Methods for determining blood gas or metabolic parameters

Abstract

To provide a method for simultaneously determining many diagnostic parameters using the same blood sample and the same automated blood analyzer.SOLUTION: The present invention relates to methods for determining a blood gas parameter and / or a basic metabolic panel parameter in a blood sample, the methods comprising: combining the blood sample with an anti-coagulant and an anti-platelet agent; and determining the blood gas parameter and / or parameter in the sample. In some aspects, the invention relates to determining the parameters in samples that have been subjected to pre-analytical stress.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to the field of diagnostic blood sample analysis. [Background technology]

[0002] Rapid access to blood tests is a mainstay in the diagnosis and treatment of acute illnesses. Oxygenation status and acid-base balance are determined by arterial blood gas (BG) analysis and constitute a central part of modern evidence-based treatment algorithms in critical care. Furthermore, devices intended for critical care testing allow the assessment of, for example, electrolytes, renal function (creatinine), inflammation (C-reactive protein) and cardiac biomarkers.

[0003] A Basic Metabolic Panel (BMP) is used to check the status of a person's kidneys as well as their electrolyte and acid / base balance, and their blood glucose levels, all of which are related to a person's metabolism. It can also be used to monitor hospitalized patients and people with certain known illnesses, such as high blood pressure and hypokalemia.

[0004] Furthermore, white blood cell counts are important biomarkers for several diseases, and differential WBC counts can distinguish different types of blood cells, such as neutrophils, lymphocytes, monocytes, eosinophils, and basophils. Each can be reported, for example, as a percentage. Changes in the percentages can indicate a pathological condition.

[0005] Additionally, platelets (also called thrombocytes) can be counted as another parameter. Platelets are small fragments of cells that are essential for normal blood clotting. Platelet counts can be used to screen or diagnose various diseases and illnesses that may cause problems with blood clot formation. It can be used as part of the workup for bleeding disorders, bone marrow disorders, or hypercoagulant disorders, to name a few. The test can be used as a monitoring tool for people who have underlying conditions or are being treated with drugs known to affect platelets. It can also be used to monitor people being treated for platelet disorders to determine if the therapy is effective.

[0006] However, blood samples usually have to be prepared differently for the diagnostic measurement of each of the above parameters. For example, for the analysis of BG and BMP parameters, the standard anticoagulant is heparin. Although heparin prevents blood clotting, it does not prevent the activation and aggregation of platelets (thrombocytes), which leads to the formation of platelet aggregates. Therefore, heparin is not currently used for complete blood count (CBC) analysis, including WBC, platelet count, 3-diff or 5-diff, red blood cell (RBC) concentration, hematocrit, hemoglobin concentration, and RBC descriptive parameters. The measured platelet count in heparinized blood will be underestimated, especially when using state-of-the-art automated hematology analyzers that cannot distinguish between single platelets and clumps of aggregated platelets. Instead, platelet aggregates may be erroneously classified as white blood cells by the hematology analyzer, thus resulting in a falsely high WBC count, which may result in a faulty diagnosis or flag and error messages making the results unusable.

[0007] Ethylenediaminetetraacetic acid (EDTA) is another standard anticoagulant commonly used in hematology in either the disodium, dipotassium or tripotassium salts. The use of A is generally accepted as safe and reliable for obtaining complete blood counts. In addition, EDTA salts are compatible with, i.e. do not interfere with, standard staining protocols for blood smears. In the event of problems with EDTA-dependent pseudothrombocytopenia, citrate is used as an alternative anticoagulant. However, EDTA or citrate cannot be used for the analysis of BG and BMP parameters, since these anticoagulants strongly interfere with the measurement of electrolytes. For example, EDTA and citrate are unable to detect Ca 2+ and Ca 2+ These anticoagulants can interfere with calcium measurements and can even destroy calcium sensors in automated analyzers.

[0008] Currently, hematology analyses, and CBCs in particular, are performed on blood samples that are anticoagulated with EDTA or citrate, but not on heparinized blood samples. As a result, previous comprehensive analyses of CBC, BG and BMP parameters must be performed on separate instruments using separate blood samples that are anticoagulated in different ways.

[0009] Schnuff-Wernet et al. (Br. J. Haematol. 162, 684, 2013) reported that MgSO4 was It is described that it can be used as an anticoagulant for blood samples from patients with pseudothrombocytopenia as an alternative to rifampicin A or citrate.

[0010] US Patent Application Publication No. 2010 / 0280412 discloses blood clotting factor Xa inhibitors and methods for anticoagulation of human blood, where blood calcium levels remain the same, thrombin is not formed and thrombocyte function is not affected.

[0011] US Pat. No. 6,880,384 B2 describes an automated blood analyzer for measuring blood gas parameters, metabolic parameters and electrolytes, as well as a blood sampler that accommodates a blood sample and a mixing element.

[0012] WO2019096598 describes an in vitro method for preparing a blood sample, in which blood is combined with: a) at least one anticoagulant for the determination of blood gas and basic metabolic panel parameters; and b) At least one antiplatelet agent.

[0013] The blood sample prepared according to the above-mentioned method is suitable for BG and BMP parameter analysis and platelet counting.Therefore, all parameters can be determined using the same blood sample and the same automated hematology analyzer.WO2019096598 also describes an in vitro method for determining blood gas and BMP parameters and platelet count in blood samples prepared according to the above-mentioned sample preparation method. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] US Patent Application Publication No. 2010 / 0280412 [Patent Document 2] U.S. Patent No. 6,880,384 B2 [Patent Document 3] International Publication No. 2019096598 [Non-patent literature]

[0015] [Non-Patent Document 1] Schnuff-Wernet et al. (Br. J. Haematol. 162, 684, 2013) Summary of the Invention

[0016] In a first aspect, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: i) combining the blood sample with an anticoagulant and an antiplatelet agent; and ii) determining said blood gas parameters and / or BMP parameters in the sample; wherein said blood sample has been subjected to a pre-analytical stress prior to the determination in step ii), such as a stress caused by exposure to a temperature below 20° C., by contact with air and / or by shear forces.

[0017] Similarly, in a second aspect, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: i) combining a blood sample with an anticoagulant and an antiplatelet agent; ii) exposing said blood sample to a temperature of less than 20° C.; and iii) determining said blood gas parameters and / or BMP parameters in the sample; The present invention relates to an in vitro method for determining blood gas parameters and / or BMP parameters in a blood sample, comprising:

[0018] In a third aspect, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: i) combining the blood sample with an anticoagulant and an antiplatelet agent; and ii) determining said blood gas parameters in the sample; wherein step ii) is performed in a sensor assembly comprising two or more analyte sensors, wherein said two or more analyte sensors are not all located 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 BMP parameter. [Brief description of the drawings]

[0019] [Figure 1]Single platelet counts and platelet aggregate counts quantified in blood collected with a PICO70 sampler containing liquid heparin only (LiHep, no gold-coated (Au) ball, gentle hand mixing), PICO70 mixed on a SAM mixer (PicoSAM), liquid heparin in combination with EDTA anticoagulant and two concentration levels of the antiplatelet drugs eptifibatide, tirofiban or iloprost or full PicoSAM. The three antiplatelet drugs show no or low single platelet counts and aggregates comparable to standard EDTA anticoagulated blood. [Diagram 2] Single platelet counts and platelet aggregate counts quantified in blood collected using a PICO70 sampler containing liquid heparin only (LiHep, no Au balls, gentle manual mixing), PICO70 mixed on a SAM mixer (PicoSAM), liquid heparin in combination with EDTA anticoagulant and two concentration levels of the antiplatelet drug MgSO4, or complete PicoSAM. [Diagram 3] Single platelet counts and platelet aggregate counts quantified in blood collected with a PICO70 sampler containing liquid heparin only (LiHep, no Au balls, gentle manual mixing), PICO70 mixed on a SAM mixer (PicoSAM), liquid heparin in combination with EDTA anticoagulant and the antiplatelet drug ticlopidine at two concentration levels, or complete PicoSAM. [Figure 4] Single platelet counts and platelet aggregate counts quantified in blood collected with a PICO70 sampler containing liquid heparin only (LiHep, no Au balls, gentle manual mixing), PICO70 mixed on a SAM mixer (PicoSAM), liquid heparin in combination with EDTA anticoagulant and the antiplatelet drug L-arginine at two concentration levels, or complete PicoSAM. [Diagram 5]Single platelet counts and platelet aggregate counts quantified in blood collected with a PICO70 sampler containing liquid heparin only (LiHep, no Au balls, gentle manual mixing), PICO70 mixed on a SAM mixer (PicoSAM), liquid heparin in combination with EDTA anticoagulant and the antiplatelet drug dipyridamole at two concentration levels, or complete PicoSAM. [Figure 6] Microscopic images of blood samples anticoagulated with (A) heparin and (B) EDTA. EDTA prevents platelet activation and maintains single platelets in the blood sample, while heparin allows or even enhances platelet aggregation induced by other agonists or foreign substances. This effect of heparin cannot be observed in the microscopic images when (C) 20 μM eptifibatide is added to the heparin. [Figure 7] Blood sample manually mixed with staining / hemolysis reagent. Stained WBCs (white blood cells) and platelets prepared by wet mount on a coverslip were imaged in bright field mode with a 40x objective on a Leica microscope. (A) Example of leukocytes showing interaction with platelets. (B) Example of leukocyte aggregates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] In a first aspect, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: i) combining the blood sample with an anticoagulant and an antiplatelet agent; and ii) determining said blood gas parameters and / or BMP parameters in the sample; wherein said blood sample has been subjected to a pre-analytical stress prior to the determination in step ii), e.g. a stress caused by exposure to a temperature below 20° C., by contact with air and / or by shear forces. Pre-analytical stress as used herein denotes a stress applied to the sample prior to the analysis, i.e. prior to the determination of the parameters in step ii).

[0021] Similarly, in a second aspect, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: i) combining a blood sample with an anticoagulant and an antiplatelet agent; ii) exposing said blood sample to a temperature of less than 20° C.; and iii) determining said blood gas parameters and / or BMP parameters in the sample; The present invention relates to an in vitro method for determining blood gas parameters and / or BMP parameters in a blood sample, comprising:

[0022] Surprisingly, it has been found that the presence of both an anticoagulant and an antiplatelet agent in a blood sample improves the accuracy and robustness of the determination of BG and BMP parameters. Thus, even samples that have been subjected to stress, for example by low temperature, contact with air or high shear forces, can be used reliably for the determination of these parameters. The inventors have observed that under such stress conditions, the presence of both an anticoagulant and an antiplatelet agent in the sample results in less clump-induced measurement errors than if only an anticoagulant was added. Without being bound by any theory, it is believed that the presence of an antiplatelet agent prevents platelet activation and thus the formation of platelet aggregates. Platelet aggregates have not previously been known as a source of error in the determination of BG and BMP parameters. In conclusion, the method of the present invention allows for a more accurate determination of BG and BMP parameters in samples that have been subjected to stress. In addition, the blood sample prepared according to step i) of the method is suitable for BG and BMP parameter analysis as well as platelet counting, so that all parameters can be determined using the same blood sample and the same automated hematology analyzer. It is therefore a further advantage of the method of the present invention that it allows a "3-in-1" analysis with one blood sample.

