasma from different mammalian species at different mixing ratios to adjust the clotting time of the whole blood control. This is because the plasma from different species have different clotting times. In certain embodiments, activators such as cephalin, thromboplastin or kaolin can be added into plasma base. These activators accelerate the initiation process of coagulation, thereby reducing the clotting time of the surrogate whole blood control. In other embodiments, a coagulation modulator such as a serine protease inhibitor (e.g. benzamidine) is added into the activation solution at different concentrations. Benzamidine inhibits the activity of coagulation factors such as factor Xa and IIa, thereby prolonging the clotting time of the surrogate whole blood control. Significantly, it has been found that by varying the concentration of benzamidine, the clotting time of the resulting surrogate whole blood control can be adjusted to represent the range of clotting times seen in patients receiving different anticoagulants at different doses. If desired, the coagulation modulator may be included in the lyophilizate or even the diluent. Most preferably, however, the coagulation modulator is included in the activation solution.

[0023] It should be understood that the whole blood and plasma discussed above is collected into containers with anticoagulants such as citrate in the form of sodium citrate or acid-citrate-dextrose, or EDTA which chelate calcium to disrupt the coagulation cascade and prevent clotting to allow for storage and processing.

[0024] C. Activation Solution

[0025] Activation solution comprises calcium chloride, which neutralizes sodium citrate in plasma base and leads to activation of clotting of surrogate whole blood controls. In one embodiment, the concentration of calcium chloride is varied to adjust the clotting time of surrogate whole blood controls. In particular embodiments, a serine protease inhibitor (e.g. benzamidine) is added into the activation solution at different concentrations. By varying the concentration of the serine protease inhibitor, the clotting time of the surrogate whole blood control can be adjusted to represent the range of clotting times seen in patients receiving different anticoagulants at different doses.

[0026] When preparing the whole blood control system of the invention, generally the ratio of fixed red blood cells to plasma in the lyophilizate will be in a range of about 0.2: 0.8 to about 0.5: 0.5, 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 by weight. The diluent is generally water and preferably water for injection (WFI). The activation solution will contain calcium chloride generally at a concentration in an amount 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 modulator is a serine protease inhibitor such as benzamidine, which may present in the lyophilizate, diluent or activation solution, more preferably the lyophilizate or activation solution, and most preferably the activation solution, the serine protease inhibitor such as benzamidine will generally be present in a range of about 0.11 mM to about 11 mM, preferably about 0.22 mM to about 8.8 mM, and more preferably about 0.33 mM to about 6.6 mM. When the coagulation modulator is rabbit brain cephalin, which may present in the lyophilizate, diluent or activation solution, more preferably the lyophilizate or activation solution, and most preferably the lyophilizate, the rabbit brain cephalin will generally be 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 and more preferably about 0.1 mg/mL to 0.36 mg/mL.

[0027] In one embodiment the whole blood control sample system will include an aliquot of lyophilizate, an aliquot of diluent and an aliquot of activation solution which are mixed together prior to introduction into the coagulometer. Preferably the lyophilizate is added first to the diluent and then the activation solution is added to the mixture after the lyophilizate is substantially rehydrated by the diluent. Typically, the ratio of lyophilizate to diluent to activation solution will be in a range of about 1: 0.2: 0.8 to about 1: 0.8: 0.2, preferably about 1: 0.3: 0.7 to about 1: 0.7: 0.3, most preferably 1: 0.4: 0.6 to about 1: 0.6: 0.4 by volume. It should be understood that each aliquot of lyophilizate, diluent and activation solution of the whole blood control sample system may be supplied in a separate container that is meant to be mixed at the time of use to provide a whole blood control system. It would also be possible to provide a single container with three separate compartments that had means to cause all three compartments to mix. In one embodiment of the invention, the whole blood control sample system will contain two aliquots of lyophilizate, two aliquots of diluent and two aliquots of activation solution, with the concentration of coagulation modulator that is either in the lyophilizate, diluent or activation solution being different so that upon combination of an aliquot of each of the lyophilizate, diluent and activation solution, the resulting clotting time of the resulting whole blood control system from one combination is different than that of the other combination. In another embodiment, the whole blood control sample system may include 3, or 4 or 5 or more of each aliquot of lyophilizate, diluent and activation solution, with each combination having a different concentration of coagulation modulator. Preferably, the whole blood control system having a plurality of aliquots of lyophilizate, diluent and activation solutions will have the coagulation modulator present in the activation solution and not present in the lyophilizate and diluent. Such whole blood control systems are preferably provided as a kit with instructions for mixing the lyophilizate, diluent and activation solution. When the coagulation modulator is present in activation solution it is preferable to first mix the lyophilizate and diluent, followed by introduction of the activation solution.

