WO2014131784A1 - In vitro method, use of an agent and collection device for the inhibition of coagulation in blood - Google Patents

In vitro method, use of an agent and collection device for the inhibition of coagulation in blood Download PDF

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Publication number
WO2014131784A1
WO2014131784A1 PCT/EP2014/053691 EP2014053691W WO2014131784A1 WO 2014131784 A1 WO2014131784 A1 WO 2014131784A1 EP 2014053691 W EP2014053691 W EP 2014053691W WO 2014131784 A1 WO2014131784 A1 WO 2014131784A1
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WO
WIPO (PCT)
Prior art keywords
blood
acid
agent
mixed
collection device
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PCT/EP2014/053691
Other languages
French (fr)
Inventor
Emmanuel BISSÈ
Original Assignee
WESER-BISSÉ, Petra
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Publication date
Application filed by WESER-BISSÉ, Petra filed Critical WESER-BISSÉ, Petra
Priority to US14/770,576 priority Critical patent/US10969396B2/en
Priority to CA2901649A priority patent/CA2901649C/en
Priority to EP14706615.3A priority patent/EP2943796B1/en
Priority to ES14706615.3T priority patent/ES2675402T3/en
Priority to JP2015559487A priority patent/JP2016508613A/en
Publication of WO2014131784A1 publication Critical patent/WO2014131784A1/en
Priority to US17/164,080 priority patent/US20210231690A1/en

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Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61BDIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00Measuring for diagnostic purposes; Identification of persons
    • A61B5/15Devices for taking samples of blood
    • A61B5/150007Details
    • A61B5/150015Source of blood
    • A61B5/15003Source of blood for venous or arterial blood
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61BDIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00Measuring for diagnostic purposes; Identification of persons
    • A61B5/15Devices for taking samples of blood
    • A61B5/150007Details
    • A61B5/150343Collection vessels for collecting blood samples from the skin surface, e.g. test tubes, cuvettes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61BDIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00Measuring for diagnostic purposes; Identification of persons
    • A61B5/15Devices for taking samples of blood
    • A61B5/150007Details
    • A61B5/150755Blood sample preparation for further analysis, e.g. by separating blood components or by mixing
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61BDIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00Measuring for diagnostic purposes; Identification of persons
    • A61B5/15Devices for taking samples of blood
    • A61B5/150992Blood sampling from a fluid line external to a patient, such as a catheter line, combined with an infusion line; blood sampling from indwelling needle sets, e.g. sealable ports, luer couplings, valves
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61BDIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00Measuring for diagnostic purposes; Identification of persons
    • A61B5/15Devices for taking samples of blood
    • A61B5/153Devices specially adapted for taking samples of venous or arterial blood, e.g. with syringes

Definitions

  • the present invention relates to the field of in vitro inhibition of coagulation in blood.
  • the present invention relates to a method and a use of an agent for the inhibi- tion of coagulation in blood in vitro, and a blood collection device provided for said method and use.
  • Blood sampling and testing are routinely carried out for various diagnostic purposes.
  • the accurate and precise determination of blood constituents or parameters is oftentimes required to be able to draw valid and reliable diagnostic and prognostic conclusions .
  • results from diagnostic tests of blood can be strongly affected by preanalytical circumstances and conditions .
  • Coagulation and platelet aggregation in blood leading to clotting can occur when blood is withdrawn by puncturing blood ves- sels and when it is collected in and comes in contact with sample containers due to the activation of platelets and coagulation factors. Further, this process can result in for example lysis of cells and changes of cellular and extracellular concen- trations of blood constituents. Moreover, subsequent in vitro coagulation tests and blood count are precluded.
  • additives which inhibit blood and/or plasma from coagulation and clotting and which are generally called anticoagulants are commonly added to and mixed with blood immediately after sample collection.
  • anticoagulant refers to inhibitors of plasmatic blood clotting or coagulation, distinguishable from inhibitors of platelet aggregation. Both calcium ions and thrombin are required for the clotting process. Therefore, salts of ethylenediaminetetraacetic acid (EDTA salts) or citrate salts which can chelate calcium ions, and heparin salts which can inhibit thrombin activity are typically used as anticoagulants.
  • EDTA salts ethylenediaminetetraacetic acid
  • citrate salts which can chelate calcium ions
  • heparin salts which can inhibit thrombin activity
  • sodium, lithium or ammonium salt of heparin, K 2 EDTA, K 3 EDTA, Na 2 EDTA and trisodium citrate are commonly used as anticoagulants in diagnostic laboratory investigations (see e.g. WHO document "Use of Anticoagulants in Diagnostic Laborato- ry Investigations” (WHO/DIL/LAB/99.1 Rev. 2, 2002)).
  • Buffering and adjustment of H, for example in the physiological range may optionally be performed, for example for citrate by adding citric acid.
  • stabilizing agents may be comprised besides the anticoagulant. The above-mentioned anticoagulants are used to obtain whole blood samples for hematologic analyses
  • chelation of e.g. Ca 2+ , Mg 2+ or Zn 2+ can interfere with the analysis of these cations, and enzymes - also in subsequent as- says - whose structure/function depend on these ions can also be affected, especially when the divalent cations are strongly depleted by a high concentration of chelating anticoagulant.
  • the polyanionic heparin may inhibit metabolic or catalytic reactions.
  • the determination of several laboratory parame- ters by hematologic tests, coagulation tests as well as clinical chemistry analyses from a single sample is precluded. The need to provide several samples with different anticoagulants or different concentrations of anticoagulant entails extra time, cost and potential for errors in the preanalytical and analytical procedures.
  • the object is solved by the in vitro method according to claim 1, the use of a blood collection device according to claim 10, the use of an agent according to claim 12, the blood collection device according to claim 14 and the kit according to claim 15, while preferred embodiments are set forth in dependent claims and will be further described below.
  • the present invention in particular provides the following items including main aspects and preferred embodiments, which respectively alone and in combination particularly contribute to solving the above object and eventually provide additional advantages :
  • blood wherein blood is mixed after its withdrawal with an agent comprising as the only anticoagulant a substance provided as free acid having a pK a of ⁇ 0.9.
  • free acid having a pK a of ⁇ 0.9 denotes a substance provided as free carboxylic acid having a pK a of ⁇ 0.9.
  • concentration of said substance provided as free acid having a pK a of > 0.9 is at least 0.1 mmol/L of blood to be mixed with, preferably is in the range from 0.1 to 100 mmol/L of blood to be mixed with, more preferably in the range from 1 to 50 mmol/L of blood to be mixed with, even more preferably in the range from 2 to 32 mmol/L of blood to be mixed with, and most preferably in the range from 2 to 10 mmol/L of blood to be mixed with.
  • steps (b) to (d) are carried out at a temperature from 0 to 37°C, and wherein steps (b) and (c) are preferably carried out at room temperature .
  • step (b) is whole blood.
  • step (e) the determination of the amount of the at least one blood component is carried out using conventional physical, chemical, enzymatic and/or immunological methods, including combinations thereof.
  • citric acid is anhydrous citric acid and/or citric acid monohydrate, preferably is citric acid monohydrate.
  • step (a) wherein in step (a) the concentration of said substance provided as free acid having a pK a of ⁇ 0.9 is as set forth in item (6) .
  • a blood collection device wherein the agent as set forth in any one of items (1) - (4) , (6), (8), (9), (16) and (17) is provided in the device, and wherein the substance provided as free acid having a pK a of > 0.9 is the only provided anticoagulant to be mixed with blood.
  • a kit comprising:
  • test substance (s) for the determination of at least one blood component in collected blood.
  • a first aspect of the present invention is an in vitro method for the inhibition of coagulation in blood, wherein blood is mixed after its withdrawal with an agent comprising as the only anticoagulant a substance provided as free acid having a pK a of
  • the logarithmic constant pK a is equal to -logi 0 K a , wherein K a is the acid dissociation constant.
  • K a is the acid dissociation constant.
  • pK a here denotes the logarithmic constant for dissociation of the first proton, i.e. pK a i .
  • the substance provided as free ac- id has a pK a of ⁇ 1.4, more preferably has a pK a of ⁇ 2, even more preferably has a pK a of ⁇ 2.5 and most preferably has a pK a of > 3. It was surprisingly found that coagulation in blood could be effectively inhibited in vitro when blood was mixed with
  • a free acid substance having a pK a of ⁇ 0.9 with the mixing preferably occurring simultaneously with or immediately or expe- ditiously after sample collection, wherein the sample preferably is withdrawn or collected whole blood.
  • an efficient method is provided when free acid having a pK a of ⁇ 0.9 is provided as the only anticoagulant, i.e. the only substance provided for the purpose of and effective in inhibiting coagulation.
  • the free acid substance having a pK a of ⁇ 0.9 is a free carboxylic acid substance having a pK a of ⁇ 0.9.
  • a substance provided as free acid having a pK a of ⁇ 0.9 according to the invention preferably is an organic Bronsted acid having a pK a of ⁇ 0.9.
  • the substance provided as free acid having a pK a of > 0.9 is an organic Bronsted ac- id comprising at least one carboxyl group per molecule, preferably at least two carboxyl groups per molecule, more preferably at least three carboxyl groups per molecule, wherein in the acid as provided (all of) the carboxyl group (s) in the molecule is (are) protonated, that is to say the carboxyl group (s) is (are) not dissociated. It is understood that in the substance provided as free acid having a pK a of ⁇ 0.9 conjugate base may only be present as minor impurity.
  • carboxylate groups may only be present as minor impurity, preferably the substance is substantially free of carboxylate, more preferably the substance is free of carboxylate.
  • a carboxylate salt let alone the salt of the respectively given carboxylic acid, shall not be used as anticoagulant and is preferably entirely excluded from the agent, and especially salts of EDTA and citrate are excluded.