[0023] In a third aspect, the present invention provides a method for producing a pharmaceutical composition comprising the steps of: i) combining the blood sample with an anticoagulant and an antiplatelet agent; and ii) determining said blood gas parameters in the sample; In an in vitro method for determining a blood gas parameter selected from the group consisting of pO2 and pCO2 in a blood sample comprising: wherein step ii) is performed in a sensor assembly comprising two or more analyte sensors, wherein said two or more analyte sensors are not all located 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 BMP parameter. A multi-sensor assembly in which the sensors are not all on the same plane, e.g. in a sandwich configuration or in a tube, allows for the analysis of smaller samples. Again, one advantage of the method of the present invention is that multiple parameters can be measured from one sample.

[0024] Pre-analysis stress As mentioned above, in the method of the present invention, the blood sample is combined with an anticoagulant and an antiplatelet agent before the determination of the BG and / or BMP parameters in said sample. Combining the blood sample with an anticoagulant and an antiplatelet agent makes the sample more robust against pre-analysis stresses, such as those caused by exposure to temperatures below 20°C, by contact with air, and / or by shear forces.

[0025] Stress caused by exposure to temperatures below 20°C may occur, for example, when the sample is stored or incubated on ice. Temperature reduction may also occur during handling or transportation of the sample prior to analysis. It is understood that exposure would have actually caused stress for a minimal period of time sufficient to cause a significant reduction in the temperature of the blood sample itself. In one embodiment, the blood sample is subjected to stress caused by exposure to temperatures between -5°C and 20°C, such as temperatures between -5°C and 15°C, for example temperatures between 0°C and 10°C, for example stress caused by temperatures between 0°C and 5°C. In further embodiments, the blood sample is stressed by exposure to a temperature of -5°C, -4°C, -3°C, -2°C, -1°C, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C ​​or 19°C to -4°C, -3°C, -2°C, -1°C, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C or 20°C. In one embodiment, the exposure time was at least 5 seconds, such as at least 10 seconds, at least 30 seconds, at least 1 minute, at least 2 minutes, at least 5 minutes, at least 10 minutes or at least 30 minutes.

[0026] The ability to incubate or store blood samples at low temperatures, e.g., below 10°C, e.g., on ice, may be desirable for certain purposes, particularly for determination of BMP parameters where incubation at low temperatures prevents further metabolism in the sample during handling prior to analysis.

[0027] Stress caused by contact with air can occur, for example, when a sample is handled suboptimally before analysis, for example, when a blood sample is drawn in a syringe, air enters the syringe and is not removed before the subsequent step in the analytical procedure. In particular, mixing of a blood sample in the presence of air can increase such stress. Thus, in one embodiment, the stress caused by contact with air is the stress caused by contact with air during mixing of the sample. Thus, in one embodiment, the method of the present invention includes a mixing step before determination, where the sample is in contact with air during the mixing step.

[0028] Stress caused by shear forces can occur in several ways, for example, when a sample is exposed to an average shear rate above 10000 s-1 for a period of time, for example, greater than 0.1 s, greater than 0.2 s, greater than 0.5 s, greater than 1 s, greater than 2 s, or greater than 5 s. Shear rates above 10000 s-1 have been described as "pathological" because they can lead to the formation of large rolling aggregates (Nesbitt et al. (2009) Nature Medicine 15: 665; Ruggeri et al. (2006) Blood 108:1903). The average shear rate can be determined, for example, using simulation techniques. For example, For example, COMSOL Multiphysics (v. 5.2. www.comsol.com. COMSOL AB, Stockholm, Sweden) is known for its ability to solve physics-based problems. The commercially available software is used to analyze the shear rate. In another embodiment, the sample is exposed to an average shear rate of more than 12000 s-1, more than 15000 s-1, or more than 20000 s-1 for a period of time, such as more than 0.1 s, more than 0.2 s, more than 0.5 s, more than 1 s, more than 2 s, or more than 5 s. Stress caused by shear forces can also occur when the sample is handled suboptimally before analysis, for example when the sample is subjected to vigorous shaking or vortexing (as opposed to mixing, which is a gentle procedure that does not cause stress). Furthermore, stress caused by shear forces can also occur during automated handling or transport of the sample during the procedure. Automated sample management systems can, for example, bump the sample onto a surface or into a sample receiving device. In one embodiment of the method of the present invention, the blood sample is handled by an automated system (i.e. without manual handling by humans) in all steps following the blood withdrawal or in all steps following step i) of the method.

[0029] parameter As mentioned above, the present invention relates to an in vitro method for determining BG and / or BMP parameters.

[0030] In a preferred embodiment, the method is for determining a BG parameter. In a further embodiment, the blood gas parameter is selected from the group consisting of pH, pCO2, pO2, oxygen saturation (sO2), total hemoglobin concentration (ctHb), percentage of oxyhemoglobin (FO2Hb), percentage of carboxyhemoglobin (FCOHb), percentage of methemoglobin (FMetHb), percentage of deoxyhemoglobin (FHHb) and percentage of fetal hemoglobin (FHbF). In a preferred embodiment, the blood gas parameter is pO2 or pCO2.

[0031] In another embodiment, the method is for determining pH, and the blood sample has been subjected to stress caused by exposure to a temperature below 20°C, or below 19°C, or below 18°C, or below 17°C, or below 16°C, or below 15°C, or below 14°C, or below 13°C, or below 12°C, or below 11°C, or below 10°C, or below 9°C, or below 8°C, or below 7°C, or below 6°C, or below 5°C, or below 4°C, or below 3°C, or below 2°C, or below 1°C. In another embodiment, the method is for determining pCO2 and the blood sample is stressed by exposure to a temperature below 20° C. or below 19° C., or below 18° C., or below 17° C., or below 16° C., or below 15° C., or below 14° C., or below 13° C., or below 12° C., or below 11° C., or below 10° C., or below 9° C., or below 8° C., or below 7° C., or below 6° C., or below 5° C., or below 4° C., or below 3° C., or below 2° C., or below 1° C. In another embodiment, the method is for determining pO2 and the blood sample is stressed by exposure to a temperature below 20° C. or below 19° C., or below 18° C., or below 17° C., or below 16° C., or below 15° C., or below 14° C., or below 13° C., or below 12° C., or below 11° C., or below 10° C., or below 9° C. or less than 8° C., or less than 7° C., or less than 6° C., or less than 5° C., or less than 4° C., or less than 3° C., or less than 2° C., or less than 1° C. In another embodiment, the method is for determining sO2 and the blood sample is stressed by exposure to a temperature of less than 20° C., or less than 19° C., or less than 18° C., or less than 17° C., or less than 16° C., or less than 15° C., or less than 14° C., or less than 13° C., or less than 12° C., or less than 11° C., or less than 10° C., or less than 9° C., or less than 8° C., or less than 7° C., or less than 6° C., or less than 5° C., or less than 4° C., or less than 3° C., or less than 2° C., or less than 1° C. In another embodiment, the method is for determining ctHb, wherein the blood sample has been subjected to stress caused by exposure to a temperature below 20°C, or below 19°C, or below 18°C, or below 17°C, or below 16°C, or below 15°C, or below 14°C, or below 13°C, or below 12°C, or below 11°C, or below 10°C, or below 9°C, or below 8°C, or below 7°C, or below 6°C, or below 5°C, or below 4°C, or below 3°C, or below 2°C, or below 1°C. In another embodiment, the method is for determining FO2Hb, wherein the blood sample has been subjected to stress caused by exposure to a temperature below 20°C, or below 19°C, or below 18°C, or below 17°C, or below 16°C, or below 15°C, or below 14°C, or below 13°C, or below 12°C, or below 11°C, or below 10°C, or below 9°C, or below 8°C, or below 7°C, or below 6°C, or below 5°C, or below 4°C, or below 3°C, or below 2°C, or below 1°C.In another embodiment, the method is for determining FCOHb, wherein the blood sample has been subjected to stress caused by exposure to a temperature below 20°C, or below 19°C, or below 18°C, or below 17°C, or below 16°C, or below 15°C, or below 14°C, or below 13°C, or below 12°C, or below 11°C, or below 10°C, or below 9°C, or below 8°C, or below 7°C, or below 6°C, or below 5°C, or below 4°C, or below 3°C, or below 2°C, or below 1°C. In another embodiment, the method is for determining FMetHb, wherein the blood sample has been subjected to stress caused by exposure to a temperature below 20°C, or below 19°C, or below 18°C, or below 17°C, or below 16°C, or below 15°C, or below 14°C, or below 13°C, or below 12°C, or below 11°C, or below 10°C, or below 9°C, or below 8°C, or below 7°C, or below 6°C, or below 5°C, or below 4°C, or below 3°C, or below 2°C, or below 1°C. In another embodiment, the method is for determining FHHb, wherein the blood sample has been subjected to stress caused by exposure to a temperature below 20°C, or below 19°C, or below 18°C, or below 17°C, or below 16°C, or below 15°C, or below 14°C, or below 13°C, or below 12°C, or below 11°C, or below 10°C, or below 9°C, or below 8°C, or below 7°C, or below 6°C, or below 5°C, or below 4°C, or below 3°C, or below 2°C, or below 1°C. In another embodiment, the method is for determining FHbF, wherein the blood sample has been subjected to stress caused by exposure to a temperature below 20°C, or below 19°C, or below 18°C, or below 17°C, or below 16°C, or below 15°C, or below 14°C, or below 13°C, or below 12°C, or below 11°C, or below 10°C, or below 9°C, or below 8°C, or below 7°C, or below 6°C, or below 5°C, or below 4°C, or below 3°C, or below 2°C, or below 1°C.

[0032] In another embodiment, the method is for determining a BMP parameter. In a further embodiment, the BMP parameter is Na + , K + , Mg 2+ , Cl - , HCO 3- , urea, creatinine, glucose, Ca 2+ , lactate and total bilirubin.

