Blood collection device comprising an inhibitor of hexokinase, a glycolysis-inhibiting agent, and an anticoagulant or plasma stabilizer

Abstract

The present invention relates to a composition for the stabilization of glucose, lactate and homocysteine in blood after collection, to a use of the provided compositions and a method for the stabilization of glucose, lactate and homocysteine in blood after collection, as well as optionally the in vitro determination of glucose, lactate and homocysteine in blood, and a blood collection device provided for said use and method.

Claims

1. A blood collection device comprising a composition comprising in combination and commonly placed in the blood collection device: i) at least one inhibitor of hexokinase, selected from the group consisting of 2-deoxy-D-glucose, 2-fluoro-2-deoxy-D-glucose, 2-amino-2-deoxy-D-glucose and 3-bromopyruvic acid or salt thereof; ii) at least one glycolysis-inhibiting agent having activity for another enzyme involved in glucose catabolism, selected from the group consisting of fluoride salt, iodoacetic acid or salt therof, oxamic acid or salt thereof and dichloroacetic acid or salt therof; and optionally iii) an anticoagulant and/or a plasma stabilizer, selected from the group consisting of EDTA salt, citrate salt, oxalate salt and heparin salt.

2. The blood collection device according to claim 1, wherein the composition further comprises iv) ammonium salt NR.sub.4X, wherein each R independently is hydrogen, linear C.sub.1-C.sub.6 alkyl, branched C.sub.3-C.sub.6 alkyl, unsubstituted phenyl or substituted phenyl, and X is halide, hydroxide, C.sub.1-C.sub.4 alkoxide and acetate.

3. The blood collection device according to claim 1, wherein the at least one glycolysis-inhibiting agent ii) has antiglycolytic activity for any one of the enzymes in the glycolytic pathway downstream of hexokinase.

4. The blood collection device according to claim 1, further comprising a blood sample which is preserved for further analysis.

5. The blood collection device according to claim 4, wherein one and/or more of glucose, lactate and homocysteine in the blood sample is stabilized.

6. The blood collection device according to claim 4, wherein the mass concentration of the at least one inhibitor of hexokinase i) is at least 0.01 mg/mL blood sample and wherein the mass concentration of the at least one glycolysis-inhibiting agent ii) is at least 0.01 mg/mL blood sample.

7. The blood collection device according to claim 1, wherein the composition is substantially free of agents for the lysis of blood cells.

8. The blood collection device according to claim 1, comprising a device which is capable of being connected with a conventional blood withdrawal device.

9. The blood collection device according to claim 1, which is a blood collection tube, an evacuated blood collection tube, a vacutainer or an aspiration system respectively enclosing said composition.

10. The blood collection device according to claim 1, combined within a kit further comprising test substances for the determination of at least one of, optionally all simultaneously, glucose, lactate and homocysteine and optionally a further blood component in collected blood.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows glucose stability at room temperature in whole blood containing A: 2-deoxy-D-glucose, ammonium fluoride and ammonium heparinate; B: 2-deoxy-D-glucose, ammonium fluoride and sodium heparinate; C: 2-deoxy-D-glucose, ammonium fluoride and potassium salt of EDTA; and D: potassium salt of EDTA.

(2) FIG. 2 depicts lactate stability at room temperature in whole blood containing A: 2-deoxy-D-glucose, ammonium fluoride and ammonium heparinate; B: 2-deoxy-D-glucose, ammonium fluoride and sodium heparinate; C: 2-deoxy-D-glucose, ammonium fluoride and potassium salt of EDTA; and D: potassium salt of EDTA.

(3) FIG. 3 shows homocysteine stability at room temperature in whole blood containing A: 2-deoxy-D-glucose, ammonium fluoride and ammonium heparinate; B: 2-deoxy-D-glucose, ammonium fluoride and sodium heparinate; C: 2-deoxy-D-glucose, ammonium fluoride and potassium salt of EDTA; and D: potassium salt of EDTA.

EXAMPLES AND COMPARATIVE EXAMPLES

(4) Materials Used and Method

(5) Materials

(6) Iodoacetate, ammonium fluoride, dichloroacetic acid, 3-bromopyruvic acid, 2-deoxy-D-glucose, and sodium oxamate were purchased from SIGMA (SIGMA-ALDICH, Germany). Blood collection tubes with anticoagulants were provided by KABE (KABE Labortechnik GmbH, Germany)

(7) Blood Collection and Sampling Protocol

(8) Before blood collection, particular mixtures of antiglycolytic agents were added into blood collection tubes containing anticoagulant. The respective concentrations of the antiglycolytic agents in the mixtures were in a range from 0.02 mg to 4 mg/mL of blood to be mixed with. Anticoagulant used was ammonium, lithium or sodium heparinate at a concentration of 12.5 IU/mL or dipotassium ethylene diamine tetraacetic acid (K2EDTA) at a concentration of 2 mg/mL.