  • heparin salt is not provided as anticoagulant nor is it comprised in the agent.
  • inhibition of coagulation means that in a conventional automated analysis system a clot- detection-system detects same or fewer positive events, i.e. clots, compared to conventional anticoagulation sampling. More preferably, in a conventional automated analysis system a clot- detection-system detects considerably fewer events, i.e. clots, compared to conventional anticoagulation sampling.
  • the acidic protons from the provided free acid having a pK a of ⁇ 0.9 specifically and/or non-specifically (e.g. through electrostatics, hydrogen bonding, acid-base reaction and proton transfer, etc.) , but in a manner at least in part distinct from other cations, interact with blood components, such as proteins (e.g. enzymes) and membrane constituents (e.g. phospholipids), and reversibly or irreversibly interfere with the proper bio- molecular structure and function of said blood components.
  • proteins e.g. enzymes
  • membrane constituents e.g. phospholipids
  • the protons may suitably interact with proteins to effect particular structural and physicochemical changes and thus specifically affect protein function, for example of serine proteases and also in terms of interactions between protein complexes.
  • free carboxylic acid is preferably used furthermore the protons and the anionic carboxylates formed in the blood may interfere with the Ca 2+ - dependent association of coagulation factors and phospholipids. Overall an inhibitory effect on the enzymatic coagulation cascade is observed.
  • the term anticoagulant as used herein thus preferably denotes inhibitors of plasmatic blood clotting or co- agulation.
  • the agent consists of the sub- stance provided as free acid having a pK a of > 0.9, or the substance provided as free acid having a pK a of ⁇ 0.9 dissolved in a solvent.
  • the agent consists of a substance provided as free carboxylic acid having a pK a of ⁇ 0.9, or a substance provided as free carboxylic acid having a pK a of > 0.9 dissolved in a solvent. It is preferred that the agent contains the free acid having a pK a of ⁇ 0.9, preferably the free carboxylic acid, as the only acid.
  • the solvent is selected from the group consisting of an aqueous solution, water, and alcohol and mixtures of water and alcohol, more preferably is water or ethanol, and even more preferably is water.
  • Additional additive commonly used for preserving blood, wherein said additional additive is not an anti- coagulant and does not comprise acid, may optionally be comprised, for example an antiglycolytic agent.
  • At least one test is carried out for the determination of at least one blood component subsequent to mixing blood with the agent according to the present invention. More preferably, at least two tests are carried out for the determination of at least two blood components subsequent to mixing blood with the agent according to the present invention.
  • the present invention allows the determination of several laboratory parameters by hematologic tests, coagulation tests and/or clinical chemistry analyses from a single sample. This way the present method provides for effective anticoagulation while making the blood amenable to downstream diagnostic analyses in a generally applicable and broadly useful manner, in particular diagnostic analysis suitable to assess pathological and/or therapeutic implications.
  • the determination of the amount of the at least one blood component is preferably carried out using conventional physical, chemical, enzymatic and/or immunological methods, including combinations thereof.
  • substantially no cations preferably no cations other than protons from the free acid having a pK a of > 0.9, preferably the free carboxylic acid having a pK a of ⁇ 0.9, are added to the blood. Therefore, in this case contamination with and interference from other cations, including clinically relevant cations, is favourably avoided.
  • there are substantially no inorganic anions preferably no inorganic anions of mineral acid provided. Fur- thermore, hyperosmolarity and the lysis of blood cells are preferably essentially avoided, more preferably are avoided. Therefore, the determination of a multitude of laboratory parameters by hematologic, coagulation as well as clinical chemistry anal- yses from a single sample becomes advantageously possible, avoiding the detrimental effects of interfering ions.
  • Changes in the intracellular and extracellular ion concentra- tions can generally lead to hyperosmolarity and hemolysis.
  • a disturbance in the electrochemical balance between the inside and outside of cells may, for example, results in a change of the plasmatic chloride concentration, or the concentration of uric acid.
  • free carboxylic acid the extent of such a possible change depends on the number of carboxyl groups. Without wishing to be bound by theory, this may be caused by an increase of hydrogen ions in the extracellular fluid of the blood along with the presence of carboxylate ions generated.
  • hy- drogen ions may react with extracellular bicarbonate anions and the flow of ions from the outside to the inside of the cells, and vice versa, may be affected, resulting for example in a chloride ion shift.
  • chloride ions may be kept in the cells functioning as counterions for intracellular cations. In such situations hemolysis of the red blood cells may be induced, which in turn could negatively influence the determination of not only chloride ions but also of haemoglobin.
  • changes in osmolarity and the outflow of water from the blood cells could lead to a possible dilution effect and possible impairment of cellular integrity due to the desiccation of blood cells.
  • an ammonium salt NR 4 X and/or a pH modifying agent may be provided together with the anticoagulant, preferably concomitantly but alternatively also slightly time-displaced. This can provide significant further benefits in terms of stabilization and preservation of a blood sample and of further facilitating the reliable determination of blood components.
  • ammonium salt NR 4 X wherein each R independently is hydrogen, linear Ci-C 6 alkyl, branched C3-C6 alkyl, unsubstituted phenyl or substituted phenyl, and X is preferably halide and more preferably is chloride. Tetrame- thylammonium chloride and/or tetraethylammonium chloride are even more preferred, and tetramethylammonium chloride is particularly preferred.
  • the concentration of the ammonium salt NR 4 X is 0.01 to 100 mol/mL blood, preferably is 5 to 25 mol/mL blood, and more preferably is 15 to 20 pmol/mL blood. Tetramethylammonium chloride in a concentration of 15 to 20 mol/mL blood is particularly preferred.
  • pH modifying agent favourably does not interfere with the laboratory parameters to be tested.
  • organic amine is preferably used, more preferably triethanolamine , ethanola- mine and/or 2-amino-2-methylpropanol are used.
  • a pH modifying agent is added and free acid is provided in solution, preferably a pH value between 2.2 and 3.0 is set.
  • Inorganic bases such as sodium hydroxide are suitably avoided.
  • hemolysis can be detrimental to blood testing and diagnostics because, for example, mixing of plasma components with cellular components from the lysed cells can lead to spurious results for both plasma and cell analyses or even prevent such analyses altogether.
  • inhibiting hemolysis is advantageous for the reliable determination of blood components. This further improved stabilization and preservation of a blood sample can provide longer storage and a more reliable determination of blood components and thus improved diagnostics .
  • the concentration of the substance provided as free acid having a pK a of ⁇ 0.9 is preferably at least 0.1 mmol/L of blood to be mixed with, more preferably is in the range from 0.1 to 100 mmol/L of blood to be mixed with, even more preferably in the range from 1 to 50 mmol/L of blood to be mixed with, yet even more preferably in the range from 2 to 32 mmol/L of blood to be mixed with, and most preferably in the range from 2 to 10 mmol/L of blood to be mixed with.
  • the concentration of the substance provided as free acid having a pK a of ⁇ 0.9 is set such that after mixing of said substance with blood the blood sample exhibits a pH in the range from 6.0 to 7.4.
  • the amount of the substance provided as free acid having a pKa of ⁇ 0.9 is set such that if dissolved in a desired amount of water the obtained aqueous solution exhibits a pH of ⁇ 1.5.
  • the amount of the substance provided as free acid having a pKa of ⁇ 0.9 is set such that if dissolved in a desired amount of water the obtained aqueous solution exhibits a pH of ⁇ 1.5 and that when mixed with blood the blood sample exhibits a pH in the range from 6.0 to 7.4.
  • the free acid is provided such that its con- centration is adjusted to below 20 mmol/L of blood to be mixed with, preferably below 15 mmol/L of blood to be mixed with.
  • Particularly preferred is a concentration in the range from 2 to 10 mmol/L of blood to be mixed with. This allows to suitably adjust the pH value in the blood sample .
  • the substance provided as free acid having a pK a of ⁇ 0.9 is selected from the group consisting of citric acid, tri- carballylic acid, ethylenediaminetetraacetic acid, diethylene- triaminepentaacetic acid, benzenepentacarboxylic acid, mellitic acid, and tetrahydrofuran-2 , 3 , 4 , 5-tetracarboxylic acid or mixtures thereof, more preferably is citric acid, tricarballylic acid or ethylenediaminetetraacetic acid, and even more preferably is citric acid.
  • citric acid is anhydrous citric acid and/or citric acid monohydrate, and citric acid mon- ohydrate is most preferred.
  • the substance provided as free acid having a pK a of ⁇ 0.9 is preferably provided as a powder or in lyophilized form.
  • the amount of at least one blood component is determined by comprising the following steps: a blood collection device is provided which contains, placed in the device, the agent according to the invention, and optionally further contains an ammonium salt NR 4 X and/or a pH modifying agent; blood, preferably whole blood, is placed in said blood collection device; said agent is mixed with blood in the blood collection device; optionally the blood is stored in the blood collection device for a desired period of time, for example until assigned tests are carried out; and the amount of the at least one blood component in the blood sample is determined.
  • placing of the blood in the blood collection device, mixing and the optional storage are carried out at a temperature from 0 to 37°C, and placing of the blood in the blood collection device and mixing more preferably are carried out at room temperature.
  • a step may be carried out which comprises placing blood in a blood collection device and subsequently placing the agent in the blood collection device.
  • an ammonium salt NR 4 X and/or a pH modifying agent may be added.
  • the amounts of at least two blood components are determined.
  • Another aspect of the invention is the use of a blood collection device for collecting and optionally storing blood in vitro, wherein in the device blood is mixed after its withdrawal with the agent according to the present invention, and wherein coagulation in blood is thereby inhibited.
  • the agent which comprises as the only antico- agulant a substance provided as free acid having a pK a of ⁇ 0.9.