[0033] In another embodiment, the method comprises the steps of: + The blood samples were stored at 20°C for 30 min. In another embodiment, the method comprises the step of: subjecting the cell to stress caused by exposure to a temperature of less than 10° C., or less than 19° C., or less than 18° C., or less than 17° C., or less than 16° C., or less than 15° C., or less than 14° C., or less than 13° C., or less than 12° C., or less than 11° C., or less than 10° C., or less than 9° C., or less than 8° C., or less than 7° C., or less than 6° C., or less than 5° C., or less than 4° C., or less than 3° C., or less than 2° C., or less than 1° C. + In another embodiment, the method comprises determining Mg, wherein the blood sample has been subjected to stress caused by exposure to a temperature below 20° C. or below 19° C., or below 18° C., or below 17° C., or below 16° C., or below 15° C., or below 14° C., or below 13° C., or below 12° C., or below 11° C., or below 10° C., or below 9° C., or below 8° C., or below 7° C., or below 6° C., or below 5° C., or below 4° C., or below 3° C., or below 2° C., or below 1° C. 2+ In another embodiment, the method comprises determining a Cl concentration in a blood sample, the blood sample being stressed by exposure to a temperature below 20° C. or below 19° C., or below 18° C., or below 17° C., or below 16° C., or below 15° C., or below 14° C., or below 13° C., or below 12° C., or below 11° C., or below 10° C., or below 9° C., or below 8° C., or below 7° C., or below 6° C., or below 5° C., or below 4° C., or below 3° C., or below 2° C., or below 1° C. -In another embodiment, the method is for determining HCO2, wherein the blood sample has been subjected to stress caused by exposure to a temperature below 20°C or below 19°C, or below 18°C, or below 17°C, or below 16°C, or below 15°C, or below 14°C, or below 13°C, or below 12°C, or below 11°C, or below 10°C, or below 9°C, or below 8°C, or below 7°C, or below 6°C, or below 5°C, or below 4°C, or below 3°C, or below 2°C, or below 1°C. 3-wherein the blood sample has been subjected to stress caused by exposure to a temperature below 20°C, or below 19°C, or below 18°C, or below 17°C, or below 16°C, or below 15°C, or below 14°C, or below 13°C, or below 12°C, or below 11°C, or below 10°C, or below 9°C, or below 8°C, or below 7°C, or below 6°C, or below 5°C, or below 4°C, or below 3°C, or below 2°C, or below 1°C. In another embodiment, the method is for determining urea, wherein the blood sample has been subjected to stress caused by exposure to a temperature below 20°C, or below 19°C, or below 18°C, or below 17°C, or below 16°C, or below 15°C, or below 14°C, or below 13°C, or below 12°C, or below 11°C, or below 10°C, or below 9°C, or below 8°C, or below 7°C, or below 6°C, or below 5°C, or below 4°C, or below 3°C, or below 2°C, or below 1°C. In another embodiment, the method is for determining creatinine, and the blood sample has been subjected to stress caused by exposure to a temperature below 20°C, or below 19°C, or below 18°C, or below 17°C, or below 16°C, or below 15°C, or below 14°C, or below 13°C, or below 12°C, or below 11°C, or below 10°C, or below 9°C, or below 8°C, or below 7°C, or below 6°C, or below 5°C, or below 4°C, or below 3°C, or below 2°C, or below 1°C. In another embodiment, the method is for determining glucose, and the blood sample has been subjected to stress caused by exposure to a temperature below 20° C. or below 19° C., or below 18° C., or below 17° C., or below 16° C., or below 15° C., or below 14° C., or below 13° C., or below 12° C., or below 11° C., or below 10° C., or below 9° C., or below 8° C., or below 7° C., or below 6° C., or below 5° C., or below 4° C., or below 3° C., or below 2° C., or below 1° C. 2+and the blood sample is maintained at a temperature below 20°C or below 19°C, or below 18°C, or below 17°C, or below 16°C, or below 15°C, or below 14°C, or below 13°C, or below 12°C, or below 11°C, or below 10°C, or below 9°C, or below 8°C, or below 7°C, or below 6°C, or below 5°C, or below 4°C, or below 3°C, or below 2°C. In another embodiment, the method is for determining lactate, and the blood sample is stressed by exposure to a temperature of less than 20° C., or less than 19° C., or less than 18° C., or less than 17° C., or less than 16° C., or less than 15° C., or less than 14° C., or less than 13° C., or less than 12° C., or less than 11° C., or less than 10° C., or less than 9° C., or less than 8° C., or less than 7° C., or less than 6° C., or less than 5° C., or less than 4° C., or less than 3° C., or less than 2° C., or less than 1° C. In another embodiment, the method is for determining total bilirubin, and the blood sample has been subjected to stress caused by exposure to a temperature below 20°C, or below 19°C, or below 18°C, or below 17°C, or below 16°C, or below 15°C, or below 14°C, or below 13°C, or below 12°C, or below 11°C, or below 10°C, or below 9°C, or below 8°C, or below 7°C, or below 6°C, or below 5°C, or below 4°C, or below 3°C, or below 2°C, or below 1°C.

[0034] The method may or may not further comprise determining the platelet count and / or white blood cell count in the sample obtained in step i). In one embodiment, the white blood cell count is a total white blood cell count. In another embodiment, the white blood cell count is a count of five different types of blood cells ("5-part differential" or "5-part diff"), namely, neutrophils, lymphocytes, monocytes, eosinophils and basophils, or one, two or more of these types. In one embodiment, neutrophils, basophils and eosinophils are reported as a group as granulocytes. Each can be reported, for example, as a percentage. A change in the percentage may be indicative of a pathological condition.

[0035] In one embodiment the method is for determining two or more of the above parameters in the same sample, such as 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more, 12 or more, 14 or more, 16 or more, 18 or more or 19 or more of the above parameters, such as for determining these parameters simultaneously in the same sample.

[0036] In one embodiment, the method is for determining two or more of the above parameters in the same sample, said two or more parameters comprising the following parameters: pH and K + or pO2 and K + or pCO2 and K + or pH and glucose or pO2 and glucose or pCO2 and glucose.

[0037] Sensor Assembly In principle, any suitable method can be used for determining said blood gas parameters and / or BMP parameters in a sample.

[0038] In one embodiment, the determination is performed with a sensor assembly that uses one or more analyte sensors. Preferably, the determination is performed with a sensor assembly that includes two or more analyte sensors, thus facilitating the simultaneous determination of two or more parameters.

[0039] Advantageously, the two or more analyte sensors are not all located in the same plane, and one of the analyte sensors analyses the blood gas parameter or the BMP parameter, and another analyte sensor, not located in the same plane, analyses a different blood gas parameter or BMP parameter. The assembly allows for the analysis of smaller amounts of sample. The term "planar" as used herein means a flat two-dimensional surface. In a preferred embodiment, the two or more analyte sensors are arranged on two or more planes that are not positioned at an angle of 180° relative to each other. In another preferred embodiment, the two or more analyte sensors are not all positioned at an angle of 180° relative to each other.

[0040] In a preferred embodiment, the determination comprises: a) a first electronic wiring substrate having a first and a second surface and at least one analyte sensor formed on the first surface, the at least one analyte sensor being connected to one or more electrical contacts; b) a second electronic wiring substrate having a first and a second surface and at least one analyte sensor formed on a portion of the first surface, the at least one analyte sensor being connected to one or more electrical contacts; and c) a spacer having a through-going recess with first and second openings; wherein a first substrate, a second substrate and a spacer are arranged in a layered structure, where a first surface of the first substrate blocks a first opening of the spacer and a first surface of the second substrate blocks a second opening of the spacer, thereby forming a measurement cell, which faces at least one analyte sensor from each of the substrates. Such a sensor assembly is described in WO 2008 / 131767 (Radiometer Medical ApS). In a further embodiment thereof the volume of the measuring cell is less than 1 ml, such as less than 0.5 ml, such as less than 200 microliters, such as less than 100 microliters, such as less than 50 microliters, such as less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 microliters. In further embodiments, the volume of the measurement cell is between 2 and 50 microliters, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 microliters to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 microliters.Thus, in one embodiment, the volume used for the determination, i.e. the volume contained in the measuring cell, is less than 1 ml, such as less than 0.5 ml, such as less than 200 microliters, such as less than 100 microliters, such as less than 50 microliters, such as between 2 and 50 microliters, such as between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 , 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 microliters to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 microliters. The measurement cell provided by the recess in the spacer and the first surface of the first and second substrates preferably provides a volume of about 25 to 45 microliters, more preferably a volume of about 30 to 40 microliters. Such a volume requires very little sample for measurement by the analyte sensor in the measurement cell. Preferably, the dimensions of the spacer are within the following ranges: The recess in the spacer may have dimensions within the following ranges: length 20-60 mm, width 5-20 mm and thickness 0.2-0.6 mm. The dimensions of the first and second substrates and the spacer, and therefore the dimensions of the sensor assembly, may be adapted depending on the intended use. However, in a preferred embodiment, the first substrate has dimensions within the following ranges: length about 20-60 mm, width about 5-20 mm and thickness about 0.3-0.8 mm. The width and / or length of the second substrate may be somewhat larger than the width and / or length of the first substrate. This is due to the fact that for some preferred embodiments it is preferred that the first surface of the second substrate protrudes beyond the edge of the spacer and the first substrate in the sensor assembly. The second substrate preferably has dimensions within the following ranges: length about 20-60 mm, width about 5-40 mm and thickness 0.3-0.8 mm. The length and width of the second substrate may provide an extension beyond the edges of the first substrate and spacer in the range of about 4-20 mm.

[0041] In another embodiment, the determination is performed in a sensor array that includes: a housing having a base, an upper portion spaced above the base, and an outer wall extending from the base to the upper portion; an inlet in the housing that is sized to receive a sample of fluid; a plurality of compartments arranged around the fluid inlet and substantially isolated from one another, each compartment having a port in the fluid inlet for receiving a portion of the sample of fluid received by the fluid inlet; and at least one sensor in each compartment, where the at least one sensor is responsive to a fluid when the fluid contacts the at least one sensor, where the sensor array is configured to selectively direct a sample of fluid received by one or more of the plurality of compartments from the fluid inlet to contact the at least one sensor. Such a sensor array is described in WO2018 / 112017. In a further embodiment thereof the volume used for the determination in each compartment, i.e. the volume contained within the compartment, is less than 1 ml, such as less than 0.5 ml, for example less than 200 microliters, such as less than 100 microliters, for example less than 50 microliters, such as between 2 and 50 microliters, for example between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 microliters to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 microliters.

