METHODS FOR DETERMINING BLOOD GAS ON METABOLIC PARAMETERS
20220205975 · 2022-06-30
Inventors
- Thomas Steen HANSEN (Brønshøj, DK)
- Ida HOLLESEN (Brønshøj, DK)
- Melanie Andrea BURKHARDT (Brønshøj, DK)
Cpc classification
International classification
Abstract
The present invention relates to methods for determining a blood gas parameter and/or a basic metabolic panel parameter in a blood sample comprising combining the blood sample with an anti-coagulant and an anti-platelet agent, and determining said blood gas parameter and/or parameter in the sample. In some aspects, the invention relates to determining said parameters in samples that have been subjected to pre-analytical stress.
Claims
1. An in vitro method for determining a blood gas parameter and/or a basic metabolic panel (BMP) parameter in a blood sample comprising: i) combining a blood sample with an anti-coagulant and an anti-platelet agent, and ii) determining said blood gas parameter and/or BMP parameter in the blood sample, wherein the blood sample, prior to the determination in ii), has been subjected to pre-analytical stress.
2. The in vitro method according to claim 1, wherein ii) is performed in a sensor assembly comprising two or more analyte sensors.
3. The in vitro method according to claim 2, wherein said two or more analyte sensors are not all positioned in the same plane, and wherein one of said analyte sensors analyzes said blood gas parameter or said BMP parameter, and wherein another analyte sensor not located in the same plane analyzes a different blood gas parameter or different BMP parameter.
4. An in vitro method for determining a blood gas parameter selected from the group consisting of pO.sub.2 and pCO.sub.2 in a blood sample comprising: i) combining the blood sample with an anti-coagulant and an anti-platelet agent, and ii) determining said blood gas parameter in the sample, wherein ii) is performed in sensor assembly comprising two or more analyte sensors, wherein said two or more analyte sensors are not all positioned in the same plane, and wherein one of said analyte sensors analyzes said blood gas parameter and wherein another analyte sensor not located in the same plane analyzes a different blood gas parameter or a different BMP parameter.
5. The in vitro method according to claim 4, wherein said blood sample, prior to the determination in ii), has been subjected to pre-analytical stress.
6. The in vitro method according to claim 1, wherein said blood sample has been subjected to stress caused by exposure to temperature between −5° C. and 20° C.
7. The in vitro method according to claim 1, wherein ii) is performed in a sensor assembly comprising: a) a first electronic wiring substrate having a first and a second surface and at least one analyte sensor formed on the first surface thereof, the at least one analyte sensor being connected with one or more electrical contact points, b) a second electronic wiring substrate having a first and a second surface and at least one analyte sensor formed on the first surface part thereof, the at least one analyte sensor being connected with one or more electrical contact points, and c) a spacer having a through-going recess with a first and a second opening, wherein the first substrate, the second substrate and the spacer are arranged in a layered structure, where the first surface of the first substrate closes the first opening of the spacer and the first surface of the second substrate closes the second opening of the spacer, thereby forming a measuring cell which is faced by at least one analyte sensor from each of the substrates.
8. The in vitro method according to claim 1, wherein the volume used for the determination in ii) is less than 1 ml.
9. The in vitro method according to claim 1, wherein the anti-coagulant is heparin.
10. The in vitro method according to claim 1, wherein the anti-platelet agent is selected from the group consisting of: glycoprotein IIb/IIIa inhibitors, ADP receptors/P2Y12 inhibitors, prostaglandin analogs, COX inhibitors, thromboxane inhibitors, phosphodiesterase inhibitors, cloricromen, ditazole, vorapraxar and combinations thereof.
11. The in vitro method according to claim 1, wherein the anti-platelet agent is iloprost.
12. The in vitro method according to claim 1, wherein i) comprises mixing the blood sample.
13. The in vitro method according to claim 1, wherein the method is for determining a blood gas parameter and wherein the blood gas parameter is selected from the group consisting of pH, pCO.sub.2, pO.sub.2, oxygen saturation (SO.sub.2), the concentration of total hemoglobin (ctHb), the fraction of oxyhemoglobin (FO.sub.2Hb), the fraction of carboxyhemoglobin (FCOHb), the fraction of methemoglobin (FMetHb), the fraction of deoxyhemoglobin (FHHb), and the fraction of fetalhemoglobin (FHbF).
14. The in vitro method according to claim 1, wherein the method is for determining a BMP parameter and wherein the BMP parameter is selected from the group consisting of Na.sup.+, K.sup.+, Mg.sup.2+, Cl.sup.−, HCO.sub.3.sup.−, urea, creatinine, glucose, Ca.sup.2+, lactate, and total bilirubin.