(9) Blood was drawn from healthy volunteers by venipuncture of the antecubital vein by aspiration. Blood samples were collected into tubes with or without the particular mixtures of antiglycolytic agents. Caution was taken that all tubes were filled to the mark and that the blood was well mixed with the agents by inverting the tubes 4 times immediately after blood collection.

(10) After whole blood was mixed with different antiglycolytic agents and/or anticoagulants in the tubes, the tubes were stored at room temperature (from 20 to 25 C.) for different time intervals before centrifugation. The tubes were centrifuged 4, 15, 24, 48 and 50 hours after blood collection, and the separated plasma was stored at 20 C. until analysis. Concentrations of glucose, lactate and homocysteine were determined for the samples centrifuged after the different time intervals of storage.

(11) As a control and for comparison, a blood sample without any antiglycolytic agent was centrifuged immediately after blood collection and the plasma was separated within 10 min and stored at 20 C. until analysis. Glucose, lactate and homocysteine concentrations were determined for this sample, said concentrations being defined as the reference concentrations at time 0. Percentages for the time-dependent glucose, lactate and homocysteine concentrations relative to the reference concentrations (baseline) were computed.

(12) Measurement of Glucose

(13) The hexokinase method on cobas c (Roche Diagnostics) was used according to the protocol of the manufacturer.

(14) Measurement of Lactate

(15) A colorimetric method on cobas c (Roche Diagnostics) was used. The method is based on the oxidation of lactate to pyruvate by lactate oxidase.

(16) Measurement of Total Homocysteine (tHcy)

(17) tHcy was measured by a competitive immunoassay on IMMULITE (SIEMENS, Germany) and by the Diazyme Homocysteine Enzymatic Assay Kit on cobas c (Roche Diagnostics).

Example 1

(18) Whole blood was added to 2-deoxy-D-glucose (2.9 mg/mL blood), ammonium fluoride (2.9 mg/mL blood) and an anticoagulant in different aqueous solutions (solvents were Solvent 1: H.sub.2O; and Solvent 2: aq. ac.=weakly acidified aqueous solution, respectively). After different time intervals glucose, lactate and homocysteine concentrations were determined. The determined values are shown in Table 1 as percentages of the baseline. The combination of 2-deoxy-D-glucose and ammonium fluoride stabilized glucose, lactate and homocysteine fast, efficiently and continuously.

(19) TABLE-US-00001 TABLE 1 changes in plasma analyte concentration time intervals (hours) analyte solvent 0 4 15 24 48 glucose H.sub.2O 100 101 101 100 101 glucose aq. ac. 100 102 100 102 102 lactate H.sub.2O 100 107 107 108 107 lactate aq. ac. 100 102 104 104 104 homocysteine H.sub.2O 100 100 100 102 100 homocysteine aq. ac. 100 101 101 100 100

(20) For whole blood with 2-deoxy-D-glucose and ammonium fluoride, ammonium heparinate (A), sodium heparinate (B) and potassium salt of EDTA (C) were used respectively as anticoagulant. For the anticoagulants A-C it was found that glucose, lactate and homocysteine were stabilized fast, efficiently and continuously, as can be seen in FIGS. 1-3.

Example 2

(21) Whole blood was added to 2-deoxy-D-glucose (2.9 mg/mL blood), sodium iodoacetate (1.13 mg/mL blood) and an anticoagulant in different aqueous solutions (solvents were Solvent 1: H.sub.2O; and Solvent 2: aq. ac.=weakly acidified aqueous solution, respectively). After different time intervals glucose, lactate and homocysteine concentrations were determined. The determined values are shown in Table 2 as percentages of the baseline. The combination of 2-deoxy-D-glucose and sodium iodoacetate stabilized glucose, lactate and homocysteine fast, efficiently and continuously.

(22) TABLE-US-00002 TABLE 2 changes in plasma analyte concentration time intervals (hours) analyte solvent 0 4 15 24 48 glucose H.sub.2O 100 100 100 101 100 glucose aq. ac. 100 101 102 101 101 lactate H.sub.2O 100 97 97 98 97 lactate aq. ac. 100 101 102 101 101 homocysteine H.sub.2O 100 100 100 101 100 homocysteine aq. ac. 100 103 103 101 103

Example 3

(23) Whole blood was added to 2-deoxy-D-glucose (2.9 mg/mL blood), sodium oxamate (1.25 mg/mL blood) and an anticoagulant in weakly acidified aqueous solution as solvent. After different time intervals glucose and homocysteine concentrations were determined. The determined values are shown in Table 3 as percentages of the baseline. The combination of 2-deoxy-D-glucose and sodium oxamate stabilized glucose and homocysteine fast, efficiently and continuously.