  • blood is collected in a single device and mixed with the agent according to the invention and is subsequently subjected to at least one test for the determination of at least one blood component, preferably to multiple tests comprising at least one hematologic test, at least one coagulation test and at least one further clinical chemistry analysis.
  • the number of collection tubes required for the different fields of laboratory medicine can be considerably reduced, which in turn allows to optimize and render more efficient laboratory activity and workflow and to reduce sample volume and turnaround time.
  • the use of only a single effective anticoagulant for the different fields in clinical laboratories, i.e. hematology, clinical chemistry and hemostase- ology can expedite the process in emergency situations.
  • the invention relates to the use of an agent comprising as the only anticoagulant a substance provided as free acid having a pK a of ⁇ 0.9 for effecting inhibition of coagulation of blood in vitro.
  • Another aspect of the invention is a blood collection device, wherein the agent according to the present invention is provided in the device, and wherein the substance provided as free acid having a pK a of ⁇ 0.9 is the only provided anticoagulant to be mixed with blood.
  • a device is comprised which is capable of being connected with a conventional blood withdrawal device.
  • Conventional blood collection tubes including evacuated blood collection tubes such as vacutainer and aspiration systems such as monovette are known in the art.
  • the present invention can provide further economic advantages with respect to the manufacture of blood collection tubes as well as stocking logistics of collections tubes at the manufacturers and clinical laboratories .
  • kits which comprises the blood collection device of the present invention and test substance (s) for the determination of at least one blood component in collected blood.
  • Citric acid monohydrate (CA) tricarballylic acid (TCA) , eth- ylenediaminetetraacetic acid (EDTA) , diethylenetriaminepent- aacetic acid (DTPA) , mellitic acid (MELA) , and benzenepentacar- boxylic acid (BPCA) were from Sigma-Aldrich, Germany.
  • Tetrahy- drofuran-2, 3 , 4 , 5 -tetracarboxylic acid (THTCA) was from TCI, Germany.
  • Pristine blood collection tubes i.e.
  • tubes as originally provided not containing or respectively not filled or coated with additive were from KABE Labortechnik GmbH, Germany, denoted KABEVETTE (GR) .
  • KABEVETTE GR
  • pristine tubes were respectively placed: 5.4 mmol of CA per L of blood to be mixed with, 31.2 mmol of TCA per L of blood to be mixed with, 2.3 mmol of EDTA per L of blood to be mixed with, 2.2 mmol of DTPA per L of blood to be mixed with, 3.1 mmol of MELA per L of blood to be mixed with, 4.6 mmol of THTCA per L of blood to be mixed with, and 8.0 mmol of BPCA per L of blood to be mixed with.
  • GR KABEVETTE
  • Blood was collected from the antecubital vein from volunteers who had previously given their informed consent. For each subject, freshly drawn blood was collected into the corresponding tubes such that respectively a blood sample according to the present invention, a serum sample (SST (BD) ) , a citrate blood sample (citrate (BD) ) , and a potassium EDTA blood sample
  • SST serum sample
  • BD citrate blood sample
  • potassium EDTA potassium EDTA blood sample
  • K 3 EDTA (BD) were obtained. Tubes were filled with blood completely to the mark (4.9 mL) , and for tubes containing anticoagulant the blood was mixed well with the respective anticoagulant by inverting the tubes 4 times immediately after blood collection. Serum samples were treated according to the protocol of the manufacturer and used for biochemical analysis. Whole blood potassium EDTA ( K3 EDTA (BD) ) and inventive samples were used first for hematologic determinations and were then centrifuged. The inventive and citrate plasma samples (citrate (BD) ) were used for both haemostasis and biochemical determinations. Potassium EDTA plasma was also used for biochemical determinations. It was found that coagulation was effectively inhibited in the tubes according to the present invention.
  • Example 1 shows results for various free acids having a pK a of ⁇ 0.9, in particular free carboxylic acids, according to the invention.
  • WBC white blood cells
  • RBC red blood cells
  • Hb hemoglobin
  • Pit platelets
  • Lympho lymphocytes
  • Neutro neutrophils
  • Mono monocytes
  • Eos eosinophils
  • Baso basophils.
  • Example 1 Example 1
  • Example 1 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using citrate (in Bec- ton Dickinson tubes (BD) ) for the determination of activated partial thromboplastin time (APTT) and prothrombin time (PT) .
  • Example 2 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and conventional sampling using K 3 EDTA (in Becton Dickinson tubes (BD) ) for the determination of white blood cells (WBC) .
  • Example 3 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and conventional sampling using K 3 EDTA (in Becton Dickinson tubes (BD) ) for the determination of white blood cells (WBC) .
  • Example 3 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and conventional sampling using K 3 EDTA (in Becton Dickinson tubes (BD) ) for the determination of white blood cells (WBC) .
  • Example 3 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and conventional sampling using K 3 EDTA (in Becton Dickinson tubes (BD) ) for the determination of lymphocytes (LYM- PHO) .
  • Example 4 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using K 3 EDTA (in Becton Dickinson tubes (BD) ) for the determination of basophils (Baso Etude) and monocytes (MONO) .
  • Example 5 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and conventional sampling using K 3 EDTA (in Becton Dickinson tubes (BD) ) for the determination of eosinophils
  • Example 6 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using K3EDTA (in Becton Dickinson tubes (BD) ) for the determination of neutrophils (NEU- TRO) .
  • Example 7 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using K3EDTA (in Becton Dickinson tubes (BD) ) for the determination of haemoglobin (Hb) .
  • Example 8 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and conventional sampling using K3EDTA (in Becton Dickinson tubes (BD) ) for the determination of red blood cells (RBC) .
  • Example 9 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and conventional sampling using citrate (in Becton Dickinson tubes) for the determination of fibrinogen (FIB ETUDE) and a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using K 3 EDTA (in Becton Dickinson tubes (BD) ) for the determination of platelets (Ptl) .
  • FIB ETUDE fibrinogen
  • K 3 EDTA in Becton Dickinson tubes
  • Example 10 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and sampling using conventional serum samples (SST (BD) ) for the determination of cholesterol (CHOL) .
  • Example 11 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and sampling using conventional serum samples (SST (BD) ) for the determination of creatinine (CREA) .
  • Example 12 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and sampling using conventional serum samples (SST (BD) ) for the determination of iron (Fe) .
  • Example 13 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and sampling using conventional serum samples (SST (BD) ) for the determination of ferritin (Fer) .
  • Example 14 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and sampling using conventional serum samples (SST (BD) ) for the determination of ferritin (Fer) .
  • Example 14 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and sampling using conventional serum samples (SST (BD) ) for the determination of triiodothyronine, free (FT3) .
  • Example 15 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and sampling using conventional serum samples (SST (BD) ) for the determination of glucose (Glue) .
  • Example 16 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and sampling using conventional serum samples (SST (BD) ) for the determination of aspartate aminotransferase (AST) .
  • Example 17 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and sampling using conventional serum samples (SST (BD) ) for the determination of alanine aminotransferase (ALT) .
  • Example 18 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and sampling using conventional serum samples (SST (BD) ) for the determination of alanine aminotransferase (ALT) .
  • SST serum samples
  • Example 18 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using BD tubes for the determination of magnesium (Mg ETUDE) and calcium (Ca ETUDE) .
  • Example 19 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and sampling using conventional serum samples (SST (BD) ) for the determination of alkaline phosphatase (ALP) .
  • Example 20 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and sampling using conventional serum samples (SST (BD) ) for the determination of triglycerides (TG) .
  • Example 21 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using BD tubes for the determination of thyrotropin (TSH) and thyroxine, free (FT4 ETUDE) .
  • Example 22 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using BD tubes for the determination of C-reactive protein (CRP ETUDE) .
  • Example 23 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using BD tubes for the determination of ⁇ -glutamyltransferase (GGT ETUDE) .
  • Example 24 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using BD tubes for the determination of ⁇ -glutamyltransferase (GGT ETUDE) .
  • Example 24 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and conventional sampling using BD tubes for the determination of potassium (K ETUDE) .
  • Example 25 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using BD tubes for the determination of sodium (Na ETUDE) .
  • Example 26 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection ABEVETTE tubes (GR 15)) and conventional sampling using BD tubes for the determination of albumin (Alb ETUDE) .
  • Example 27 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection ABEVETTE tubes (GR 15)) and conventional sampling using BD tubes for the determination of albumin (Alb ETUDE) .
  • Example 27 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and sampling using conventional serum samples (SST (BD) ) for the determination of total protein (TP) .
  • HCT hematocrit
  • MCV mean corpuscular volume
  • MCH mean corpuscular hemoglobin
  • MCHC mean corpuscular hemoglobin concentration
  • Hb haemoglobin
  • Example 29A shows a comparison between sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR15)) and conventional sampling using K3EDTA (in Becton Dick- inson tubes (BD) ) for the determination of haemoglobin (Hb) .
  • the citric acid solution contained no alkylammonium compound and the pH 1.8 was not adjusted. See Figure 1 for results.
  • Example 29B shows a comparison between sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (HCT) ) and conventional sampling using K 3 EDTA (in Becton Dickinson tubes (BD) ) for the determination of haemoglobin (Hb) .
  • the citric acid solution contained alkylammonium salt, in particular tetramethylammonium chloride in a concentration of 16 ⁇ /ml of blood, and the pH was adjusted to 2.4. See Figure 1 for results.
  • Example 3OA shows a comparison between sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR15) ) and conventional sampling using K 3 EDTA (in Becton Dickinson tubes (BD) ) for the determination of chloride ions (CI) .
  • the citric acid solution contained no alkylammonium compound and the pH 1.8 was not adjusted. See Figure 2 for results.