15. The in vitro method according to claim 1, wherein the method further comprises determining platelet count and/or white blood cell count in the blood sample obtained in i).
16. An in vitro method for determining a blood gas parameter and/or a BMP parameter in a blood sample comprising: i) combining the blood sample with an anti-coagulant and an anti-platelet agent, ii) exposing the blood sample to a temperature below 20° C., and iii) determining said blood gas parameter and/or BMP parameter in the blood sample.
17. (canceled)
18. The in vitro method according to claim 1, wherein the pre-analytical stress is stress caused by exposure to temperature below 20° C., by contact with air, and/or by shear forces.
19. The in vitro method according to claim 5, wherein the pre-analytical stress is stress caused by exposure to temperature below 20° C., by contact with air, and/or by shear forces.
20. The in vitro method according to claim 6, wherein said blood sample has been subjected to stress caused by exposure to temperature between −5° C. and 15° C.
21. The in vitro method according to claim 6, wherein said blood sample has been subjected to stress caused by exposure to temperature between 0° C. and 5° C.
Description
BRIEF DESCRIPTION OF FIGURES
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EXAMPLES
Example 1 Analysis of Platelet Aggregation Using Heparin and Different Anti-Platelet Agents
[0153] Each experiment was conducted on a separate day with blood from one voluntary donor testing one anti-platelet drug candidate. For each experiment PICO70 syringe samplers (Radiometer Medical ApS) were prepared just before sample drawing. For “LiHep” (Liquid Heparin) conditions, PICO70 samplers were emptied of the gold ball and heparin brick and 15 μl of aqueous liquid balanced heparin comprising heparin lithium (Celsus Laboratories) and heparin sodium (Celsus Laboratories) (final heparin conc. 60 IU/mL blood) and 15 μl of the solvent of the tested drug were added. For “PicoSAM” conditions, unmodified PICO70 samplers (with gold ball and heparin brick) were used and 15 μl of the solvent of the respective tested drug was added. For “LiHep xxx drug” conditions, PICO70 samplers were emptied as described for LiHep and 15 μl of liquid balanced heparin (final heparin conc. 60 IU/mL blood) and 15 μl of the dissolved tested drug were added. For “PicoSAM xxx drug” conditions, unmodified PICO70 samplers (with gold ball and heparin brick) were used and 15 μl of the dissolved tested drug was added. The tested anti-platelet drug candidates and the corresponding solvents were eptifibatide acetate (Sigma, SML1042; dissolved in saline, final conc. 5 and 20 μM), MgSO.sub.4 (Sigma, M7506; dissolved in saline, final conc. 3 and 12 mM), tirofiban hydrochloride monohydrate (Sigma, SML0246; dissolved in 1:200 DMSO in saline, final conc. 0.5 and 1 μM), iloprost (Sigma, SML1651; dissolved in 1:1000 or 1:10000 ethanol in saline, final conc. 10 and 100 nM (later also 1 μM)), ticlopidine hydrochloride (dissolved in saline, final conc. 60 and 600 μM), L-Arginine (Sigma, A5006; dissolved in saline, final conc. 600 μM and 6 mM) and dipyridamole (Sigma, D9766; dissolved in 1:10 or 1:100 DMSO in saline, final conc. 10 and 100 μM). An EDTA tube (BD Vacutainer with spray-coated K.sub.2EDTA, 10 ml) and duplicate samples of all conditions of PICO70 syringe samplers were filled by drawing venous blood via a butterfly needle using a sealed VTC (vented tip cap) to fill the otherwise self-aspirating PICO70 samplers with 1.5 mL venous whole blood. Samplers were inverted 8 times immediately after drawing to insure proper anti-coagulation of the samples. The blood samples were mixed gently by hand (samplers without gold ball) or on a SAM mixer (Radiometer Medical ApS) for 15 minutes after drawing the sample. Mixed samples were fixed immediately with 10% formalin solution (1:1 dilution of blood in formalin) for at least 10 minutes and further diluted 1:10 in platelet dilution solution to hemolyze red blood cells (RBCs) to assessed manual platelet count using a hemocytometer. Manual platelet counts of single platelets (not aggregated), number of platelet aggregates and if possible, size (number of platelets in an aggregate) of platelet aggregates were quantified in duplicate on each sample by counting platelets in a hemocytometer using a Leica 750 microscope with 10× and 20× phase-contrast air objectives. Single platelet counts were adjusted for dilution with liquid heparin and or dissolved drug solution to calculate single platelet concentrations.