[0028] Typically the difference in the amount of coagulation modulator, such as benzamidine, in each whole blood control system that has more than one aliquot of each of lyophilizate, diluent and activation solution will be about 0.55 mM to about 4.4 mM, preferable about 1.1 mM to about 3.3 mM, most preferably about 1.1 mM to about 2.2 mM.

[0029] In a particularly preferred embodiment, having multiple activation solutions with differing coagulation modulator concentrations allows for each system to employ a single lyophilizate and single diluent, i.e., where each aliquot of lyophilizate in the whole blood control system is identical and each aliquot of diluent in the whole blood control system is identical. Multiple activation solutions in the system advantageously allows for a range of resulting clotting times. Additionally, with coagulation activator in the activation solution being separate from the diluent, more time is allowed after the addition of the diluent to the lyophilizate providing for a more complete and consistent rehydration of the lyophilizate. This provides a significant advantage compared to existing coagulation controls which have a single diluent with activator in the diluent, and so, must be introduced to the measuring instrument quickly after reconstitution. In particular, the above described invention wherein the whole blood control system is provided as three separate components, i.e., lyophilizate, diluent and activation solution, provides a whole blood control system that has improved accuracy and precision over prior art systems because of the ability to achieve a more complete and consistent rehydration of the lyophilizate.

[0030] In yet another embodiment of the invention, the whole blood control sample system of this invention consists essentially of: (a) a single lyophilizate comprising, (i) fixed red blood cells sourced from one or more mammalian species; (ii) and plasma sourced from one or more mammalian species; (b) a single diluent; (c) and at least one activation solution, wherein the lyophilizate and diluent are substantially free of coagulation activator. Preferably, the activation solution further comprises a coagulation modulator. The absence of coagulation activator from the lyophilizate and diluent allows for a more consistent and complete rehydration of the lyophilizate with diluent prior to addition of the activation solution when preparing the whole blood control samples.

Examples

Example 1. Adjusting the Surrogate Whole Blood Control Clotting Time by Using Plasma Base Prepared with Plasma from Different Mammalian Species at Different Mixing Ratios

[0031] Plasma was prepared by mixing sheep plasma and horse plasma at different mixing ratios of 9:1, 7:3, 3:7 and 1:9. Plasma base was 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) saline. Whole blood control, containing 35% (w/v) glutaraldehyde-fixed horse red blood cells and 65% (w/v) plasma base, was recalcified by 0.2 M calcium chloride at the volume ratio of 17:1, and the samples were tested on Perosphere Technologies' PoC Coagulometers immediately. As shown in Table 1, a higher percentage of horse plasma resulted in a longer clotting time.

TABLE-US-00001 TABLE 1 Effect of sheep/horse plasma mixing ratio on the clotting time of surrogate whole blood control. Fixed horse Sheep/horse plasma Clotting Standard red blood cells mixing ratio time (sec) deviation (sec) 1% 9:1 343.67 5.86 glutaraldehyde-fixed 7:3 354.33 12.58 3:7 371.67 16.17 2% 9:1 392.00 16.97 glutaraldehyde-fixed 1:9 493.67 9.45

Example 2. Adjusting the Clotting Time of Surrogate Whole Blood Control by Varying the Concentration of Benzamidine in the Lyophilizate