  • Example 3 OB shows a comparison between sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (HCT) ) and conventional sampling using K 3 EDTA (in Becton Dickinson tubes (BD) ) for the determination of chloride ions (Cl) .
  • the citric acid solution contained alkylammonium salt, in particular tetramethylammonium chloride in a concentration of 16 ⁇ / ⁇ of blood, and the pH was adjusted to 2.4. See Figure 2 for results.

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Abstract

The present invention relates to a method and a use of an agent for the in vitro inhibition of coagulation in blood and a blood collection device provided for said method and use.

Description

In vitro method, use of an agent and collection device for the inhibition of coagulation in blood Field of the Invention
The present invention relates to the field of in vitro inhibition of coagulation in blood. In particular, the present invention relates to a method and a use of an agent for the inhibi- tion of coagulation in blood in vitro, and a blood collection device provided for said method and use.
Background of the Invention Blood sampling and testing are routinely carried out for various diagnostic purposes. The accurate and precise determination of blood constituents or parameters is oftentimes required to be able to draw valid and reliable diagnostic and prognostic conclusions . In this respect results from diagnostic tests of blood can be strongly affected by preanalytical circumstances and conditions . In many cases it is desirable to maintain blood after withdrawal in a substantially physiologically native state. Depending on the particular situation and the type(s) of (extra) cellular component (s) and constituent (s) to be determined, it may be necessary to use whole blood, or also preferable to use plasma samples instead of serum.
Coagulation and platelet aggregation in blood leading to clotting can occur when blood is withdrawn by puncturing blood ves- sels and when it is collected in and comes in contact with sample containers due to the activation of platelets and coagulation factors. Further, this process can result in for example lysis of cells and changes of cellular and extracellular concen- trations of blood constituents. Moreover, subsequent in vitro coagulation tests and blood count are precluded. In order to prepare and provide whole blood and plasma samples for in vitro diagnostic investigations additives which inhibit blood and/or plasma from coagulation and clotting and which are generally called anticoagulants are commonly added to and mixed with blood immediately after sample collection. More specifically, the term anticoagulant refers to inhibitors of plasmatic blood clotting or coagulation, distinguishable from inhibitors of platelet aggregation. Both calcium ions and thrombin are required for the clotting process. Therefore, salts of ethylenediaminetetraacetic acid (EDTA salts) or citrate salts which can chelate calcium ions, and heparin salts which can inhibit thrombin activity are typically used as anticoagulants. For a general description of the processes and components involved in coagulation and primary and secondary hemostasis, comprising the coagulation cascade and the intrinsic and extrinsic pathway, it is referred to "Textbook of Biochemistry with Clinical Correlations", 5th edition, T. M. Devlin (ed.), Wiley, 2002.
In particular, sodium, lithium or ammonium salt of heparin, K2EDTA, K3EDTA, Na2EDTA and trisodium citrate are commonly used as anticoagulants in diagnostic laboratory investigations (see e.g. WHO document "Use of Anticoagulants in Diagnostic Laborato- ry Investigations" (WHO/DIL/LAB/99.1 Rev. 2, 2002)). Buffering and adjustment of H, for example in the physiological range, may optionally be performed, for example for citrate by adding citric acid. Moreover, stabilizing agents may be comprised besides the anticoagulant. The above-mentioned anticoagulants are used to obtain whole blood samples for hematologic analyses
(e.g. full blood cell counts and white blood cell differential analysis) or to obtain plasma samples for hemostasis and clinical chemistry analyses . However, for diagnostic applications these typically used anticoagulants have certain drawbacks and shortcomings due to the interference with certain analytical methods and the changing of the concentration of certain constituents to be measured. For example, sample contamination with clinically relevant cations such as Na+, Li+, K+ and NH4 + from these anticoagulants, which are added in comparatively high concentrations, can be problematic in view of determination of these ions as well as hyperos- molarity/change in osmolarity. The latter can lead to water outflow from cells, loss of cellular integrity, ion leakage and thus also to interference in subsequent analyte testing. Likewise chelation of e.g. Ca2+, Mg2+ or Zn2+ can interfere with the analysis of these cations, and enzymes - also in subsequent as- says - whose structure/function depend on these ions can also be affected, especially when the divalent cations are strongly depleted by a high concentration of chelating anticoagulant. The polyanionic heparin may inhibit metabolic or catalytic reactions. Moreover, the determination of several laboratory parame- ters by hematologic tests, coagulation tests as well as clinical chemistry analyses from a single sample is precluded. The need to provide several samples with different anticoagulants or different concentrations of anticoagulant entails extra time, cost and potential for errors in the preanalytical and analytical procedures.
There is a need in the art to simplify the inhibition of coagulation in blood during preanalytics and to make it more robust and economical while enabling the subsequent testing of blood constituents and providing benefits in terms of sample handling, storage, transport and throughput and, as far as possible, accuracy, without however affecting anticoagulation efficacy. Summary of the Invention
The object is solved by the in vitro method according to claim 1, the use of a blood collection device according to claim 10, the use of an agent according to claim 12, the blood collection device according to claim 14 and the kit according to claim 15, while preferred embodiments are set forth in dependent claims and will be further described below. The present invention in particular provides the following items including main aspects and preferred embodiments, which respectively alone and in combination particularly contribute to solving the above object and eventually provide additional advantages :
(1) An in vitro method for the inhibition of coagulation in
blood, wherein blood is mixed after its withdrawal with an agent comprising as the only anticoagulant a substance provided as free acid having a pKa of ≥ 0.9.
(2) The method according to item (1), wherein the substance
provided as free acid having a pKa of ≥ 0.9 denotes a substance provided as free carboxylic acid having a pKa of ≥ 0.9.
(3) The method according to item (1) or (2) , wherein said sub- stance provided as free acid having a pKa of > 0.9 comprises at least two carboxyl groups per molecule, preferably comprises at least three carboxyl groups per molecule.
(4) The method according to any one of the preceding items, wherein said agent consists of
(i) said substance provided as free acid having a pKa of ≥
0.9; or
(ii) said substance provided as free acid having a pKa of ≥ 0.9 dissolved in a solvent. Optionally, no any further substance than the specified agent is mixed with the withdrawn blood.
(5) The method according to any one of the preceding items,
wherein subsequently at least one test is carried out for the determination of at least one blood component, preferably at least two tests are carried out for the determination of at least two blood components.
(6) The method according to any one of the preceding items,
wherein the concentration of said substance provided as free acid having a pKa of > 0.9 is at least 0.1 mmol/L of blood to be mixed with, preferably is in the range from 0.1 to 100 mmol/L of blood to be mixed with, more preferably in the range from 1 to 50 mmol/L of blood to be mixed with, even more preferably in the range from 2 to 32 mmol/L of blood to be mixed with, and most preferably in the range from 2 to 10 mmol/L of blood to be mixed with.
(7) The method according to any one of the preceding items, wherein the lysis of blood cells is essentially avoided.
(8) The method according to any one of the preceding items, wherein said substance provided as free acid having a pKa of ≥ 0.9 is provided as a powder or in lyophilized form.
(9) The method according to any one of items (4) to (8) , where¬ in in case (ii) the solvent is selected from the group consisting of an aqueous solution, water, and alcohol and mix- tures of water and alcohol, preferably is water or ethanol, and more preferably is water.
(10) The method according to any one of the preceding items, wherein the withdrawn blood is whole blood.
(11) The method according to any one of items (1) to (9) , where- in the amount of at least one blood component is determined, preferably the amounts of at least two blood compo¬ nents are determined, by comprising the following steps: (a) providing a blood collection device containing, placed in the device, the agent as set forth in any one of items (l)-(4), (6), (8) and (9), optionally further containing a pH modifying agent and/or an ammonium salt NR4X, wherein each R independently is hydrogen, linear Ci-Cs alkyl, branched C3-C6 alkyl, unsubstituted phenyl or substituted phenyl, and X is halide and preferably is chloride;
(b) placing blood in the blood collection device;
(c) mixing said agent with blood in the blood collection device;
optionally (d) storing the blood in the blood collection device for a desired period of time; and
(e) determining the amount of the at least one blood component, preferably of the at least two blood components, in the blood sample.
(12) The method according to item (11) , wherein steps (b) to (d) are carried out at a temperature from 0 to 37°C, and wherein steps (b) and (c) are preferably carried out at room temperature .
(13) The method according to item (11) or (12) , wherein, instead of steps (a) and (b) , a step is carried out which comprises placing blood in a blood collection device and subsequently placing the agent in the blood collection device.
(14) The method according to any one of items (11) to (13) ,
wherein the blood in step (b) is whole blood.
(15) The method according to any one of items (11) to (14) ,
wherein in step (e) the determination of the amount of the at least one blood component is carried out using conventional physical, chemical, enzymatic and/or immunological methods, including combinations thereof.
(16) The method according to any one of the preceding items, wherein said substance provided as free acid having a pKa of ≥ 0.9 is selected from the group consisting of citric acid, tricarballylic acid, ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, benzenepentacarboxylic acid, mellitic acid, and tetrahydrofuran-2 , 3 , , 5- tetracarboxylic acid or mixtures thereof, preferably is citric acid, tricarballylic acid or ethylenediaminetetraacetic acid, and more preferably is citric acid.
(17) The method according to item (16), wherein citric acid is anhydrous citric acid and/or citric acid monohydrate, preferably is citric acid monohydrate.
(18) The method according to any one of items (11) to (17),
wherein in step (a) the concentration of said substance provided as free acid having a pKa of ≥ 0.9 is as set forth in item (6) .
(19) Use of a blood collection device for collecting and option- ally storing blood in vitro, wherein in the device blood is mixed after its withdrawal with the agent as set forth in any one of items (l)-(4), (6), (8), (9), (16) and (17), and wherein coagulation in blood is thereby inhibited.