[0154] Eptifibatide, tirofiban and iloprost reduced the formation of platelet aggregates compared to “LiHep” and “PicoSAM” references (see
[0155] MgSO.sub.4 also reduced the formation of platelet aggregates (see
[0156] Ticlopidine LiHep showed a similar count of single platelets compared to EDTA reference and a slightly reduced number of aggregates compared to the LiHep and PicoSAM controls (see
[0157] Dipyridamole, a phosphodiesterase inhibitor and thromboxane inhibitor, showed a slightly higher platelet number compared to the LiHep and PicoSAM controls (see
Example 2 Platelet Aggregation Studies with Heparinized Blood and EDTA-Treated Blood
[0158] Venous blood samples drawn, mixed and fixed with 10% formalin solution, as described before in Example 1 were used to prepare wet mounts on a glass slide and covered with a glass coverslip. Wet mount samples of fixed blood cells were then imaged with a Leica 750 microscope using a 40× phase-contrast air objective. Aggregated platelets in heparinized blood versus non-aggregated single platelets in EDTA anti-coagulated blood and heparinized blood with e.g. eptifibatide are seen between abundant RBCs.
[0159] The results are shown in
Example 3 Wet Mount Images of Stained Blood Samples
[0160] Venous blood samples were drawn and mixed as described before in Example 1 and used to prepare stained wet mounts images. Blood samples were mixed with a staining and hemolyzing agent (methylene blue and deoxycholic acid, respectively) and incubated at 47° C. in a water bath for 30 seconds. Stained and hemolyzed blood samples were then prepared in wet mount samples on a glass slide and covered with a glass coverslip. Images of stained samples were then acquired with a Leica 750 microscope using a 40× air objective in bright field mode. An image processing software (FIJI, ImageJ) was used to select regions of interest (ROIs) of representative stained WBCs from the images.
[0161] PicoSAM samples (heparinized blood without an anti-platelet drug) showed many aggregated platelets (small roundish cells).
[0162] PicoSAM samples with 1 μM tirofiban or 20 μM eptifibatide showed single platelets but also platelet satellinism, i.e. platelets binding to WBCs. This is for example shown in
[0163] PicoSAM samples with 100 nM iloprost (or samples with balanced LiHep and 100 nM Iloprost) showed single platelets, not platelet satellinism or WBC aggregates. The same results were obtained using EDTA anti-coagulated blood.
Example 4 Single Platelet Concentrations of Iloprost Heparin Samples Compared to EDTA Reference Samples
[0164] Manual Platelet Count
[0165] Samples were prepared as described in Example 1.
[0166] ABX Platelet Count
[0167] Blood samples drawn and mixed after 15 minutes as described above were assessed with an automated hematology analyzer (Horiba, ABX Pentra 60C+) (ABX) for complete blood count (CBC) with 5-diff including WBC concentration, platelet concentration, mean platelet volume (MPV), concentrations and fractions of neutrophils, lymphocytes, monocytes, eosinophils and basophils, RBC concentration, hematocrit, hemoglobin concentration, RBC descriptive parameters (MCV, MCH, MCHC, RDW). Samples were prepared from several donors. Measured platelet concentration was used to compare to manually assessed platelet concentrations of iloprost heparin samples (in PICO70 samplers) and EDTA anti-coagulated samples. For control, EDTA samples with no platelet aggregates are used as reference measurement for the measured automated ABX platelet concentration.
[0168] For one donor, the average performance of platelet count of PicoSAM 100 nM iloprost samples compared to EDTA samples as reference was 97% for the manual count.
[0169] For eight donors, the average performance of platelet count of PicoSAM 1 μM iloprost samples compared to EDTA samples as reference was 93% for the manual count.
[0170] The respective PicoSAM sample, i.e. without iloprost but with heparin, in contrast showed a performance of less than 40% compared to the EDTA reference.
[0171] For seven donors, the average performance of platelet count of PicoSAM 1 μM iloprost samples compared to EDTA samples as reference was 97% for the ABX platelet count.
[0172] Table 1 shows the results of platelet counts for donors samples where both types of platelet counts (manual and ABX platelet count) were performed.