[0042] In another embodiment, the determination is performed in a sensor assembly including: a first microsensor having a first outer sheath, a first membrane core within the first outer sheath, and a first conductive element at least partially enclosed by and in contact with the first membrane core, where the first conductive element detects a first electrical response signal when the first membrane core contacts a fluid; and a second microsensor adjacent to an outer surface of the first microsensor, the second microsensor having a second outer sheath, a second membrane core within the second outer sheath, and a second conductive element at least partially enclosed by and in contact with the second membrane core, where the second conductive element detects a second electrical response signal when the second membrane core contacts a fluid. Such a sensor assembly is described in WO 2018 / 112012. In a further embodiment thereof the volume used for the determination, i.e. the volume contained in the measuring cell, is less than 1 ml, such as less than 0.5 ml, such as less than 200 microliters, such as less than 100 microliters, such as less than 50 microliters, such as between 2 and 50 microliters, such as between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 9, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 microliters to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 microliters.

[0043] In another embodiment, the determination is performed in a microcapillary sensor array that includes a sensor body elongated along a longitudinal axis, the sensor body having a first end, a second end spaced apart from the first end along the longitudinal axis, an outer surface, and an inner surface, where the inner surface defines a hollow capillary extending from the first end to the second end along the longitudinal axis; a sensing element extending through the sensor body from the outer surface to the hollow capillary; and a conductive element in contact with the sensing element; where the conductive element detects a response signal generated by a reaction between the sensing element and a fluid as the fluid flows through the hollow capillary and contacts the sensing element. Such a sensor array is described in WO 2018 / 112008. In a further embodiment thereof the volume used for the determination, i.e. the volume contained in the measuring cell, is less than 1 ml, such as less than 0.5 ml, such as less than 200 microliters, such as less than 100 microliters, such as less than 50 microliters, such as between 2 and 50 microliters, such as between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 2, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 microliters to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 microliters.

[0044] In another embodiment, the determination is performed in a test device that includes: a first planar substrate having a first planar surface; a second planar substrate having a second planar surface; a first sensing area and a second sensing area, the first sensing area and the second sensing area being disposed between the first and second planar surfaces, both the first sensing area and the second sensing area containing chemicals and / or reagents electrically connected to the first and second electrodes, respectively; a first planar intermediate isolation layer having a flow path, where the first sensing area is opposite the second sensing area, and the flow path is disposed between the first and second sensing areas; and a first heating element disposed between the first and second planar surfaces. Such a test device is described in WO 2017 / 120464. In a further embodiment thereof the volume used for the determination, i.e. the volume contained in the measuring cell, is less than 1 ml, such as less than 0.5 ml, such as less than 200 microliters, such as less than 100 microliters, such as less than 50 microliters, such as between 2 and 50 microliters, such as between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 2, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 microliters to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 microliters.

[0045] In another embodiment, the determination is performed in a test device that includes: a first planar intermediate insulating layer having at least a first sensing area; a second planar intermediate insulating layer having at least a second sensing area; a third planar intermediate isolation layer having a flow path 14, where the first sensing region is opposite the second sensing region and the flow path is disposed between the first sensing region and the second sensing region; a first planar conductive layer disposed adjacent to the first intermediate isolation layer opposite the third planar intermediate isolation layer; a first planar substrate disposed adjacent to the first planar conductive layer opposite the first intermediate isolation layer; a second planar substrate disposed adjacent to the second planar intermediate isolation layer opposite the third planar intermediate isolation layer, the second substrate having a first conductive via in electrical contact with at least the second sensing region; A second planar conductive layer is disposed in contact with the planar substrate, the second planar conductive layer being in electrical contact with the first conductive via, where each of the first planar intermediate isolation layer, the second planar intermediate isolation layer, the third planar intermediate isolation layer, the first planar conductive layer, the first planar substrate, the second planar substrate and the second planar conductive layer has two planar surfaces separated by a thickness, each of the two respective planar surfaces having approximately equal planar areas, where the planar area of ​​the first conductive layer is greater than each of the planar areas of the first planar intermediate isolation layer, the second planar intermediate isolation layer, the third planar intermediate isolation layer, the second planar substrate and the second planar conductive layer. Such a test device is described in WO 2017 / 019609. In a further embodiment thereof the volume used for the determination, i.e. the volume contained in the measuring cell, is less than 1 ml, such as less than 0.5 ml, such as less than 200 microliters, such as less than 100 microliters, such as less than 50 microliters, such as between 2 and 50 microliters, such as between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 2, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 microliters to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 microliters.

[0046] In another embodiment, the determination is performed on a test device that includes: a single substrate, the substrate having a first surface, the first surface having a first region and a second region separated by a line, the first region being opposite the second region of the first surface of the single substrate; a conductor layer disposed on the single substrate, the conductor layer including a first group of electrodes printed on the first region and a second group of electrodes printed on the second region; a dielectric layer disposed on the conductor layer, the dielectric layer including a first region of dielectric material disposed on the first group of electrodes and a second region of dielectric material disposed on the second group of electrodes, the first region of dielectric material and the second region of dielectric material each including a respective first group of reaction wells and a second group of reaction wells formed in the dielectric layer, at least one reaction well is electrically coupled to a respective electrode and contains a chemical; and a spacer layer adjacent the first region of dielectric material and the second region of dielectric material, the spacer layer forming a flow path between the first group of reaction wells and the second group of reaction wells. Such a test device is described in WO2016 / 106320. In a further embodiment thereof the volume used for the determination, i.e. the volume contained in the measuring cell, is less than 1 ml, such as less than 0.5 ml, such as less than 200 microliters, such as less than 100 microliters, such as less than 50 microliters, such as between 2 and 50 microliters, such as between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 2, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 microliters to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 microliters.

[0047] In another embodiment, the determination is performed on a test strip that includes a first planar substrate having a coplanar electrode on a first planar surface and a second planar substrate having a coplanar electrode on a second planar surface, the first planar substrate and the second planar substrate being arranged such that the first surface of the first planar substrate is opposite the second planar substrate; an intermediate layer disposed between the opposite first surface of the first planar substrate and the second planar substrate; the first planar substrate having a first sensing area electrically connected to a first electrical contact; and the second planar substrate having a second electrical contact electrically connected to the first electrical contact via a conductive element, the conductive element extending between the first surface of the first planar substrate and the second surface of the second planar substrate without passing through the first planar substrate or the second planar substrate. Such a test strip is described in WO 2016 / 011308. In a further embodiment thereof the volume used for the determination, i.e. the volume contained in the measuring cell, is less than 1 ml, such as less than 0.5 ml, such as less than 200 microliters, such as less than 100 microliters, such as less than 50 microliters, such as between 2 and 50 microliters, such as between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 2, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 microliters to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 microliters.

[0048] In another embodiment, the determining is performed in a sensor assembly including: a first planar substrate having a base layer, a conductive layer formed on the first planar surface of the base layer, and a dielectric layer formed on at least one of the first planar surface of the conductive layer or the first planar surface of the base layer, the dielectric layer having a first planar surface disposed a distance away from the first planar surface of the conductive layer, the conductive layer including at least a first electrical contact and a second electrical contact electrically insulated from the first electrical contact, the dielectric layer defining a liquid flow path through the dielectric layer, the flow path having two side walls and a bottom surface extending between the two side walls, extends between the first planar surface of the base layer and the first planar surface of the dielectric layer, and the dielectric layer further defines a first sensing area and a second sensing area on the first electrical contact and the second electrical contact of the conductive layer, respectively, the first sensing area and the second sensing area allowing the liquid in the flow path to contact the first electrical contact and the second electrical contact, respectively; and a second planar substrate, the second substrate being bonded to the first substrate, and when bonded to the first substrate, the second substrate defining an upper surface of the liquid flow path, the upper surface of the liquid flow path extending between the two side walls and positioned a short distance away from the bottom surface of the liquid flow path. Such a sensor assembly is described in WO 2016 / 007716. In a further embodiment thereof the volume used for the determination, i.e. the volume contained in the measuring cell, is less than 1 ml, such as less than 0.5 ml, such as less than 200 microliters, such as less than 100 microliters, such as less than 50 microliters, such as between 2 and 50 microliters, such as between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 2, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 microliters to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 microliters.

[0049] In another embodiment, the determining is performed in a sensor assembly that includes: a substrate having a first surface and a second surface opposite the first surface; at least one analyte sensor located on at least one of the first and second surfaces; and at least one electrical contact located on the substrate in electrical communication with a corresponding one of the at least one analyte sensor, where the substrate is configured to define a tube having an interior surface and an exterior surface, at least a portion of the first surface of the substrate defining the interior surface of the tube, and where the at least one analyte sensor is disposed on at least one of the interior and exterior surfaces of the tube. Such a sensor assembly is described in WO 2013 / 163120. In a further embodiment thereof the volume used for the determination, i.e. the volume contained in the measuring cell, is less than 1 ml, such as less than 0.5 ml, such as less than 200 microliters, such as less than 100 microliters, such as less than 50 microliters, such as between 2 and 50 microliters, such as between 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 2, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 microliters to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 microliters.

[0050] In a preferred embodiment, the determination of BG and BMP parameters and platelet count is performed using an automated hematology analyzer. Suitable hematology analyzers are described, for example, in U.S. Patent No. 5,564,419 or U.S. Patent No. 6,880,384.

[0051] sample The blood sample is typically whole blood. In a preferred embodiment, the blood of the blood sample is not a purified fraction of a particular type of blood cell, such as a preparation or population of platelets, such as a population of washed platelets.

[0052] In one embodiment, all steps of the method are performed on whole blood, i.e. step i) (combination with an anticoagulant and an antiplatelet agent) is performed on whole blood, the pre-analytical stressed blood sample is a whole blood sample and step ii) (determination of the parameter(s)) is performed on whole blood.

[0053] In other embodiments, step i) is performed on whole blood, the pre-analytical stressed blood sample is a whole blood sample and step ii) is performed on a blood fraction, such as serum or plasma.

[0054] In yet other embodiments, step i) is performed on whole blood, which is then fractionated, e.g. into serum or plasma samples, and the fractionated samples, e.g. serum or plasma samples, are subjected to pre-analytical stress prior to the determination in step ii).

[0055] The blood can be venous or arterial. Preferably, the blood is arterial. However, when the method of the present invention is used to prepare blood samples in an emergency department setting, the use of venous blood may be preferred. In one embodiment, the blood can be capillary blood. This embodiment is particularly preferred when the blood sample is obtained from a neonate.

[0056] Anticoagulants and antiplatelet agents As noted above, the methods of the present invention involve combining a blood sample with an anticoagulant and an antiplatelet agent. The terms "a" or "an" as used herein, unless otherwise specified, have the meaning of "at least one." Thus, a blood sample may be combined with one or more anticoagulants. The anticoagulant used in the method of the present invention may be combined with one or more antiplatelet agents and / or one or more anticoagulants. It is understood that the anticoagulant used in the method of the present invention is an anticoagulant suitable for the determination of BG and BMP parameters, i.e. a substance that is suitable as an anticoagulant in a blood sample so that the blood sample can be used for the analysis of BG and BMP parameters.