[0032] Plasma was prepared by mixing sheep plasma and horse plasma at a mixing ratio of 1:9. Sheep fibrinogen solution (45.72 mg/mL in 20 mM sodium citrate-HCl, pH 7.4) was diluted to 40 mg/mL with saline. Plasma base was further prepared by mixing 77% (w/v) plasma and 23% (w/v) sheep fibrinogen solution (40 mg/mL). Whole blood control was prepared by resuspending 35% (w/v) 2% glutaraldehyde-fixed horse red blood cells in 65% (w/v) plasma base, with addition of rabbit brain cephalin to a final concentration of 0.144 mg/mL. Benzamidine of varying concentrations were added to the preparation. The whole blood control was then freeze-dried in glass vials. To test the effect of benzamidine in lyophilizate on clotting time, the freeze-dried surrogate whole blood control was reconstituted in a diluent solution to half of its original volume, incubated at room temperature for 6 minutes, and mixed by swirling the glass vial gently for an additional 1 minute. The control was then activated by addition of 22 mM calcium chloride at the volume ratio of 1:1, followed by six times of rapid inversion. The samples were tested on Perosphere Technologies' PoC Coagulometers immediately after activation. As shown in Table 2, the clotting time increased with the increase in benzamidine concentration and the dose-response curve was linear (FIG. 1).

TABLE-US-00002 TABLE 2 Effect of benzamidine concentration in lyophilizate on the clotting time of surrogate whole blood control. Benzamidine concentration in Clotting Standard lyophilizate (mM) time (sec) deviation (sec) 0 154.33 10.07 1.1 339.33 10.02 2.2 495.00 14.11

[0033] As illustrated in FIG. 1, the clotting time of surrogate whole blood control showed a linear response to the benzamidine concentration in lyophilizate.

Example 3. Adjust the Whole Blood Control Clotting Time by Varying the Calcium Chloride Concentration in Activation Solution

[0034] Plasma was prepared by mixing sheep plasma and horse plasma at a mixing ratio of 1:9. Sheep fibrinogen solution (45.72 mg/mL in 20 mM sodium citrate-HCl, pH 7.4) was diluted to 40 mg/mL by saline. Plasma base was further prepared by mixing 77% (w/v) plasma and 23% (w/v) sheep fibrinogen solution (40 mg/mL). Whole blood control was prepared by resuspending 35% (w/v) 2% glutaraldehyde-fixed horse red blood cells in 65% (w/v) plasma base, with addition of rabbit brain cephalin to a final concentration of 0.144 mg/mL. The whole blood control was then freeze-thawed once and freeze-dried in glass vials. To test the effect of calcium chloride concentration in activation solution on clotting time, the freeze-dried surrogate whole blood control was reconstituted in a diluent solution to half of its original volume, incubated at room temperature for 6 minutes, and mixed by swirling the glass vial gently for an additional 1 minute. The control was then activated by addition of calcium chloride solution of varying concentrations at the volume ratio of 1:1, followed by six times of rapid inversion. The samples were tested on Perosphere Technologies' PoC Coagulometers immediately after activation. As shown in Table 3, the clotting time can be adjusted by varying the concentration of calcium chloride in activation solution.

TABLE-US-00003 TABLE 3 Effect of CaCl.sub.2 concentration in activation solution on the clotting time of surrogate whole blood control. CaCl.sub.2 concentration in activation Clotting Standard solution (mM) time (Sec) deviation (sec) 14 199.00 10.54 22 154.00 8.54 30 166.00 8.54 40 203.33 10.07

Example 4: Adjusting the Clotting Time of Surrogate Whole Blood Controls by Varying Benzamidine Concentration in Activation Solution

[0035] Plasma was prepared by mixing sheep plasma and horse plasma at a mixing ratio of 1:9. Sheep fibrinogen solution (45.72 mg/mL in 20 mM sodium citrate-HCl, pH 7.4) was diluted to 40 mg/mL with saline. Plasma base was further prepared by mixing 77% (w/v) plasma and 23% (w/v) sheep fibrinogen solution (40 mg/mL). Whole blood control was prepared by resuspending 35% (w/v) 3% glutaraldehyde-fixed horse red blood cells in 65% (w/v) plasma base, with addition of rabbit brain cephalin to a final concentration of 0.144 mg/mL. The whole blood control was then freeze-thawed once and freeze-dried in glass vials. To test the effect of benzamidine concentration in activation solution on clotting time, the freeze-dried surrogate whole blood control was reconstituted in a diluent solution to half of its original volume, incubated at room temperature for 6 minutes, and mixed by swirling the glass vial gently for an additional 1 minute. The control was then activated by addition of an activation solution containing 22 mM CaCl.sub.2) and varying concentrations of benzamidine at the volume ratio of 1:1, followed by six times of rapid inversion. The samples were tested on Perosphere Technologies' PoC Coagulometers immediately after activation. As shown in Table 4, the clotting time demonstrated a linear response to the benzamidine concentration in activation solution.