TABLE-US-00001 TABLE 1 Comparison platelet count Mean ABX Mean Manual Donor PLT count PLT count ID (PLT/nl) (PLT/nl) 4 PicoSAM Iloprost 1 μM 204 198 EDTA 200 200 5 PicoSAM Iloprost 1 μM 253 242 EDTA 259 242 6 PicoSAM Iloprost 1 μM 173 154 EDTA 185 189 7 PicoSAM Iloprost 1 μM 233 247 EDTA 240 249 8 PicoSAM Iloprost 1 μM 191 203 EDTA 202 207 9 PicoSAM Iloprost 1 μM 216 202 EDTA 236 255
Example 5 Anti-Platelet Drug Interference Test on ABL90 Parameters
[0173] Each experiment was conducted on a separate day with blood from one voluntary donor. For each experiment three PICO70 syringe samplers (Radiometer Medical ApS) (not modified, containing a gold ball and a heparin brick) were prepared. One PICO70 was used unmodified (Ctrl), one PICO70 was filled with 15 μl of dissolved anti-platelet drug to reach the indicated final concentration in the blood sample. A third PICO70 sampler was filled with 15 μl of solvent (specific solvent, which is used to dissolve the tested anti-platelet drug: reference sample). Venous blood samples were drawn into PICO70 syringe samplers via a butterfly needle and a sealed vented tip cap (VTC) to fill the self-aspirating PICO70 samplers with 1.5 ml whole blood. Samplers were inverted eight times immediately after drawing to insure proper heparinization of the sample. The blood samples were mixed on a SAM mixer (Radiometer Medical ApS), moving the gold-coated steel ball through the blood sample in order to ensure homogenous and reproducible mixing for 15 minutes after drawing of the blood, and analyzed on an ABL90 blood gas analyzer (Radiometer Medical ApS). Samples were measured in alternating order (Ctrl, Reference sample, Drug sample) five times each (5 replicate measurements) with gentle manual inversion of the samples in order to keep blood samples homogenously mixed through the measurements. All calculated values are described in Table 3. Afterwards blood samples were centrifuged to separate the plasma fraction. Plasma of all samples was measured in triplicate in alternating order on the ABL90 instrument to assess the free Hemoglobin concentration in the plasma, which indicates a possible problematic hemolyzation of red blood cells (RBCs) in the whole blood sample. Hemolyzed samples show interference on e.g. the measured K.sup.+ concentration.
[0174] Measured parameters are described in Table 2.
TABLE-US-00002 TABLE 2 Parameters measured with ABL90 blood gas analyzer Parameter Description pH pH tHb (g/dL) total Hemoglobin concentration COHb (%) Carboxyhemoglobin (carboxylated Hemoglobin) MetHb (%) Methemoglobin (methylated Hemoglobin) K.sup.+ (mmol/L) Potassium concentration Na.sup.+ (mmol/L) Sodium concentration Ca.sup.2+ (mmol/L) Calcium concentration, ionized Cl.sup.− (mmol/L) Chloride concentration Glu (mmol/L) Glucose concentration Lac (mmol/L) Lactate concentration tBil (μmol/L) Bilirubin concentration
[0175] The nomenclature of how the calculations have been made is described in Table 3 and the nomenclature of the measured samples is described in Table 4.
TABLE-US-00003 TABLE 3 Calculated values Mean Mean value of 5 replicate measurements performed on the same whole blood sample or 3 replicate measurements of Hemoglobin measured on plasma sample (data not shown) SD Standard deviation calculated from 5 replicate measurements (data not shown) Difference Difference in measured parameter between indicated samples (test sample with drug − reference sample with solvent only) (data not shown) Fraction of Difference divided by maximum allowed interference for maximum each parameter (a value >1 indicates an interference, interference whereas <1 are acceptable)
TABLE-US-00004 TABLE 4 Sample names Ctrl Control sample drawn into unmodified PICO70 syringe sampler Saline Reference sample containing solvent saline (physiological NaCl solution) DMSO/Saline Reference sample containing solvent DMSO in saline (1:200) (1:200 dissolved) EtOH/Saline Reference sample containing solvent ethanol (EtOH) (1:1000) in saline (1:1000 dissolved) EtOH/Saline Reference sample containing solvent ethanol (EtOH) (1:100) in saline (1:100 dissolved) Eptifibatide 20 Eptifibatide dissolved in saline to reach final conc. of uM in saline 20 μM eptifibatide in the blood sample Tirofiban 1 uM Tirofiban dissolved inDMSO/saline solvent (1:200) to in DMSO/Saline reach final conc. of 1 μM tirofiban in the blood sample Iloprost 100 nM Iloprost dissolved in ethanol/saline solvent (1:1000) to in EtOH/Saline reach final conc. of l00 nM iloprost in the blood sample Iloprost 1 uM Iloprost dissolved in ethanol/saline solvent (1:100) to in EtOH/Saline reach final conc. of 1 μM iloprost in the blood sample MgSO.sub.4 12 mM MgSO.sub.4 dissolved in saline to reach final conc. of 12 in saline mM MgSO.sub.4 in the blood sample
[0176] Reference range for adults and maximum interference allowed in the examples are shown in Table 5.