[0057] In a preferred embodiment, the inhibitor is selected from the group consisting of an indirect factor Xa inhibitor or a direct factor Xa inhibitor or a combination thereof. In a preferred embodiment, the anticoagulant is selected from the group consisting of heparinates or heparinoids or combinations thereof.Preferred embodiments of heparinates are heparin, such as unfractionated, high molecular weight heparin (HMWH), low molecular weight heparin (LMWH) (including bemiparin, certoparin, dalteparin, enoxaparin, nadroparin, parnaparin, reviparin, tinzaparin); and oligosaccharides, such as fondaparinux, idraparinux.Preferred embodiments of heparinoids include danaparoid, dermatan sulfate and sulodexide.

[0058] Preferred embodiments of direct factor Xa inhibitors include apixaban, betrixaban, darexaban, edoxaban, otamixaban and rivaroxaban.

[0059] In a preferred embodiment, the anticoagulant suitable for BG and BMP parameter analysis includes heparin. As mentioned above, heparin is the standard anticoagulant for BG and BMP parameter analysis. Heparin is a naturally occurring polysaccharide, which inhibits coagulation, the process that leads to thrombosis. Natural heparin consists of molecular chains of various lengths or molecular weights. It is also used as an anticoagulant (blood thinner). It binds to the enzyme inhibitor antithrombin III (AT) and causes a conformational change, which results in its activation due to an increase in the flexibility of the loop at its reactive site. The activated AT then inactivates thrombin, factor Xa and other proteases.

[0060] In a preferred embodiment, the anticoagulant is electrolyte-balanced heparin (also called "balanced heparin"). Heparin is known to bind positively charged electrolytes, which can interfere with electrolyte measurements. It is preferred that the electrolyte-balanced heparin formulations include lithium, zinc, sodium, potassium, or ammonium salts of heparin. In a preferred embodiment, the electrolyte-balanced heparin formulations include lithium heparin and sodium heparin.

[0061] In another embodiment, the anticoagulant heparin is human heparin, porcine heparin or synthetic heparin. In a preferred embodiment, the anticoagulant is porcine heparin. In another preferred embodiment, the anticoagulant is unfractionated heparin.

[0062] In a preferred embodiment, the final concentration of the anticoagulant, such as heparin, is about 10 IU / mL to about 200 IU / mL, preferably about 20 IU / mL to about 100 IU / mL. In a particularly preferred embodiment, the final concentration of the anticoagulant, such as heparin, is about 60 IU / mL.

[0063] In one embodiment, the anticoagulant used in accordance with the present invention can be in liquid form (also referred to as "liquid heparin") or can be in a dry form, such as, for example, when combined with blood, dry balanced heparin. One example of a dry form of anticoagulant is a lyophilized anticoagulant, such as lyophilized heparin or lyophilized balanced heparin.

[0064] In one embodiment, the anticoagulant is in lyophilized form when combined with the blood. In a preferred embodiment, the antiplatelet agent is a glycoprotein IIb / IIIa inhibitor, an ADP receptor / P2Y 12 inhibitors, prostaglandin analogs, COX inhibitors, thromboxane inhibitors, phosphodiesterase inhibitors, cloricromene, dithazol, vorapaxar or combinations thereof.

[0065] Glycoprotein IIb / IIIa, also known as integrin αIIbβ3, is an integrin complex found on platelets. It is a receptor for fibrinogen and von Willebrand factor and helps activate platelets. Glycoprotein IIb / IIIa inhibitors can be used to prevent blood clots to lower the risk of heart attack or stroke. Examples of glycoprotein IIb / IIIa include, but are not limited to, abciximab, eptifibatide (also called integrillin), orbofiban, lotrafiban, roxifiban, sibrafiban, and tirofiban (also called aggrastat) or salts thereof. Preferred glycoprotein IIb / IIIa inhibitors are eptifibatide and tirofiban or salts thereof.

[0066] Adenosine diphosphate (ADP) receptor / P2Y 12 Inhibitors are a class of antiplatelet drugs that are used in the treatment of acute coronary syndromes or as prophylaxis in patients at risk for thromboembolism, myocardial infarction or stroke. 12antagonism to proteins, hence ADP to P2Y 12 The reason is to prevent platelets from binding to the receptor. This results in decreased platelet aggregation and prevents the formation of blood clots. 12 The receptors are surface-bound proteins present on platelets. They belong to the G protein-coupled purinergic receptors (GPCRs) and are chemoreceptors for ADP. ADP Receptor / P2Y 12 Examples of inhibitors include, but are not limited to, thienopyridines, such as clopidogrel, prasugrel and ticlopidine or salts thereof; and nucleotide / nucleoside analogues / receptor antagonists, such as cangrelor, elinogrel, ticagrelor, suramin sodium and 2-MeSAMP. Thienopyridines are compounds that inhibit the ADP receptor / P2Y receptor in vitro. 12 The less preferred ADP receptor / P2Y inhibitors according to the present invention are prodrugs that do not exhibit inhibitory activity. 12 Thus, nucleotide / nucleoside analogues / receptor antagonists, such as cangrelor, elinogrel, ticagrelor or salts thereof, sodium suramin and 2-MeSAMP, are preferred ADP receptor / P2Y inhibitors according to the present invention. 12 It is an inhibitor.

[0067] Prostaglandins can induce or inhibit platelet aggregation and stenosis, and dilate blood vessels.Prostaglandin analogues are a class of drugs that bind to prostaglandin receptors.Examples include, but are not limited to, beraprost, iloprost (also known as ZK36374), prostacyclin, epoprostenol and treprostinil.

[0068] Cyclooxygenase (COX), formally known as prostaglandin-endoperoxide synthase (PTGS), is an enzyme responsible for the formation of prostanoids, including thromboxanes and prostaglandins. Examples of COX inhibitors include, but are not limited to, acetolisalicylic acid, aloxypurine, carbasalate calcium, ibuprofen, trifusal, sulfinpyrazone, and nitroaspirin (NCX-4016).

[0069] Thromboxanes are members of a family of lipids known as eicosanoids. The two major thromboxanes are thromboxane A2 and thromboxane B2. The distinguishing feature of thromboxanes is the six-membered ether-containing ring. Thromboxane A synthase is named for its role in blood clot formation. Thromboxane A synthase is an enzyme present in platelets that converts prostaglandin H2, an arachidonic acid derivative, to thromboxane. Thromboxane inhibitors include thromboxane synthase inhibitors such as dipyridamole, picotamide, terbogrel, daltroban, seratrodast, SQ-29548, and ramatroban; and thromboxane receptor antagonists such as terbogrel and terutroban.

[0070] Phosphodiesterases are enzymes that cleave phosphodiester bonds. Phosphodiesterase enzymes (PDEs) are frequent targets for pharmacological inhibition due to their unique tissue distribution, structural and functional properties. Inhibitors of phosphodiesterases can prolong or enhance the action of physiological processes mediated by cAMP or cGMP by inhibiting their degradation by phosphodiesterases. PDE inhibitors have been identified as novel potential therapies in areas such as pulmonary arterial hypertension, coronary heart disease, dementia, depression, asthma, COPD, protozoal infections including malaria, and schizophrenia. Furthermore, cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) are important intracellular second messengers and are endowed with strong inhibitory activity on fundamental platelet functions. PDEs limit intracellular levels of cyclic nucleotides by catalyzing the hydrolysis of cAMP and cGMP, thus controlling platelet function. Thus, inhibition of PDEs can exert a strong platelet inhibitory effect (Gresele et al. Br. J. Clin. Pharmacol. 2011 Oct;72(4):634-46). Examples of PDEs include, but are not limited to, cilostazol, dipyridamole, trifusal, milrinone, anagrelide, and theophylline.

[0071] Antiplatelet agents not known to belong to one of the above groups include, but are not limited to, cloricromene, dithazol, vorapaxar, and L-arginine or salts thereof.

[0072] Cloricromene is an antiplatelet agent with vasodilatory activity used in the treatment of thromboembolic disorders. Ditazol is a nonsteroidal anti-inflammatory drug with analgesic and antipyretic activity similar to phenylbutazone. In addition, Ditazol is a platelet aggregation inhibitor and is marketed in Spain and Portugal under the trade name Ageroplas®.

[0073] Vorapraxar (formerly known as SCH 530348) is a thrombin receptor (protease-activated receptor, PAR-1) antagonist based on the natural product himbacine.

[0074] Oral L-arginine has been shown to inhibit platelet aggregation via the nitric oxide pathway (Adams et al., J. Am. Coll. Cardiol. 1995 Oct;26(4):1054-61). Antiplatelet agents include glycoprotein IIb / IIIa inhibitors, ADP receptor / P2Y 12 Preferably, the inhibitor is selected from the group consisting of prostaglandin analogs, cloricromene, dithazol, vorapaxar, or a combination thereof.

[0075] In another preferred embodiment, the antiplatelet agent is selected from the group consisting of glycoprotein IIb / IIIa inhibitors, prostaglandin analogs, cloricromene, dithazol, vorapaxar, or combinations thereof.

[0076] In another preferred embodiment, the antiplatelet agent is selected from the group consisting of glycoprotein IIb / IIIa inhibitors and prostaglandin analogs. In a preferred embodiment, the antiplatelet agent comprises a prostaglandin analog.

[0077] In yet another preferred embodiment, the antiplatelet agent is a glycoprotein IIb / IIIa inhibitor or a salt thereof selected from the group consisting of abciximab, eptifibatide, orbofiban, lotrafiban, roxifiban, sibrafiban and tirofiban. In a preferred embodiment, the antiplatelet agent comprises eptifibatide and / or tirofiban or a salt thereof. In a preferred embodiment, eptifibatide is used as eptifibatide acetate. It is also preferred that tirofiban is used as tirofiban hydrochloride, more preferably as tirofiban hydrochloride monohydrate salt.

[0078] In yet another preferred embodiment, the antiplatelet agent is a prostaglandin analog selected from the group consisting of beraprost, iloprost, prostacyclin, epoprostenol, treprostinil, or a salt thereof.

[0079] In particularly preferred embodiments, the antiplatelet agent comprises eptifibatide, tirofiban, iloprost, salts thereof, or combinations thereof. Particularly preferred is that the antiplatelet agent comprises iloprost.Iloprost is particularly preferred as antiplatelet agent, because it is not only suitable for preparing blood samples for BG, BMP and platelet counting, but also surprisingly found that it inhibits the activation of white blood cells, and therefore causes very little clumping of WBC in blood samples.