(24) TABLE-US-00003 TABLE 3 changes in plasma analyte concentration time intervals (hours) analyte 0 4 15 24 48 glucose 100 100 100 100 100 homocysteine 100 102 108 112 112

Example 4

(25) Whole blood was added to 3-bromopyruvic acid (2.5 mg/mL blood), sodium iodoacetate (1.13 mg/mL blood) and an anticoagulant in water and weakly acidified aqueous solution as solvent respectively. After different time intervals glucose concentrations were determined. The determined values are shown in Table 4 as percentages of the baseline. The combination of 3-bromopyruvic acid and sodium iodoacetate stabilized glucose fast, efficiently and continuously.

(26) TABLE-US-00004 TABLE 4 changes in plasma glucose concentration time intervals (hours) solvent 0 4 15 24 48 H.sub.2O 100 100 101 100 100 aq. ac. 100 104 104 104 104

Example 5

(27) Whole blood was added to 2-deoxy-D-glucose (2.9 mg/mL blood), ammonium fluoride (2.9 mg/mL blood), sodium oxamate (0.81 mg/mL blood) and an anticoagulant in weakly acidified aqueous solution as solvent. After different time intervals glucose, lactate and homocysteine concentrations were determined. The determined values are shown in Table 5 as percentages of the baseline. The combination of 2-deoxy-D-glucose, ammonium fluoride and sodium oxamate stabilized glucose, lactate and homocysteine fast, efficiently and continuously.

(28) TABLE-US-00005 TABLE 5 changes in plasma analyte concentration time intervals (hours) analyte 0 4 15 24 48 glucose 100 102 102 103 102 lactate 100 96 96 96 97 homocysteine 100 104 103 104 103

Example 6

(29) Whole blood was added to 2-deoxy-D-glucose (2.9 mg/mL blood), sodium iodoacetate (1.2 mg/mL blood), sodium oxamate (0.65 mg/mL blood) and an anticoagulant in weakly acidified aqueous solution as solvent. After different time intervals glucose and lactate concentrations were determined. The determined values are shown in Table 6 as percentages of the baseline. The combination of 2-deoxy-D-glucose, sodium iodoacetate and sodium oxamate stabilized glucose and lactate fast, efficiently and continuously.

(30) TABLE-US-00006 TABLE 6 changes in plasma analyte concentration time intervals (hours) analyte 0 4 15 24 48 glucose 100 102 101 102 102 lactate 100 98 97 98 98

Comparative Example 1

(31) When whole blood was mixed with potassium salt of EDTA only, i.e. no antiglycolytic agent was added, glucose, lactate and homocysteine were not stabilized. The glucose concentration decreased rapidly and continuously (see FIG. 1), while the lactate and homocysteine concentrations increased rapidly and continuously (see FIGS. 2-3).

Comparative Example 2

(32) Whole blood was added to Sarstedt blood collection tubes containing sodium fluoride and potassium oxalate. After different time intervals glucose and lactate concentrations were determined. The determined values are shown in Table 7 as percentages of the baseline. Glucose and lactate concentrations were not sufficiently stabilized, in any event remarkably less than in the Examples according to the present invention. The glucose concentration decreased significantly and continuously, while the lactate concentration increased significantly and continuously.

(33) TABLE-US-00007 TABLE 7 changes in plasma analyte concentration time intervals (hours) analyte 0 4 15 24 48 glucose 100 96 94 92 91 lactate 100 106 110 113 116

Comparative Example 3

(34) Whole blood was added to 2-deoxy-D-glucose (2.9 mg/mL blood) and an anticoagulant in weakly acidified aqueous solution as solvent. After different time intervals glucose and homocysteine concentrations were determined. The determined values are shown in Table 8 as percentages of the baseline. Glucose and homocysteine concentrations were not sufficiently stabilized. The glucose concentration decreased significantly and continuously, while the homocysteine concentration increased significantly and continuously.