[0177] The results of the measurements (as fraction of maximum interference) are summarized in Table 6.
TABLE-US-00005 TABLE 5 Reference values for determining maximum allowed interference. tHb COHb MetHb K.sup.+ Na.sup.+ pH (g/dL) (%) (%) (mmol/L) (mmol/L) Reference range 7.32-7.43 11.4-17.5 2-3% (non-smokers) 1-2% 3.4-4.5 136-145 adult 7-9% (smokers) max. Interference <0.010 <0.5 <1% <1% <0.1 <1 allowed Ca.sup.2+ Cl— Glu Lac tBil (mmol/L) (mmol/L) (mmol/L) (mmol/L) (μmol/L) Reference range 1.15-1.33 98-107 3.6-5.3 0.56-1.39 0-34 adult max. Interference 0.02 1 0.1 0.1 30 allowed
TABLE-US-00006 TABLE 6 Fraction of maximum interference of parameter for antiplatelet drugs Eptifibatide Tirofiban Iloprost MgSO.sub.4 pH 0.18 0.18 0.32 5.56 tHb (g/dL) 0.04 0.12 0.12 0.08 COHb (%) 0.02 0.06 0.08 0.04 MetHb (%) 0.08 0.04 0.04 0.04 K.sup.+ (mmol/L) 0 0 0.18 2.0 Na.sup.+ (mmol/L) 0.4 0 0.14 5.6 Ca.sup.2+ (mmol/L) 0.2 0.1 0.1 2.7 Cl.sup.− (mmol/L) 0 0 0.06 8.6 Glu (mmol/L) 0 0.8 0.86 0.2 Lac (mmol/L) 0.6 0 0.62 4.6 tBil (μmol/L) 0.013 0.000 0.013 0.140
[0178] Eptifibatide, tirofiban and iloprost showed no interference on assessed ABL parameters, whereas MgSO.sub.4 shows interference (Difference Fraction max. Interf. >2) on multiple parameters (pH, N.sup.+, K.sup.+, Ca.sup.2+, Cl.sup.−, and Lac).
[0179] Eptifibatide, tirofiban and iloprost should therefore be safe to add to heparinized blood used for measurement of blood gas and basic metabolic panel parameters at the tested concentrations.
Example 6 Robustness of Samples Against Pre-Analytical Stress
[0180] A test of sample robustness against stress conditions was performed, comparing blood samples with heparin (lithium heparin 18 IU/ml) only and blood samples with heparin (lithium heparin 18 IU/ml)+ iloprost (1 microM).
[0181] Shortly after blooddraw, the blood from the vacutainer tubes was aspirated into marked SafePicoAsp syringes (1.5 ml), placed in a plastic bag and fully emerged into icecubes for 30 minutes. After 30 minutes, the syringes were removed and shaken for 30 seconds by fast lengthwise movements back and forth, approx. 4/sec. Every syringe was mixed gently (by slow inversions by twisting the wrist, approx. 1/sec) prior to aspiration on analyzers. The resulting samples were aspirated on nine ABL90 analyzers approximately 400 times each and instances of clots were recorded. All samples were aspirated from picosafe samplers. The results are shown in Table 7.
TABLE-US-00007 TABLE 7 Clots detected in samples exposed to low temperature and shaking. Total number of Clots (Error aspirations # 1271) Frequency Heparin 452 5 1.11% Heparin + Iloprost 416 1 0.24%
[0182] The clot frequency was 4-5 times higher in the samples without Iloprost.
[0183] A further experiment was conducted with larger blood volumes using 9 ml Greiner Bio-one Vaceutte vacuum tubes. Shortly after blooddraw, the vacutainer tubes were placed on ice for 30 minutes. Subsequently, the vacutainer tubes were tempered to ambient temperature and mixed gently before centrifugation for 3 minutes at 1500 G to imitate sedimentation. After centrifugation, the blood was pooled and distributed into 2×3 marked 20 ml syringes and tested on 10 ABL90 analyzers via robot. The samples were aspirated using large 20 ml samples (robots). The results are shown in Table 8.
TABLE-US-00008 TABLE 8 Clots detected in samples exposed to low temperature and shaking. Total number of Clots (Error aspirations # 1271) Frequency Heparin 707 7 0.99% Heparin + Iloprost 659 0 0%
[0184] Again, the clot frequency was higher in the sample without Iloprost.