[0080] In another embodiment, the antiplatelet agent is an ADP receptor / P2Y 12 Inhibitors, wherein ADP receptor / P2Y 12 Inhibitors are nucleotide / nucleoside analogues / receptor antagonists. ADP receptor / P2Y 12 The inhibitor is preferably selected from the group consisting of cangrelor, elinogrel, ticagrelor or a salt thereof, suramin sodium, 2-MeSAMP.

[0081] In another preferred embodiment, the method of the present invention does not include combining MgSO4, EDTA or citrate with the blood of the blood sample. These anticoagulants are not suitable for preparing blood samples for BG and BMP parameter analysis.

[0082] In one embodiment of the method of the invention, the sample is subjected to a pre-analytical stress after step i), ie after the sample has been combined with the anticoagulant and antiplatelet agents. In another preferred embodiment, the method of the present invention further comprises a step of mixing the blood sample. Mixing the blood sample is advantageous because the blood sample may otherwise clot or settle, or the sample may react with air in the blood sample before analysis. Mixing can be performed, for example, by stirring. Mixing can be performed by repeatedly inverting the blood sample manually or by rolling it horizontally. A stirring element can also be included in the blood sample. The sample can then be stirred, for example, by using a moving means, such as those described in U.S. Pat. No. 6,880,384 B2. Mixing the blood sample promotes dissolution of the anticoagulant, such as heparin, which prevents settling. If the anticoagulant does not dissolve properly, it can lead to the formation of microscopic clumps, which can bias the results and / or damage the analysis device. Sedimentation can lead to sample heterogeneity and misleading analysis results.

[0083] In one embodiment, the method of the invention involves the use of a blood sampler that contains: a) an anticoagulant as described above; and b) Antiplatelet agents as above.

[0084] The blood sampler can be used to carry out the method of the present invention described above. The anticoagulant for BG and BMP parameter analysis can be present in the blood sampler in liquid form, or it can be present in a dry formulation.

[0085] In one embodiment, the blood sampler contains an additional element comprising an anticoagulant and / or antiplatelet agent. For example, the additional element can be a "brick" as known for "heparin bricks". "Brick" in this context means that the anticoagulant is prepared in a "puff" of inert filler material, where the puff dissolves and the heparin is dispersed throughout the sample by appropriate mixing, where the puff can be distributed during manufacture to deliver a reproducible amount of heparin in each sampler. An example of such a brick is, for example, a cellulose strip soaked in an anticoagulant and / or antiplatelet agent. However, other additional elements that can release the anticoagulant and / or antiplatelet agent upon contact with blood are also possible, for example, a blood sampler wall coating matrix sprayed onto the inner sampler surface.

[0086] In one embodiment, the blood sampler is constructed of plastic or glass. In another preferred embodiment, the blood sampler comprises a sampler cap. The sampler cap is a cap that should be connected to the open end of the blood sampler, for example to the tip of a syringe, or to the open end of a capillary or test tube. The gas exchange with the surroundings, which may bias the BG analysis result, can be avoided by using the sampler cap. Suitable sampler caps are described, for example, in WO 2004 / 000412.

[0087] In another preferred embodiment, the blood sampler contains a mixing element. The mixing element is preferably a spherical element or a cylindrical element with rounded ends. It is particularly preferred that the mixing element has the shape of a ball. The ball can be made of, for example, steel or plastic.

[0088] In another embodiment, the mixing element includes a coating with an inert material. The inert material preferably does not interfere or does not substantially interfere with blood analysis. For example, the inert material can be selected from the group consisting of gold, platinum, palladium, or rhodium. In a preferred embodiment, the inert material is gold.

[0089] In a particularly preferred embodiment, it is particularly preferred that the mixing elements are gold coated balls, preferably gold coated steel balls. In one embodiment, the mixing element is a mixing element as described in US Patent No. 6,880,384 B2. The movement of the mixing element is also described in US Patent No. 6,880,384 B2. Thus, it is preferred that the mixing element is moved by a moving means, e.g., by mechanical means. The moving means can be a robotic arm or a support for moving, e.g., tilting or rotating, the sample handler and / or the sampler bed, thereby moving by gravity the mixing elements held in any samplers therein.

[0090] definition As used herein, the term "parameter" refers to any piece of clinical information related to a blood sample.

[0091] As used herein, the term "blood gases," as in "blood gas parameters," refers to the gaseous parameters of blood, and specifically refers to the gaseous parameters dissolved in blood, typically arterial blood. The blood gas parameters include the amount of gases (e.g. oxygen and carbon dioxide). Blood gas parameters include pH, pCO2, pO2, oxygen saturation (sO2), total hemoglobin concentration (ctHb or tHb), percentage of oxyhemoglobin (FO2Hb or O2Hb), percentage of carboxyhemoglobin (FCOHb or COHb), percentage of methemoglobin (FMetHb or MetHb), percentage of deoxyhemoglobin (FHHb or RHb) and percentage of fetal hemoglobin (FHbF). "Blood sample suitable for blood gas analysis" means that the blood sample can be used to measure at least one blood gas parameter, but is preferably suitable for measuring all blood gas parameters: pH, pCO2, pO2, ctHb, FO2Hb, FCOHb, FMetHb, FHHb and FHbF. It may be preferred that at least pH, tHb, FCOHb and FMetHb can be measured.

[0092] As used herein, the term "basic metabolic panel" as in "basic metabolic panel parameter analysis" refers to the analysis of biochemical blood parameters, particularly electrolytes, i.e. Na + , K + , Mg 2+ , Cl - , HCO 3- , urea, creatinine, glucose (glu), Ca 2+ The term "blood sample suitable for BMP parameter analysis" refers to the concentrations of lactic acid (lac) and total bilirubin (tBil). Thus, a "blood sample suitable for BMP parameter analysis" is a blood sample that can be used to determine at least one, but preferably all, of the above BMP parameters. At least Na + , K + , Mg 2+ , Cl - , Ca 2+ It may be preferred that , glu, lac and tBil can be measured.

[0093] As used herein, the term "platelet count" or "determining the number of platelets" refers to a diagnostic test that determines the number of platelets in a patient's blood. Platelets, also called thrombocytes, are small, disk-shaped blood cells produced in the bone marrow and are involved in the process of blood clotting. Normally, there are 150,000 to 450,000 platelets in every microliter of blood. Low platelet counts or abnormally shaped platelets are associated with bleeding disorders. High platelet counts sometimes indicate bone marrow damage.

[0094] As used herein, the term "anticoagulant" refers to a substance that prevents or reduces blood clotting, i.e., the coagulation cascade that leads to fibrin polymerization and thus the formation of a fibrin clot. Anticoagulants thereby prolong clotting time by inhibiting the clotting factor cascade after the initial platelet aggregation.

[0095] As used herein, the term "anticoagulant suitable for the determination of BG and BMP parameters" means that a substance is suitable as an anticoagulant in a blood sample so that the blood sample can be used for the analysis of BG and BMP parameters. Some anticoagulants, such as EDTA, are suitable for example because they contain Ca 2+ It is known that EDTA is not suitable for the determination of BG and BMP parameters because it forms a complex with EDTA and therefore the calcium concentration cannot be reliably determined in blood samples. Therefore, EDTA is not a suitable anticoagulant for the determination of BG and BMP parameters.

[0096] As used herein, the term "antiplatelet agent" refers to a substance that reduces platelet aggregation and / or inhibits thrombus formation, i.e., inhibits platelet aggregation early in blood clotting. Antiplatelet agents therefore interfere with the platelet activation cascade, which results in activated platelets that can attach to fibrin fibers, other extracellular matrix components, or aggregate into platelet aggregates. It is emphasized that the coagulation cascade and the platelet aggregation cascade are two separate cascades, even though some proteins, e.g., thrombin, play a role in both cascades. Antiplatelet drugs reversibly or irreversibly inhibit the processes involved in platelet activation, resulting in the tendency of platelets to adhere to each other and to damaged vascular endothelium, or to foreign surfaces, such as the material of blood samplers. This can result in a decrease in the tendency

[0097] As used herein, the term "blood sample" or "blood analysis sample" refers to a sample of blood suitable for diagnostic or analytical purposes. Thus, a blood sample includes a relatively small amount of blood (20 μL to 10 mL of blood), i.e., not including the amount required for, for example, a blood donation (up to about 450 mL of blood).

[0098] As used herein, the term "blood sampler" means a device for the collection of blood, such as a syringe, capillary tube or test tube, such as an aspirating or self-aspirating sampler, e.g., a PICO syringe (Radiometer Medical ApS), evacuated test tube or similar device designated for blood collection.

[0099] As used herein, the term "leukocyte count" refers to a diagnostic test that counts the number of white blood cells in a sample of a patient's blood. The average normal range is 3,500 to 10,500 white blood cells per μL of blood.

[0100] In general, the invention includes all salts of the disclosed reagents, such as antiplatelet or anticoagulant agents, so long as they do not interfere or do not substantially interfere with blood analysis. Examples of salts include inorganic and organic acid addition salts and base salts. Salts include, but are not limited to, metal salts such as cesium salts, alkali salts such as lithium, sodium, potassium, calcium or magnesium salts, organic amine salts such as triethylamine, pyridine, picoline, ethanolamine, triethanolamine, dicyclohexylamine, N,N'dibenzylethylenediamine, etc.; inorganic acid salts such as citrate, tartrate, maleate, fumarate, mandelate, acetate, dichloroacetate, trifluoroacetate, dichloroacetate, trifluoroacetate, oxalate, formate, etc.; sulfonates such as methanesulfonate, benzenesulfonate, p-toluenesulfonate, etc.; and amino acid salts such as alginate, glutamate, etc. Acid addition salts include, but are not limited to, hydrochloric acid, fumaric acid, maleic acid, succinic acid, acetic acid, citric acid, tartaric acid, phosphoric acid, oxalic acid, dichloroacetic acid, etc. EXAMPLES