(35) TABLE-US-00008 TABLE 8 changes in plasma analyte concentration time intervals (hours) analyte 0 4 15 24 48 glucose 100 87 85 80 80 homocysteine 100 102 106 117 117

Comparative Example 4

(36) Whole blood was added to 3-bromopyruvic acid (2.5 mg/mL blood) and an anticoagulant in weakly acidified aqueous solution as solvent. After different time intervals glucose and homocysteine concentrations were determined. The determined values are shown in Table 9 as percentages of the baseline. Glucose and homocysteine concentrations were not sufficiently stabilized. The glucose concentration decreased significantly and continuously, while the homocysteine concentration increased significantly and continuously.

(37) TABLE-US-00009 TABLE 9 changes in plasma analyte concentration time intervals (hours) analyte 0 4 15 24 48 glucose 100 90 89 88.8 88 homocysteine 100 101 111 111 111

(38) A comparison of the Examples with the Comparative Examples shows that the compositions according to the present invention stabilize glucose, lactate and homocysteine fast, efficiently and continuously, whereas compositions not containing the particular combinations of agents according to the present invention are insufficient in stabilizing glucose, lactate and homocysteineeven when agents are used that inhibit hexokinase such as 2-deoxy-D-glucose (see Comparative Example 3) and 3-bromopyruvic acid (see Comparative Example 4) alone.

Examples 7-13 and Comparative Example 5

(39) In vitro hemolysis was tested in blood samples incubated at room temperature (22 C.-25 C.) by determining the free plasma hemoglobin concentration (g/L) over time (0-50 hours), wherein blood samples were mixed with different compositions.

Example 7

(40) Whole blood was added to 2-deoxy-D-glucose, sodium iodoacetate, sodium oxamate and K.sub.3EDTA.

Example 8

(41) Whole blood was added to 2-deoxy-D-glucose, ammonium salt of iodoacetic acid, sodium oxamate and K.sub.3EDTA.

Example 9

(42) Whole blood was added to 2-deoxy-D-glucose, sodium iodoacetate, ammonium salt of oxamic acid and K.sub.3EDTA.

Example 10

(43) Whole blood was added to 2-deoxy-D-glucose, sodium iodoacetate, sodium oxamate, K.sub.3EDTA and tetraethylammonium chloride.

Example 11

(44) Whole blood was added to 2-deoxy-D-glucose, sodium iodoacetate, sodium oxamate, K.sub.3EDTA and tetramethylammonium fluoride.

Example 12

(45) Whole blood was added to 2-deoxy-D-glucose, sodium iodoacetate, sodium oxamate, K.sub.3EDTA and tetramethylammonium chloride.

Example 13

(46) Whole blood was added to 2-deoxy-D-glucose, sodium iodoacetate, sodium oxamate, K.sub.3EDTA and ammonium chloride.

Comparative Example 5

(47) Whole Blood was added to K.sub.3EDTA

(48) The concentrations of antiglycolytic agents in the mixtures were 0.1-4 mg/mL blood. In the cases of Examples 10-13, 0.5-1 mol of the respective ammonium salts per mL blood were added to the mixture.

(49) TABLE-US-00010 TABLE 10 Free plasma hemoglobin concentration (g/L) time intervals (hours) 0 1.5 3.5 50 Comparative 0.290 0.290 0.300 0.370 Example 5 Example 7 0.270 0.275 0.290 0.330 Example 8 0.200 0.210 0.210 0.210 Example 9 0.245 0.250 0.250 0.250 Example 10 0.150 0.150 0.154 0.160 Example 11 0.220 0.220 0.230 0.232 Example 12 0.280 0.280 0.280 0.280 Example 13 0.290 0.290 0.290 0.290

(50) Comparative Example 5 shows that when whole blood is mixed with only K.sub.3EDTA, over time a significant degree of hemolysis occurs, as seen in the increase of the free plasma hemoglobin concentration at 3.5 hours and especially at 50 hours.

(51) In Example 7, which does not contain any ammonium salt, hemolysis is also observed, but to a lesser extent at 50 hours compared to Comparative Example 5.

(52) Examples 8-9 demonstrate that when the combination of antiglycolytic agents comprises salts, hemolysis can unexpectedly and advantageously be significantly inhibited by providing at least one salt of said agents as an ammonium salt.

(53) Surprisingly, hemolysis can also be efficiently and effectively inhibited by adding to the composition as a further component an ammonium salt, as shown in Examples 10-13.

(54) Therefore, the provision of an ammonium salt, as one or more of the antiglycolytic agents according to the invention and/or as a further additive, provides significant further benefits. One and/or more of glucose, lactate and homocysteine, preferably all, in the blood sample is (are) stabilized, while furthermore the inhibition of hemolysis can be significantly enhanced. This particularly advantageous stabilization and preservation of the blood samples in turn can facilitate an improved determination of blood components and reliable diagnostics.