TABLE-US-00004 TABLE 4 Effect of benzamidine concentration in activation solution on the clotting time of surrogate whole blood control Benzamidine concentration in activation solution Clotting Standard (mM) time (sec) deviation (sec) 0 149.67 7.51 1.1 206.00 8.72 2.2 249.00 6.56 4.4 357.67 8.33 6.6 422.50 0.71

[0036] FIG. 2 illustrates that the clotting time of surrogate whole blood control shows a linear response to the benzamidine concentration in activation solution.

Example 5: Adjusting the Clotting Time of Surrogate Whole Blood Controls by Adding Rabbit Brain Cephalin

[0037] Plasma was prepared by mixing sheep plasma and horse plasma at a mixing ratio of 1:9. Sheep fibrinogen solution (45.72 mg/mL in 20 mM sodium citrate-HCl, pH 7.4) was diluted to 40 mg/mL by saline. Plasma base was further prepared by mixing 77% (w/v) plasma and 23% (w/v) sheep fibrinogen solution (40 mg/mL). Whole blood control, containing 35% (w/v) glutaraldehyde-fixed horse red blood cells and 65% (w/v) plasma base with or without addition of rabbit brain cephalin to a final concentration of 0.144 mg/mL, was recalcified by 0.2 M calcium chloride at the volume ratio of 17:1, and the samples were tested on Perosphere Technologies' PoC Coagulometers immediately. As shown in Table 5, the addition of rabbit brain cephalin led to significantly reduced clotting times.

TABLE-US-00005 TABLE 5 Effect of adding rabbit brain cephalin on the clotting time of surrogate whole blood control. Rabbit brain cephalin Fixed horse final concentration Clotting Standard red blood cells (mg/mL) time (sec) deviation (sec) 2% 0 493.67 9.45 glutaraldehyde-fixed 0.144 307.67 7.51 3% 0 492.00 29.87 glutaraldehyde-fixed 0.144 302.00 11.27

Example 6: Preparing Activator Solutions Containing Varying Concentrations of Benzamidine to Meet Whole Blood Liquid Control Clotting Time Ranges

[0038] Four activator solutions were prepared having the following constituents: 1). 0 mM benzamidine, 22 mM CaCl.sub.2 in saline; 2) 2.5 mM benzamidine, 22 mM CaCl.sub.2) in saline; 3) 5 mM benzamidine, 22 mM CaCl.sub.2) in saline; and 4) 10 mM benzamidine, 22 mM CaCl.sub.2 in saline.

[0039] DI water was used as a diluent solution. Lyophilized 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 swirled for a minute to ensure reconstitution and then 0.5 ml of each of the activator solutions described above were individually added to separate vials of the reconstituted whole blood controls. The vials were closed and shaken before loading each sample on three Perosphere Technologies PoC coagulometers. The mean value of clotting time for each of the four activators described above was determined and the mean clotting times were plotted against the benzamidine concentration in each activator. Using the equation defined by the resulting plot, the benzamidine concentration for four clotting time ranges described below were calculated.

[0040] The clotting time ranges used were as follows: Level 1 range of 150-250 seconds clotting time; Level 2 range of 220-320 seconds clotting time; Level 3 range of 290-390 seconds clotting time; and Level 4 range of 370-470 seconds clotting time. Four activator solutions were then prepared by weighing the appropriate amount of benzamidine based on the equation calculated above so that each individual solution provides a clotting time for each desired level. One liter of each activator solution (Level 1, 2, 3 and 4) was prepared with the calculated amount of benzamidine and also containing 22 mM CaCl.sub.2).

REFERENCES

[0041] 1. A novel whole blood point-of-care coagulometer to measure the effect of direct oral anticoagulants and heparins. Semin Thromb Hemost. 2019, 45(3):259-263. [0042] 2. Studies on the chemistry of blood coagulation. III. The chemical constituents of blood platelets and their role in blood clotting, with remarks on the activation of clotting by lipids. J. biol. Chem. 1936, 116, 237. [0043] 3. Comparative Studies on the Inhibition of Trypsin, Plasmin, and Thrombin by Derivatives of Benzylamine and Benzamidine, European J. Biochem. 6 (1968) 502-506.