[0101] Example 1 Analysis of platelet aggregation with heparin and different antiplatelet agents Each experiment was performed on a separate day using blood from one volunteer donor and tested one antiplatelet drug candidate. For each experiment, a PICO70 syringe sampler (Radiometer Medical ApS) was prepared immediately prior to sample withdrawal. For the "LiHep" (liquid heparin) condition, the PICO70 sampler was emptied of the gold ball and heparin brick, and 15 μl of aqueous liquid buffered heparin containing lithium heparin (Celsus Laboratories) and sodium heparin (Celsus Laboratories) (final heparin concentration 60 IU / mL blood) and 15 μl of the test drug's solvent were added. For the "PicoSAM" condition, an unmodified PICO70 sampler (with gold ball and heparin brick) was used, and 15 μl of the respective test drug's solvent was added. For the "LiHep xxx drug" condition, the PICO70 sampler was emptied as described for LiHep and 15 μl of liquid buffered heparin (final concentration of heparin 60 IU / mL blood) and 15 μl of dissolved test drug were added. For the "PicoSAM xxx drug" condition, an unmodified PICO70 sampler (with gold ball and heparin brick) was used and 15 μl of dissolved test drug was added. Antiplatelet drug candidates and corresponding vehicles tested were eptifibatide acetate (Sigma, SML1042; dissolved in saline, final concentrations 5 and 20 μM), MgSO4 ( The drugs were: Sigma, M7506; dissolved in saline, final concentrations 3 and 12 mM), tirofiban hydrochloride monohydrate (Sigma, SML0246; dissolved in DMSO in saline 1:200, final concentrations 0.5 and 1 μM), iloprost (Sigma, SML1651; dissolved in ethanol in saline 1:1000 or 1:10000, final concentrations 10 and 100 nM (later also 1 μM)), ticlopidine hydrochloride (dissolved in saline, final concentrations 60 and 600 μM), L-arginine (Sigma, A5006; dissolved in saline, final concentrations 600 μM and 6 mM) and dipyridamole (Sigma, D9766; dissolved in DMSO in saline 1:10 or 1:100, final concentrations 10 and 100 μM). Duplicate samples of all conditions in EDTA tubes (BD Vacutainer, with spray-coated K2EDTA, 10 ml) and PICO70 syringe samplers were filled by withdrawing venous blood through a butterfly needle with a sealed VTC (vented tip cap), and 1.5 mL of venous whole blood was filled into the otherwise self-aspirating PICO70 sampler. To ensure proper anticoagulation of the sample, the sampler was inverted 8 times immediately after withdrawal. Blood samples were mixed gently by hand (sampler without gold ball) or on a SAM mixer (Radiometer Medical ApS) for 15 minutes after sample withdrawal. Mixed samples were immediately fixed in 10% formalin solution for at least 10 minutes (1:1 dilution of blood in formalin) and diluted 1:10 in platelet diluent solution to lyse red blood cells (RBCs) and manually assessed for platelet count using a hemocytometer. Manual platelet counts of single platelets (unaggregated), number of platelet aggregates, and platelet aggregate size (number of platelets in an aggregate) when possible were quantified in duplicate in each sample by counting platelets on a hemocytometer using a Leica 750 microscope with 10x and 20x phase contrast air objectives. Single platelet counts were adjusted for dilution with liquid heparin and / or dissolved drug solution to calculate single platelet concentrations.

[0102] Eptifibatide, tirofiban and iloprost reduced the formation of platelet aggregates compared to the "LiHep" and "PicoSAM" controls (see FIG. 1). MgSO4 also reduced the formation of platelet aggregates (see Figure 2).

[0103] Ticlopidine LiHep showed similar single platelet counts compared to the EDTA reference and slightly reduced aggregate counts compared to the LiHep and PicoSAM controls (see Figure 3). L-Arginine showed reduced platelet counts and higher platelet aggregate counts compared to the EDTA control, but still showed slightly higher platelet counts than the LiHep and PicoSAM controls (see Figure 4).

[0104] Dipyridamole, a phosphodiesterase inhibitor and thromboxane inhibitor, showed slightly higher platelet counts compared to the LiHep and PicoSAM controls (see FIG. 5).

[0105] Example 2 Platelet aggregation test using heparinized blood and EDTA-treated blood Wet mounts were prepared on glass slides using venous blood samples drawn, mixed, and fixed in 10% formalin solution as previously described in Example 1, and covered with a coverslip. Fixed blood cell wet mount samples were then imaged on a Leica 750 microscope using a 40x phase contrast air objective. Aggregated platelets in heparinized blood versus single, non-aggregated platelets in EDTA-anticoagulated blood and heparinized blood containing, for example, eptifibatide, are seen among the abundant RBCs.

[0106] The results are shown in Figure 6. Heparinized blood showed no significant differences in platelet counts compared to blood samples prepared with EDTA and antiplatelet drugs such as heparin and eptifibatide. The results showed platelet aggregates that were not visible in the blood samples taken.

[0107] Example 3 Wet mount images of stained blood samples Venous blood samples were drawn and mixed as previously described in Example 1 and used to make stained wet mount images. Blood samples were mixed with staining agent and hemolytic agent (methylene blue and deoxycholic acid, respectively) and incubated in a water bath at 47°C for 30 seconds. Stained and hemolyzed blood samples were then prepared as wet mount samples on glass slides and covered with coverslips. Images of stained samples were then taken in bright field mode with a Leica 750 microscope using a 40x air objective. Image processing software (FIJI, ImageJ) was used to select representative stained WBC regions of interest (ROIs) from the images.

[0108] The PicoSAM sample (heparinized blood without antiplatelet drugs) showed many aggregated platelets (small rounded cells). PicoSAM samples containing 1 μM tirofiban or 20 μM eptifibatide showed single platelets, but also platelet satelliteism, i.e., platelets bound to WBCs, as shown, for example, in Figure 7A. Additionally, WBC aggregates were also observed, as shown in Figure 7B.

[0109] PicoSAM samples containing 100 nM iloprost (or samples containing buffered LiHep and 100 nM iloprost) showed single platelets and no platelet satelliteism or WBC aggregates. The same results were obtained using EDTA-anticoagulated blood.

[0110] Example 4 Single Platelet Concentration of Iloprost Heparin Samples Compared to EDTA Reference Sample Manual Platelet Counting The samples were prepared as described in Example 1.

[0111] ABX Platelet Count Blood samples mixed 15 min after drawing as described above were evaluated using an automated hematology analyzer (Horiba, ABX Pentra 60C+) (ABX) for complete blood count (CBC) with 5 categories including WBC concentration, platelet concentration, mean platelet volume (MPV), neutrophil, lymphocyte, monocyte, eosinophil and basophil concentration and percentage, RBC concentration, hematocrit, hemoglobin concentration, RBC descriptive parameters (MCV, MCH, MCHC, RDW). Samples were prepared from several donors. The measured platelet concentrations were used to compare the manually assessed platelet concentrations of iloprost heparin samples (in a PICO70 sampler) and samples anticoagulated with EDTA. For controls, EDTA samples without platelet aggregates were used as reference measurements for the measured automated ABX platelet concentrations.

[0112] For one donor, the average platelet count performance of the PicoSAM 100 nM iloprost sample compared to the EDTA sample as reference was 97% relative to manual counts.

[0113] For the eight donors, the average platelet count performance of the PicoSAM 1 μM iloprost samples compared to the EDTA samples as reference was 93% relative to manual counts. Each PicoSAM sample, ie, without iloprost but with heparin, in contrast, performed less than 40% compared to the EDTA reference.

[0114] PicoSAM 1 μM compared to EDTA sample as reference for 7 donors The average performance of the platelet count for the iloprost samples was 97% relative to the ABX platelet count.

[0115] Table 1 shows the platelet count results for donor samples in which both types of platelet counts (manual and ABX platelet counts) were performed.

[0116] [Table 1]

[0117] Example 5 Antiplatelet drug interference test on ABL90 parameters Each experiment was performed on a separate day with blood from one volunteer donor. For each experiment, three PICO70 syringe samplers (Radiometer Medical ApS) (unmodified, containing gold balls and heparin bricks) were prepared. One PICO70 was used without modification (control) and one was filled with 15 μl of dissolved antiplatelet drug to reach the final concentration in the blood sample indicated. The third PICO70 sampler was filled with 15 μl of solvent (specific solvent used to dissolve the tested antiplatelet drug: reference sample). Venous blood samples were drawn into the PICO70 syringe sampler via a butterfly needle and a sealed apertured tip cap (VTC), and the self-aspirating PICO70 sampler was filled with 1.5 mL of whole blood. To ensure proper heparinization of the sample, the sampler was inverted eight times immediately after withdrawal. Blood samples were mixed on a SAM mixer (Radiometer Medical ApS), a gold-coated steel ball was moved through the blood sample for 15 minutes after blood withdrawal to ensure uniform and reproducible mixing, and analyzed on an ABL90 blood gas analyzer (Radiometer Medical ApS). Samples were run five times each (five replicates) in rotation (control, reference, drug sample) with gentle manual sample inversion to keep the blood sample uniformly mixed throughout the run. All calculated values ​​are listed in Table 3. Blood samples were then centrifuged to separate the plasma fraction. Plasma from all samples was run in rotation in triplicate on the ABL90 instrument to assess the free hemoglobin concentration in the plasma, which indicates possible problematic hemolysis of red blood cells (RBCs) in the whole blood sample. Hemolyzed samples were analyzed using a 5-fold increase in the measured K + The interference with concentration is shown.

[0118] The measured parameters are listed in Table 2.

[0119] [Table 2]

[0120] The nomenclature of how the calculations have been made is given in Table 3, and the nomenclature of the samples that were measured is given in Table 4.

[0121] [Table 3]

[0122] [Table 4]

[0123] The reference ranges for adults and the maximum interference permitted in the examples are given in Table 5. The results of the measurements (as a percentage of maximum interference) are summarized in Table 6.

[0124] [Table 5]

[0125] [Table 6]

[0126] Eptifibatide, tirofiban and iloprost showed no interference with the evaluated ABL parameters, while MgSO4 significantly increased the activity of several parameters (pH, N + , K + , Ca 2+ , Cl - and Lac) showed interference (difference / maximum interference >2).

[0127] Therefore, eptifibatide, tirofiban and iloprost should be safe to add at the concentrations tested to heparinized blood used for the measurement of blood gas and basic metabolic panel parameters.

[0128] Example 6 Robustness of samples to pre-analytical stress A test of the robustness of the samples to stress conditions was carried out comparing blood samples containing only heparin (lithium heparin 18 IU / ml) and blood samples containing heparin (lithium heparin 18 IU / ml) + iloprost (1 microM).

[0129] Immediately after blood was drawn, blood from the vacutainer tubes was aspirated (1.5 ml) into a marked SafePicoAsp syringe, placed in a plastic bag, and allowed to emerge completely in ice cubes for 30 minutes. After 30 minutes, the syringe was removed and shaken back and forth in a fast longitudinal motion at approximately 4 / sec for 30 seconds. All syringes were mixed gently (slow inversion by twisting the wrist, approximately 1 / sec) before aspirating on the analyzer. The resulting samples were aspirated approximately 400 times each on nine ABL90 analyzers, and any instances of clots were recorded. All samples were aspirated from picosafe samplers. The results are shown in Table 7.