(20) The use according to item (19), wherein blood collected in a single device and mixed with said agent is subsequently subjected to at least one test for the determination of at least one blood component, preferably to multiple tests comprising at least one hematologic test, at least one coagulation test and at least one further clinical chemistry analysis.
(21) Use of an agent comprising as the only anticoagulant a substance provided as free acid having a pKa of ≥ 0.9 for effecting inhibition of coagulation of blood in vitro.
(22) The use according to item (21), wherein the agent is as set forth in any one of items (2) -(4), (6), (8), (9), (16) and
(17) .
(23) A blood collection device, wherein the agent as set forth in any one of items (1) - (4) , (6), (8), (9), (16) and (17) is provided in the device, and wherein the substance provided as free acid having a pKa of > 0.9 is the only provided anticoagulant to be mixed with blood.
(24) The blood collection device according to item (23), com- prising a device which is capable of being connected with a conventional blood withdrawal device.
(25) A kit comprising:
the blood collection device according to item (23) or (24), and
test substance (s) for the determination of at least one blood component in collected blood.
Detailed Description of the Invention In the following, the present invention is described in more detail while referring to preferred embodiments and examples, which are presented however for illustrative purposes and shall not be construed to limit the invention in any way. A first aspect of the present invention is an in vitro method for the inhibition of coagulation in blood, wherein blood is mixed after its withdrawal with an agent comprising as the only anticoagulant a substance provided as free acid having a pKa of
> 0.9.
The logarithmic constant pKa is equal to -logi0Ka, wherein Ka is the acid dissociation constant. For polyprotic acids pKa here denotes the logarithmic constant for dissociation of the first proton, i.e. pKai . Preferably the substance provided as free ac- id has a pKa of ≥ 1.4, more preferably has a pKa of ≥ 2, even more preferably has a pKa of ≥ 2.5 and most preferably has a pKa of > 3. It was surprisingly found that coagulation in blood could be effectively inhibited in vitro when blood was mixed with
a free acid substance having a pKa of ≥ 0.9, with the mixing preferably occurring simultaneously with or immediately or expe- ditiously after sample collection, wherein the sample preferably is withdrawn or collected whole blood. In particular, according to the present invention an efficient method is provided when free acid having a pKa of ≥ 0.9 is provided as the only anticoagulant, i.e. the only substance provided for the purpose of and effective in inhibiting coagulation. In a preferred embodiment the free acid substance having a pKa of ≥ 0.9 is a free carboxylic acid substance having a pKa of ≥ 0.9.
Optionally, use of any substance other than the specified anti- coagulant agent to be mixed with the withdrawn blood can be omitted. A substance provided as free acid having a pKa of ≥ 0.9 according to the invention preferably is an organic Bronsted acid having a pKa of ≥ 0.9. In an embodiment the substance provided as free acid having a pKa of > 0.9 is an organic Bronsted ac- id comprising at least one carboxyl group per molecule, preferably at least two carboxyl groups per molecule, more preferably at least three carboxyl groups per molecule, wherein in the acid as provided (all of) the carboxyl group (s) in the molecule is (are) protonated, that is to say the carboxyl group (s) is (are) not dissociated. It is understood that in the substance provided as free acid having a pKa of ≥ 0.9 conjugate base may only be present as minor impurity. Accordingly in an embodiment where said substance is provided as free carboxylic acid carboxylate groups may only be present as minor impurity, preferably the substance is substantially free of carboxylate, more preferably the substance is free of carboxylate. Besides such possible impurity, according to the present invention a carboxylate salt, let alone the salt of the respectively given carboxylic acid, shall not be used as anticoagulant and is preferably entirely excluded from the agent, and especially salts of EDTA and citrate are excluded. Furthermore, heparin salt is not provided as anticoagulant nor is it comprised in the agent. Considering that conventionally salts of EDTA and citrate as well as heparin salts are used as anticoagulant, it is unexpected that a substance provided as free acid having a pKa of ≥ 0.9, preferably a substance provided as free carboxylic acid having a pKa of ≥ 0.9, when so mixed in vitro with blood after its withdrawal, ex- hibits an inhibitory effect against coagulation. Inhibition of coagulation according to the present invention means that, instead of coagulating, blood after its withdrawal and careful mixing with the inventive agent remains fluid. Furthermore, late or delayed clotting can be advantageously avoided. Preferably, inhibition of coagulation according to the present invention means that in a conventional automated analysis system a clot- detection-system detects same or fewer positive events, i.e. clots, compared to conventional anticoagulation sampling. More preferably, in a conventional automated analysis system a clot- detection-system detects considerably fewer events, i.e. clots, compared to conventional anticoagulation sampling.
Without wishing to be bound by theory, it is thought that in the blood the acidic protons from the provided free acid having a pKa of ≥ 0.9 specifically and/or non-specifically (e.g. through electrostatics, hydrogen bonding, acid-base reaction and proton transfer, etc.) , but in a manner at least in part distinct from other cations, interact with blood components, such as proteins (e.g. enzymes) and membrane constituents (e.g. phospholipids), and reversibly or irreversibly interfere with the proper bio- molecular structure and function of said blood components. Considering the specific size and the charge, the protons may suitably interact with proteins to effect particular structural and physicochemical changes and thus specifically affect protein function, for example of serine proteases and also in terms of interactions between protein complexes. When free carboxylic acid is preferably used furthermore the protons and the anionic carboxylates formed in the blood may interfere with the Ca2+- dependent association of coagulation factors and phospholipids. Overall an inhibitory effect on the enzymatic coagulation cascade is observed. The term anticoagulant as used herein thus preferably denotes inhibitors of plasmatic blood clotting or co- agulation. In the present invention complex formation via chelation of Ca2+ with the anticoagulant is believed to play, if at all, at most only a minor role. It has been found that when free carboxylic acid having a pKa of > 0.9 is used as the only anticoagulant the measured free Ca2+ plasma concentration can be similar to and slightly higher than in the corresponding serum, while Ca2+ is almost completely depleted in plasma with salts of EDTA and citrate (see also Example 18) .
In one embodiment of the method the agent consists of the sub- stance provided as free acid having a pKa of > 0.9, or the substance provided as free acid having a pKa of ≥ 0.9 dissolved in a solvent. In a preferred embodiment of the method the agent consists of a substance provided as free carboxylic acid having a pKa of ≥ 0.9, or a substance provided as free carboxylic acid having a pKa of > 0.9 dissolved in a solvent. It is preferred that the agent contains the free acid having a pKa of ≥ 0.9, preferably the free carboxylic acid, as the only acid. Preferably the solvent is selected from the group consisting of an aqueous solution, water, and alcohol and mixtures of water and alcohol, more preferably is water or ethanol, and even more preferably is water. Additional additive commonly used for preserving blood, wherein said additional additive is not an anti- coagulant and does not comprise acid, may optionally be comprised, for example an antiglycolytic agent.
According to an aspect of the invention preferably at least one test is carried out for the determination of at least one blood component subsequent to mixing blood with the agent according to the present invention. More preferably, at least two tests are carried out for the determination of at least two blood components subsequent to mixing blood with the agent according to the present invention. Advantageously the present invention allows the determination of several laboratory parameters by hematologic tests, coagulation tests and/or clinical chemistry analyses from a single sample. This way the present method provides for effective anticoagulation while making the blood amenable to downstream diagnostic analyses in a generally applicable and broadly useful manner, in particular diagnostic analysis suitable to assess pathological and/or therapeutic implications. The determination of the amount of the at least one blood component is preferably carried out using conventional physical, chemical, enzymatic and/or immunological methods, including combinations thereof. In a preferred embodiment, besides minor impurity, substantially no cations, preferably no cations other than protons from the free acid having a pKa of > 0.9, preferably the free carboxylic acid having a pKa of ≥ 0.9, are added to the blood. Therefore, in this case contamination with and interference from other cations, including clinically relevant cations, is favourably avoided. Moreover, in said preferred embodiment, besides minor impurity, there are substantially no inorganic anions, preferably no inorganic anions of mineral acid provided. Fur- thermore, hyperosmolarity and the lysis of blood cells are preferably essentially avoided, more preferably are avoided. Therefore, the determination of a multitude of laboratory parameters by hematologic, coagulation as well as clinical chemistry anal- yses from a single sample becomes advantageously possible, avoiding the detrimental effects of interfering ions.
Changes in the intracellular and extracellular ion concentra- tions can generally lead to hyperosmolarity and hemolysis. In principle when free acid is used as anticoagulant a disturbance in the electrochemical balance between the inside and outside of cells may, for example, results in a change of the plasmatic chloride concentration, or the concentration of uric acid. In the case of free carboxylic acid the extent of such a possible change depends on the number of carboxyl groups. Without wishing to be bound by theory, this may be caused by an increase of hydrogen ions in the extracellular fluid of the blood along with the presence of carboxylate ions generated. In particular, hy- drogen ions may react with extracellular bicarbonate anions and the flow of ions from the outside to the inside of the cells, and vice versa, may be affected, resulting for example in a chloride ion shift. Considering that carboxylate ions barely penetrate, if at all, into the red blood cells, chloride ions may be kept in the cells functioning as counterions for intracellular cations. In such situations hemolysis of the red blood cells may be induced, which in turn could negatively influence the determination of not only chloride ions but also of haemoglobin. Furthermore, changes in osmolarity and the outflow of water from the blood cells could lead to a possible dilution effect and possible impairment of cellular integrity due to the desiccation of blood cells.
In the present invention, optionally furthermore an ammonium salt NR4X and/or a pH modifying agent may be provided together with the anticoagulant, preferably concomitantly but alternatively also slightly time-displaced. This can provide significant further benefits in terms of stabilization and preservation of a blood sample and of further facilitating the reliable determination of blood components.
It was surprisingly found that a possible impairment of cellular integrity and even lysis of blood cells could be favourably inhibited by additionally adding an ammonium salt NR4X, wherein each R independently is hydrogen, linear Ci-C6 alkyl, branched C3-C6 alkyl, unsubstituted phenyl or substituted phenyl, and X is preferably halide and more preferably is chloride. Tetrame- thylammonium chloride and/or tetraethylammonium chloride are even more preferred, and tetramethylammonium chloride is particularly preferred. Preferably, when ammonium salt NR4X is added, the concentration of the ammonium salt NR4X is 0.01 to 100 mol/mL blood, preferably is 5 to 25 mol/mL blood, and more preferably is 15 to 20 pmol/mL blood. Tetramethylammonium chloride in a concentration of 15 to 20 mol/mL blood is particularly preferred.
Moreover, it was surprisingly found that a possible impairment of cellular integrity and even lysis of blood cells could also be favourably inhibited by adding a pH modifying agent. Said pH modifying agent favourably does not interfere with the laboratory parameters to be tested. As pH modifying agent organic amine is preferably used, more preferably triethanolamine , ethanola- mine and/or 2-amino-2-methylpropanol are used. For example, when a pH modifying agent is added and free acid is provided in solution, preferably a pH value between 2.2 and 3.0 is set. Inorganic bases such as sodium hydroxide are suitably avoided. It was particularly found that surprisingly a possible impairment of cellular integrity and even lysis of blood cells could be especially favourably inhibited by adding a combination of the ammonium salt NR4X and the pH modifying agent. This way the correct determination of both haemoglobin and chloride ions is even more surely possible (see Examples 29 and 30 and Figs. 1 and 2) . This is also an indication for a still more favourable condition in the electrochemical balance between the inside and the outside of the cells. This way hemolysis can be inhibited particularly efficiently and effectively. This advantageous effect is especially relevant in cases where occurrence of hemolysis must be significantly reduced or even safely and surely avoided. Hemolysis can for example destabilize a sample and pre- vent prolonged storage. Furthermore, hemolysis can be detrimental to blood testing and diagnostics because, for example, mixing of plasma components with cellular components from the lysed cells can lead to spurious results for both plasma and cell analyses or even prevent such analyses altogether. There- fore, inhibiting hemolysis is advantageous for the reliable determination of blood components. This further improved stabilization and preservation of a blood sample can provide longer storage and a more reliable determination of blood components and thus improved diagnostics .
The concentration of the substance provided as free acid having a pKa of ≥ 0.9, preferably the substance provided as free car- boxylic acid having a pKa of ≥ 0.9, is preferably at least 0.1 mmol/L of blood to be mixed with, more preferably is in the range from 0.1 to 100 mmol/L of blood to be mixed with, even more preferably in the range from 1 to 50 mmol/L of blood to be mixed with, yet even more preferably in the range from 2 to 32 mmol/L of blood to be mixed with, and most preferably in the range from 2 to 10 mmol/L of blood to be mixed with. In an em- bodiment the concentration of the substance provided as free acid having a pKa of ≥ 0.9 is set such that after mixing of said substance with blood the blood sample exhibits a pH in the range from 6.0 to 7.4. According to an embodiment of the present in- vention the amount of the substance provided as free acid having a pKa of ≥ 0.9 is set such that if dissolved in a desired amount of water the obtained aqueous solution exhibits a pH of ≥ 1.5. In a preferred embodiment the amount of the substance provided as free acid having a pKa of ≥ 0.9 is set such that if dissolved in a desired amount of water the obtained aqueous solution exhibits a pH of ≥ 1.5 and that when mixed with blood the blood sample exhibits a pH in the range from 6.0 to 7.4. In a preferred embodiment the free acid is provided such that its con- centration is adjusted to below 20 mmol/L of blood to be mixed with, preferably below 15 mmol/L of blood to be mixed with. Particularly preferred is a concentration in the range from 2 to 10 mmol/L of blood to be mixed with. This allows to suitably adjust the pH value in the blood sample .
Preferably the substance provided as free acid having a pKa of ≥ 0.9, more preferably the substance provided as free carboxylic acid, is selected from the group consisting of citric acid, tri- carballylic acid, ethylenediaminetetraacetic acid, diethylene- triaminepentaacetic acid, benzenepentacarboxylic acid, mellitic acid, and tetrahydrofuran-2 , 3 , 4 , 5-tetracarboxylic acid or mixtures thereof, more preferably is citric acid, tricarballylic acid or ethylenediaminetetraacetic acid, and even more preferably is citric acid. In an embodiment citric acid is anhydrous citric acid and/or citric acid monohydrate, and citric acid mon- ohydrate is most preferred. According to the invention the substance provided as free acid having a pKa of ≥ 0.9 is preferably provided as a powder or in lyophilized form. In an embodiment according to the above aspect of the invention the amount of at least one blood component is determined by comprising the following steps: a blood collection device is provided which contains, placed in the device, the agent according to the invention, and optionally further contains an ammonium salt NR4X and/or a pH modifying agent; blood, preferably whole blood, is placed in said blood collection device; said agent is mixed with blood in the blood collection device; optionally the blood is stored in the blood collection device for a desired period of time, for example until assigned tests are carried out; and the amount of the at least one blood component in the blood sample is determined. Preferably, placing of the blood in the blood collection device, mixing and the optional storage are carried out at a temperature from 0 to 37°C, and placing of the blood in the blood collection device and mixing more preferably are carried out at room temperature. Instead of providing a blood collection device containing the agent according to the invention and placing blood in said blood collection device, al- ternatively a step may be carried out which comprises placing blood in a blood collection device and subsequently placing the agent in the blood collection device. Optionally, an ammonium salt NR4X and/or a pH modifying agent may be added. In a preferred embodiment the amounts of at least two blood components are determined.
Another aspect of the invention is the use of a blood collection device for collecting and optionally storing blood in vitro, wherein in the device blood is mixed after its withdrawal with the agent according to the present invention, and wherein coagulation in blood is thereby inhibited. Unexpectedly it was found in the present invention that an efficient inhibitory effect against coagulation and clotting is obtained when blood is mixed in the device with the agent which comprises as the only antico- agulant a substance provided as free acid having a pKa of ≥ 0.9.
In an embodiment blood is collected in a single device and mixed with the agent according to the invention and is subsequently subjected to at least one test for the determination of at least one blood component, preferably to multiple tests comprising at least one hematologic test, at least one coagulation test and at least one further clinical chemistry analysis. According to this aspect of the invention the number of collection tubes required for the different fields of laboratory medicine can be considerably reduced, which in turn allows to optimize and render more efficient laboratory activity and workflow and to reduce sample volume and turnaround time. Furthermore, the use of only a single effective anticoagulant for the different fields in clinical laboratories, i.e. hematology, clinical chemistry and hemostase- ology, can expedite the process in emergency situations. The use of a single sample from a single blood collection tube is however not only very useful in emergency situations but also for pediatric patients and for patients from whom it is difficult to obtain more than one blood sample. Moreover, further biochemical analyses that were not initially anticipated or requested for the initial examination may advantageously be carried out on account of the broad test compatibility of the anticoagulant according to the invention (see also Examples 1-28) . The broad ap- plicability for ex vivo diagnostic analytics is beneficial to patient care and convenience.
In another aspect the invention relates to the use of an agent comprising as the only anticoagulant a substance provided as free acid having a pKa of ≥ 0.9 for effecting inhibition of coagulation of blood in vitro.
Another aspect of the invention is a blood collection device, wherein the agent according to the present invention is provided in the device, and wherein the substance provided as free acid having a pKa of ≥ 0.9 is the only provided anticoagulant to be mixed with blood. Preferably a device is comprised which is capable of being connected with a conventional blood withdrawal device. Conventional blood collection tubes including evacuated blood collection tubes such as vacutainer and aspiration systems such as monovette are known in the art. Besides the above- mentioned advantages in terms of use, the present invention can provide further economic advantages with respect to the manufacture of blood collection tubes as well as stocking logistics of collections tubes at the manufacturers and clinical laboratories . Another aspect of the invention relates to a kit which comprises the blood collection device of the present invention and test substance (s) for the determination of at least one blood component in collected blood. The following examples are merely illustrative of the present invention and they should not be considered as limiting the scope of the invention in any way. The examples and modifications or other equivalents thereof will become apparent to those skilled in the art in the light of the present entire disclo- sure.
Examples and Comparative Examples
Materials Used and Method
Materials
Citric acid monohydrate (CA) , tricarballylic acid (TCA) , eth- ylenediaminetetraacetic acid (EDTA) , diethylenetriaminepent- aacetic acid (DTPA) , mellitic acid (MELA) , and benzenepentacar- boxylic acid (BPCA) were from Sigma-Aldrich, Germany. Tetrahy- drofuran-2, 3 , 4 , 5 -tetracarboxylic acid (THTCA) was from TCI, Germany. Pristine blood collection tubes, i.e. tubes as originally provided not containing or respectively not filled or coated with additive, were from KABE Labortechnik GmbH, Germany, denoted KABEVETTE (GR) . In said provided pristine tubes were respectively placed: 5.4 mmol of CA per L of blood to be mixed with, 31.2 mmol of TCA per L of blood to be mixed with, 2.3 mmol of EDTA per L of blood to be mixed with, 2.2 mmol of DTPA per L of blood to be mixed with, 3.1 mmol of MELA per L of blood to be mixed with, 4.6 mmol of THTCA per L of blood to be mixed with, and 8.0 mmol of BPCA per L of blood to be mixed with. For com- parison, blood collection tubes with tripotassium EDTA (K3EDTA) , Serum Separator tubes with clot activator (SST) , and 3.2% sodium citrate tubes were used which were from Becton Dickinson (BD) .