[0130] [Table 7]

[0131] The frequency of masses was 4-5 times higher in samples without iloprost. Further experiments were performed with larger blood volumes using 9 ml Greiner Bio-one Vaceutte vacuum tubes. Shortly after blood withdrawal, the vacutainer tubes were placed on ice for 30 minutes. The vacutainer tubes were then adjusted to ambient temperature and gently mixed before centrifugation at 1500G for 3 minutes to mimic sedimentation. After centrifugation, the blood was pooled and distributed into 20 ml syringes marked 2x3 and run on 10 ABL90 analyzers by robot. Samples were aspirated using the larger 20 ml sampler (robot). The results are shown in Table 8.

[0132] [Table 8]

[0133] Again, the frequency of masses was higher in samples without iloprost. This specification includes the disclosure of the following inventions. [Item 1] An in vitro method for determining blood gas parameters and / or basic metabolic panel (BMP) parameters in a blood sample, comprising the steps of: i) combining the blood sample with an anticoagulant and an antiplatelet agent; and ii) determining said blood gas parameters and / or BMP parameters in said sample; wherein said blood sample has been subjected to a pre-analytical stress prior to the determination in step ii), such as a stress caused by exposure to a temperature below 20° C., by contact with air and / or by shear forces. [Item 2] An in vitro method according to item 1, wherein step ii) is carried out in a sensor assembly comprising two or more analyte sensors. [Item 3] An in vitro method according to item 2, wherein the two or more analyte sensors are not all located in the same plane, and one of the analyte sensors analyzes the blood gas parameter or the BMP parameter, and another analyte sensor not located in the same plane analyzes a different blood gas parameter or BMP parameter. [Item 4] pO in blood samples 2 and pCO 2 1. An in vitro method for determining a blood gas parameter selected from the group consisting of: i) combining the blood sample with an anticoagulant and an antiplatelet agent; and ii) determining said blood gas parameters in said sample; and step ii) is performed on a sensor assembly including two or more analyte sensors, the two or more analyte sensors not all being located in the same plane. and wherein one of the analyte sensors analyzes the blood gas parameter and another analyte sensor, not disposed in the same plane, analyzes a different blood gas parameter or BMP parameter. [Item 5] An in vitro method according to item 4, wherein the blood sample is subjected to a pre-analytical stress, such as a stress caused by a temperature below 20°C, by contact with air and / or by shear forces, prior to the determination in step ii). [Item 6] An in vitro method according to any one of Items 1 to 5, wherein the blood sample is subjected to stress caused by exposure to a temperature of -5°C to 20°C, for example, a temperature of -5°C to 15°C, for example, a temperature of 0°C to 10°C, for example, stress caused by a temperature of 0°C to 5°C. [Item 7] The in vitro method according to any one of Items 1 to 6, wherein step ii) comprises the following steps: a) a first electronic wiring substrate having a first and a second surface and at least one analyte sensor formed on the first surface, wherein the at least one analyte sensor is connected to one or more electrical contacts; b) a second electronic wiring substrate having a first and a second surface and at least one analyte sensor formed on a portion of the first surface, wherein the at least one analyte sensor is connected to one or more electrical contacts; and c) a spacer having a through recess with first and second openings; wherein the first substrate, the second substrate and the spacer are arranged in a layered configuration, the first surface of the first substrate blocking the first opening of the spacer and the first surface of the second substrate blocking the second opening of the spacer, thereby forming a measurement cell, the measurement cell facing at least one analyte sensor from each of the substrates. [Item 8] An in vitro method according to any one of items 1 to 7, wherein the volume used for the determination in step ii) is less than 1 ml, for example less than 0.5 ml, for example less than 200 microliters, for example less than 100 microliters, for example less than 50 microliters, for example 2 to 50 microliters. [Item 9] The in vitro method according to any one of Items 1 to 8, wherein the anticoagulant is heparin. [Item 10] An in vitro method according to any one of Items 1 to 9, wherein the antiplatelet agent is selected from the group consisting of glycoprotein IIb / IIIa inhibitors, ADP receptor / P2Y12 inhibitors, prostaglandin analogues, COX inhibitors, thromboxane inhibitors, phosphodiesterase inhibitors, cloricromene, diazepam, vorapaxar, and combinations thereof. [Item 11] The in vitro method according to any one of Items 1 to 10, wherein the antiplatelet agent is iloprost. [Item 12] An in vitro method according to any one of Items 1 to 11, wherein step i) comprises mixing the blood sample. [Item 13] The in vitro method according to any one of items 1 to 3 or 6 to 12, wherein the method is for determining a blood gas parameter, and the blood gas parameter is pH, pCO 2 , pO 2 , oxygen saturation (sO 2 ), total hemoglobin concentration (ctHb), percentage of oxyhemoglobin (FO 2 Hb), percent carboxyhemoglobin (FCOHb), percent methemoglobin (FMetHb), percent deoxyhemoglobin (FHHb) and percent fetal hemoglobin (FHbF). [Item 14] The in vitro method according to any one of items 1 to 3 or 6 to 12, wherein the method is for determining a BMP parameter, and the BMP parameter is Na + 、K + , Mg 2+ 、Cl - , HCO 3 - , urea, creatinine, glucose, Ca2+ , lactate and total bilirubin. [Item 15] The in vitro method according to any one of Items 1 to 14, further comprising determining a platelet count and / or a white blood cell count in the sample obtained in step i). [Item 16] An in vitro method for determining blood gas parameters and / or BMP parameters in a blood sample, comprising the steps of: i) combining a blood sample with an anticoagulant and an antiplatelet agent; ii) exposing said blood sample to a temperature of less than 20° C.; and iii) determining said blood gas and / or BMP parameters in said sample; An in vitro method comprising: [Item 17] An in vitro method according to Item 16, comprising any one of the features of Items 2, 3, or 6 to 15. [Explanation of symbols]

[0134] 14 Flow Path

Claims

1. 1. An in vitro method for determining blood gas parameters and / or basic metabolic panel (BMP) parameters in a blood sample, comprising the steps of: i) combining the blood sample with an anticoagulant and an antiplatelet agent; and ii) determining said blood gas parameters and / or BMP parameters in said sample; wherein the blood sample has been subjected to a pre-analytical stress, such as stress caused by exposure to temperatures below 20°C, by contact with air and / or by shear forces, prior to the determination in step ii), and wherein step i) comprises mixing the blood sample.

2. 10. The in vitro method of claim 1, wherein step ii) is performed in a sensor assembly comprising two or more analyte sensors.

3. 3. The in vitro method of claim 2, wherein the two or more analyte sensors are not all located in the same plane, and one of the analyte sensors analyzes the blood gas parameter or the BMP parameter, and another analyte sensor not located in the same plane analyzes a different blood gas parameter or BMP parameter.

4. pO in blood sample ２ and pCO ２ 1. An in vitro method for determining a blood gas parameter selected from the group consisting of: i) combining the blood sample with an anticoagulant and an antiplatelet agent; and ii) determining the blood gas parameters in the sample; wherein step ii) is performed on a sensor assembly comprising two or more analyte sensors, the two or more analyte sensors not all located in the same plane, and one of the analyte sensors analyzes the blood gas parameter and another analyte sensor not located in the same plane analyzes a different blood gas parameter or BMP parameter.

5. 5. An in vitro method according to claim 4, wherein the blood sample has been subjected to pre-analytical stress, such as stress caused by a temperature below 20°C, by contact with air and / or by shear forces, prior to the determination in step ii).

6. 6. An in vitro method according to any one of claims 1 to 5, wherein the blood sample is subjected to stress caused by exposure to a temperature of -5°C to 20°C, such as a temperature of -5°C to 15°C, such as a temperature of 0°C to 10°C, such as a stress caused by a temperature of 0°C to 5°C.

7. 7. The in vitro method according to any one of claims 1 to 6, wherein step ii) comprises: a) a first electronic wiring substrate having first and second surfaces and at least one analyte sensor formed on the first surface, wherein the at least one analyte sensor is connected to one or more electrical contacts; b) a second electronic wiring substrate having first and second surfaces and at least one analyte sensor formed on a portion of the first surface thereof, wherein the at least one analyte sensor is connected to one or more electrical contacts; and c) a spacer having a through recess with first and second openings; wherein the first substrate, the second substrate, and the spacer are arranged in a layered structure, the first surface of the first substrate blocking the first opening of the spacer and the first surface of the second substrate blocking the second opening of the spacer, thereby forming a measurement cell, and the measurement cell facing at least one analyte sensor from each of the substrates.

8. 8. The in vitro method according to any one of claims 1 to 7, wherein the volume used for the determination in step ii) is less than 1 ml.

9. 9. The in vitro method according to any one of claims 1 to 8, wherein the anticoagulant is heparin.

10. 10. The in vitro method of any one of claims 1 to 9, wherein the antiplatelet agent is selected from the group consisting of glycoprotein IIb / IIIa inhibitors, ADP receptor / P2Y12 inhibitors, prostaglandin analogs, COX inhibitors, thromboxane inhibitors, phosphodiesterase inhibitors, cloricromene, diazol, vorapaxar, and combinations thereof.

11. 11. The in vitro method according to any one of claims 1 to 10, wherein the antiplatelet agent is a prostaglandin analogue.

12. An in vitro method according to any one of claims 1 to 11, wherein the antiplatelet agent is beraprost.

13. 13. An in vitro method according to any one of claims 1 to 3 or 6 to 12, wherein the method is for determining blood gas parameters, and the blood gas parameters are pH, pCO ２ , pO ２ , oxygen saturation (sO ２ ), total hemoglobin concentration (ctHb), percentage of oxyhemoglobin (FO ２ Hb), percent carboxyhemoglobin (FCOHb), percent methemoglobin (FMetHb), percent deoxyhemoglobin (FHHb), and percent fetal hemoglobin (FHbF).

14. 13. An in vitro method according to any one of claims 1 to 3 or 6 to 12, wherein the method is for determining a BMP parameter, and the BMP parameter is determined by measuring Na ＋ , K. ＋ , Mg ２＋ , Cl － , HCO ３ － , urea, creatinine, glucose, Ca ２＋ , lactate and total bilirubin.

15. 15. The in vitro method according to any one of claims 1 to 14, further comprising determining the platelet count and / or the white blood cell count in the sample obtained in step i).

16. 1. An in vitro method for determining blood gas parameters and / or BMP parameters in a blood sample, comprising the steps of: i) combining the blood sample with an anticoagulant and an antiplatelet agent; ii) exposing said blood sample to a temperature of less than 20°C; and iii) determining said blood gas parameters and / or BMP parameters in said sample; An in vitro method comprising:

17. An in vitro method according to claim 16, comprising the features of any one of claims 2, 3 or 6 to 15.

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