Blood Collection and Sampling Protocol
Blood was collected from the antecubital vein from volunteers who had previously given their informed consent. For each subject, freshly drawn blood was collected into the corresponding tubes such that respectively a blood sample according to the present invention, a serum sample (SST (BD) ) , a citrate blood sample (citrate (BD) ) , and a potassium EDTA blood sample
( K3 EDTA (BD) ) were obtained. Tubes were filled with blood completely to the mark (4.9 mL) , and for tubes containing anticoagulant the blood was mixed well with the respective anticoagulant by inverting the tubes 4 times immediately after blood collection. Serum samples were treated according to the protocol of the manufacturer and used for biochemical analysis. Whole blood potassium EDTA ( K3 EDTA (BD) ) and inventive samples were used first for hematologic determinations and were then centrifuged. The inventive and citrate plasma samples (citrate (BD) ) were used for both haemostasis and biochemical determinations. Potassium EDTA plasma was also used for biochemical determinations. It was found that coagulation was effectively inhibited in the tubes according to the present invention. Subsequently a multitude of laboratory parameters in a sample from respectively a single blood collection device according to the present inven- tion could be determined and evaluated, and depending on the particular test, be compared to either of K3EDTA(BD) , citrate (BD) or SST (BD) samples. In Examples 1-27 results using CA (denoted GR 15) are shown. Example 28 shows results for various free acids having a pKa of ≥ 0.9, in particular free carboxylic acids, according to the invention.
Measurements
The analyses of metabolites, minerals, and enzymes were per- formed using the automated analyzers cobas 6000 C and 6000 E. Haemostasis tests and haematologic parameters were measured by STA-R (Diagnostica Stago) analyzer and LH 780 analyzer (Beckman Coulter) respectively. For results and comparative results see Examples 1-28.
Abbreviations :
WBC : white blood cells; RBC : red blood cells Hb : hemoglobin; Pit: platelets; Lympho : lymphocytes; Neutro : neutrophils; Mono: monocytes; Eos: eosinophils; Baso : basophils.
Alb: albumin; ALP: alkaline phosphatase; AST: aspartate aminotransferase; ALT: alanine aminotransferase; APTT : activated partial thromboplastin time; Ca : calcium; Choi: cholesterol; CREA: creatinine; CRP : C-reactive protein; Fe : iron; Fer : ferritin; Fib: fibrinogen; FT3 : triiodothyronine, free; FT4 : thyrox- ine, free; GG : γ- glutamyltransferase ; Glue : glucose; K: potassium; Mg : magnesium; Na : sodium; P : prothrombin time; TG: triglycerides; TP: total protein; TSH: thyrotropin. Example 1
Example 1 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using citrate (in Bec- ton Dickinson tubes (BD) ) for the determination of activated partial thromboplastin time (APTT) and prothrombin time (PT) .
Exam le 2
Example 2 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and conventional sampling using K3EDTA (in Becton Dickinson tubes (BD) ) for the determination of white blood cells (WBC) . Example 3
Example 3 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and conventional sampling using K3EDTA (in Becton Dickinson tubes (BD) ) for the determination of lymphocytes (LYM- PHO) .
Example 4
Example 4 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using K3EDTA (in Becton Dickinson tubes (BD) ) for the determination of basophils (Baso Etude) and monocytes (MONO) .
Example 5
Example 5 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and conventional sampling using K3EDTA (in Becton Dickinson tubes (BD) ) for the determination of eosinophils
(EOS) .
Example 6
Example 6 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using K3EDTA (in Becton Dickinson tubes (BD) ) for the determination of neutrophils (NEU- TRO) .
Example 7
Example 7 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using K3EDTA (in Becton Dickinson tubes (BD) ) for the determination of haemoglobin (Hb) .
Example 8
Example 8 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and conventional sampling using K3EDTA (in Becton Dickinson tubes (BD) ) for the determination of red blood cells (RBC) .
Example 9
Example 9 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and conventional sampling using citrate (in Becton Dickinson tubes) for the determination of fibrinogen (FIB ETUDE) and a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using K3EDTA (in Becton Dickinson tubes (BD) ) for the determination of platelets (Ptl) . Example 10
Example 10 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and sampling using conventional serum samples (SST (BD) ) for the determination of cholesterol (CHOL) .
Example 11
Example 11 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and sampling using conventional serum samples (SST (BD) ) for the determination of creatinine (CREA) .
Example 12
Example 12 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and sampling using conventional serum samples (SST (BD) ) for the determination of iron (Fe) .
Example 13
Example 13 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and sampling using conventional serum samples (SST (BD) ) for the determination of ferritin (Fer) . Example 14
Example 14 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and sampling using conventional serum samples (SST (BD) ) for the determination of triiodothyronine, free (FT3) .
Example 15 Example 15 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and sampling using conventional serum samples (SST (BD) ) for the determination of glucose (Glue) .
Exam le 16
Example 16 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and sampling using conventional serum samples (SST (BD) ) for the determination of aspartate aminotransferase (AST) .
Example 17
Example 17 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and sampling using conventional serum samples (SST (BD) ) for the determination of alanine aminotransferase (ALT) . Example 18
Example 18 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using BD tubes for the determination of magnesium (Mg ETUDE) and calcium (Ca ETUDE) .
Example 19
Example 19 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and sampling using conventional serum samples (SST (BD) ) for the determination of alkaline phosphatase (ALP) .
Example 20 Example 20 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and sampling using conventional serum samples (SST (BD) ) for the determination of triglycerides (TG) .
Example 21
Example 21 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using BD tubes for the determination of thyrotropin (TSH) and thyroxine, free (FT4 ETUDE) .
Exam le 22
Example 22 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using BD tubes for the determination of C-reactive protein (CRP ETUDE) .
Example 23
Example 23 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using BD tubes for the determination of γ-glutamyltransferase (GGT ETUDE) . Example 24
Example 24 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15) ) and conventional sampling using BD tubes for the determination of potassium (K ETUDE) .
Example 25
Example 25 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and conventional sampling using BD tubes for the determination of sodium (Na ETUDE) .
Example 26
Example 26 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection ABEVETTE tubes (GR 15)) and conventional sampling using BD tubes for the determination of albumin (Alb ETUDE) . Example 27
Example 27 shows a comparison between inventive sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR 15)) and sampling using conventional serum samples (SST (BD) ) for the determination of total protein (TP) .
The results and concentrations of the respective parameters as determined in the inventive and reference samples in Examples 1 to 27 are shown below. The results of the inventive samples are completely statistically and medically comparable with results and concentrations obtained by using conventional sampling.
EXAMPLE 1
Method comparison ; ΑΡΪΤ
Method comparison : PT
IfSMethods : <PT GR 15 versus <PT citrate (BD)>
Figure imgf000028_0002
Figure imgf000028_0003
Distribution of concentrations :
Figure imgf000028_0001
EXAMPLE 2 EXAMPLE 3
Method comparison : V BC Method comparison : LYMPHO
Figure imgf000029_0004
Figure imgf000029_0006
Figure imgf000029_0001
Figure imgf000029_0005
Figure imgf000029_0007
EXAMPLE 4
Method comparison : Baso Etude Method comparison : MONO
<MO O 3EDTA
Figure imgf000029_0008
Figure imgf000029_0010
Figure imgf000029_0002
Figure imgf000029_0009
Figure imgf000029_0011
EXAMPLE 5
EXAMPLE 6
Method comparison : EOS
Method comparison : NEUTRO
15.-.
Figure imgf000029_0003
EXAMPLE 7 EXAMPLE 8
Method comparison : Hb Method comparison : RBC
Figure imgf000030_0004
Figure imgf000030_0006
Figure imgf000030_0001
Figure imgf000030_0005
Figure imgf000030_0007
EXAMPLE 9
Method comparison : FIB ETUDE Method comparison : Ptl
Methods : <Ptl QR versus < I K3EDTA (BD)>
Figure imgf000030_0008
Figure imgf000030_0010
Figure imgf000030_0009
Figure imgf000030_0002
Figure imgf000030_0011
EXAMPLE 10 EXAMPLE 11
Method comparison : CREA
Method comparison : CHOL
Figure imgf000030_0003
EXAMPLE 12 EXAMPL 13
Method comparison : Fe
Figure imgf000031_0009
Figure imgf000031_0007
Figure imgf000031_0010
Figure imgf000031_0001
Figure imgf000031_0008
EXAMPLE 14 EXAMPLE 15
Method comparison : FT3 Method comparison : Glue
Figure imgf000031_0011
Figure imgf000031_0013
Figure imgf000031_0002
Figure imgf000031_0012
Figure imgf000031_0014
EXAMPLE 16
EXAMPLE 17
Method comparison : AST
Method comparison : ALT
Figure imgf000031_0015
Figure imgf000031_0003
Figure imgf000031_0017
Distribution nee ni rations
Figure imgf000031_0016
Figure imgf000031_0004
Figure imgf000031_0018
EXAMPLE 18
Method comparison : Mg ETUDE
Method comparison : Ca ETUDE
Methods : ORIS* versus BD»
Figure imgf000031_0005
Figure imgf000031_0019
Figure imgf000031_0020
Distribution o1 corc ntraiionG
Figure imgf000031_0006
EXAMPLE 20
EXAMPLE 19
Method comparison : TG
Method comparison : ALP
po
Figure imgf000032_0004
Figure imgf000032_0006
Figure imgf000032_0001
Figure imgf000032_0005
Figure imgf000032_0007
EXAMPLE 21 elhc l comparison : TSH Method comparison : FT4 ETUDE
Methods : <TUBE GR15> versus <TUBE BD>
Figure imgf000032_0009
Figure imgf000032_0010
Figure imgf000032_0008
Figure imgf000032_0002
Figure imgf000032_0011
[ EXAMPLE 22 [ EXAMPLE 23
Method comparison : CRP ETUDE
Method comparison : GGT ETUDE
Figure imgf000032_0003
[ EXAMPLE 241
EXAMPLE 25
Method comparison : K ETUDE
Method comparison : Na ETUDE
Methods : .-Tube GR15> versus <Tube BD
Methods ; <Tube GR15 versus Tube BD;>
Figure imgf000033_0001
Figure imgf000033_0005
Figure imgf000033_0007
Distribution of concentrations : Distribution of concentrations
Figure imgf000033_0008
Figure imgf000033_0002
Figure imgf000033_0006
EXAMPLE 26 EXAMPLE 27
Method comparison : Alb ETUDE Method comparison : TP
Methods : <TUBE QH15 > versus TUBE BD>
Methods : <TP OR 15 versus <TP SST BD>
Figure imgf000033_0009
Figure imgf000033_0003
Figure imgf000033_0011
Distribution of conceniraticnc ;
Distribution of concentrations
Figure imgf000033_0010
Figure imgf000033_0004
Figure imgf000033_0012
Example 28
Full blood cell counts and white blood cell differential analysis were performed for samples from blood collection tubes according to the present invention using as anticoagulant respectively CA, TCA, EDTA, DTPA, MELA, THTCA, and BPCA. For a comparison, results are shown for samples with potassium EDTA
( K3EDTA (BD) ) . The results for the inventive samples are completely statistically and medically comparable with those obtained with conventional K3EDTA as anticoagulant (see Table 1) , showing that anticoagulants according to the present invention effectively inhibit coagulation. Table 1
K3EDTA (BD) CA TCA EOT A DTPA MELA THTCA BPCA BC (103/μ1) 6 .56 6.54 6 . 6 6.23 6.53 6.3 6.42 5.9
RBC (ΐο6/μΐ) 4.58 4.53 4.49 4.44 4.52 4.53 4.49 4.55
Hb (g/dL) 14.3 14.2 14.2 13.8 14.1 14.2 14.2 14.2
HCT (%) 42.2 42.0 43.4 41.9 42.3 42.8 43.1 46.1
MCV (fl) 92.1 93.0 96.7 94.4 93.6 94.5 96.0 101
MCH (pg) 31.2 31.3 31.6 31.1 31.2 31.3 31.6 31.2
MCHC (g/dL) 33.9 33.8 32.7 32.9 33.3 33.2 32.9 30.8
PLT (103/μ1) 283 270 287 266 210 270 255 250
NEUTRO ( % ) 68.4 68.5 69.0 70.8 70.3 68.3 68.6 68.9
LYMPHO ( % ) 22.0 22.0 22.0 19.9 20.5 22.4 22.3 21.7
MONO ( % ) 7.6 8.0 7.6 7.9 7.7 7.8 7.6 7.5
EOS (%) 0.9 0.7 0.7 0.6 0.8 0.8 0.8 0.7
BASO 1.1 0.8 0.7 0.8 0.8 0.7 0.8 1.2 ix = 10
/Abbreviations: HCT: hematocrit, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular hemoglobin concentration
Example 29
Tests were carried out to determine haemoglobin (Hb) .
Example 29A shows a comparison between sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR15)) and conventional sampling using K3EDTA (in Becton Dick- inson tubes (BD) ) for the determination of haemoglobin (Hb) . The citric acid solution contained no alkylammonium compound and the pH 1.8 was not adjusted. See Figure 1 for results. Example 29B shows a comparison between sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (HCT) ) and conventional sampling using K3EDTA (in Becton Dickinson tubes (BD) ) for the determination of haemoglobin (Hb) . The citric acid solution contained alkylammonium salt, in particular tetramethylammonium chloride in a concentration of 16 μτηοΐ/ml of blood, and the pH was adjusted to 2.4. See Figure 1 for results.
Example 30
Tests were carried out to determine chloride ions (CI) .
Example 3OA shows a comparison between sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (GR15) ) and conventional sampling using K3EDTA (in Becton Dickinson tubes (BD) ) for the determination of chloride ions (CI) . The citric acid solution contained no alkylammonium compound and the pH 1.8 was not adjusted. See Figure 2 for results. Example 3 OB shows a comparison between sampling using citric acid monohydrate (in pristine blood collection KABEVETTE tubes (HCT) ) and conventional sampling using K3EDTA (in Becton Dickinson tubes (BD) ) for the determination of chloride ions (Cl) . The citric acid solution contained alkylammonium salt, in particular tetramethylammonium chloride in a concentration of 16 μτηοΐ/τηΐ of blood, and the pH was adjusted to 2.4. See Figure 2 for results.

Claims

Claims
An in vitro method for the inhibition of coagulation in blood, wherein blood is mixed after its withdrawal with an agent comprising as the only anticoagulant a substance pro vided as free acid having a pKa of ≥ 0.9.
The method according to claim 1, wherein the substance pro vided as free acid having a pKa of > 0.9 denotes a substance provided as free carboxylic acid having a pKa of ≥ 0.9.
The method according to claim 1 or 2 , wherein said substance provided as free acid having a pKa of ≥ 0.9 comprises at least two carboxyl groups per molecule, preferably comprises at least three carboxyl groups per molecule.
The method according to any one of the preceding claims, wherein said agent consists of
(i) said substance provided as free acid having a pKa of ≥ 0.9; or
(ii) said substance provided as free acid having a pKa of = 0.9 dissolved in a solvent .
The method according to any one of the preceding claims, wherein subsequently at least one test is carried out for the determination of at least one blood component, prefera bly at least two tests are carried out for the determination of at least two blood components.
The method according to any one of the preceding claims, wherein the concentration of said substance provided as free acid having a pKa of > 0.9 is at least 0.1 mmol/L of blood to be mixed with, preferably is in the range from 0.1 to 100 mmol/L of blood to be mixed with, more preferably in the range from 1 to 50 mmol/L of blood to be mixed with, even more preferably in the range from 2 to 32 mmol/L of blood to be mixed with, and most preferably in the range from 2 to 10 mmol/L of blood to be mixed with.
The method according to any one of claims 4 to 6, wherein in case (ii) the solvent is selected from the group consisting of an aqueous solution, water, and alcohol and mixtures of water and alcohol, preferably is water or ethanol, and more preferably is water.
The method according to any one of the preceding claims, wherein the amount of at least one blood component is determined, preferably the amounts of at least two blood components are determined, by comprising the following steps:
(a) providing a blood collection device containing, placed in the device, the agent as set forth in any one of claims 1-4, 6 and 7, optionally further containing a pH modifying agent and/or an ammonium salt NR4X, wherein each R independently is hydrogen, linear Ci-Cs alkyl, branched C3-C6 alkyl, unsubstituted phenyl or substituted phenyl, and X is halide and preferably is chloride;
(b) placing blood in the blood collection device;
(c) mixing said agent with blood in the blood collection device ;
optionally (d) storing the blood in the blood collection device for a desired period of time; and
(e) determining the amount of the at least one blood component, preferably of the at least two blood components, in the blood sample. The method according to any one of the preceding claims, wherein said substance provided as free acid having a pKa of ≥ 0.9 is selected from the group consisting of citric acid, tricarballylic acid, ethylenediaminetetraacetic acid diethylenetriaminepentaacetic acid, benzenepentacarboxylic acid, mellitic acid, and tetrahydrofuran-2 , 3 , 4 , 5- tetracarboxylic acid or mixtures thereof, preferably is citric acid, tricarballylic acid or ethylenediaminetetraacetic acid, and more preferably is citric acid.
0. Use of a blood collection device for collecting and option ally storing blood in vitro, wherein in the device blood i mixed after its withdrawal with the agent as set forth in any one of claims 1-4, 6, 7 and 9, and wherein coagulation in blood is thereby inhibited.
1. The use according to claim 10, wherein blood collected in single device and mixed with said agent is subsequently subjected to at least one test for the determination of at least one blood component, preferably to multiple tests comprising at least one hematologic test, at least one coagulation test and at least one further clinical chemistry analysis .
2. Use of an agent comprising as the only anticoagulant a sub stance provided as free acid having a pKa of ≥ 0.9 for effecting inhibition of coagulation of blood in vitro.
3 The use according to claim 12, wherein the agent is as set forth in any one of claims 2-4, 6, 7 and 9.
14. A blood collection device, wherein the agent as set forth in any one of claims 1-4, 6, 7 and 9 is provided in the device, and wherein the substance provided as free acid having a pKa of ≥ 0.9 is the only provided anticoagulant to be mixed with blood.
15. A kit comprising:
the blood collection device according to claim 14, and test substance (s) for the determination of at least one blood component in collected blood.
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EP14706615.3A EP2943796B1 (en) 2013-02-28 2014-02-26 In vitro method, use of an agent and collection device for the inhibition of coagulation in blood
ES14706615.3T ES2675402T3 (en) 2013-02-28 2014-02-26 In vitro method, use of an agent and collection device for blood clotting inhibition
JP2015559487A JP2016508613A (en) 2013-02-28 2014-02-26 In vitro method, drug use and recovery apparatus for coagulation inhibition in blood
US17/164,080 US20210231690A1 (en) 2013-02-28 2021-02-01 In vitro method, use of an agent and collection device for the inhibition of coagulation in blood

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US6716392B1 (en) * 1995-12-12 2004-04-06 The United States Of America As Represented By The United States National Aeronautics And Space Administration Preservation of liquid biological samples
EP1884778A1 (en) * 2005-05-23 2008-02-06 Kawasaki, Jun Method for testing efficacy of antithrombotic agent
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