WO2022016343A1 - 一种组合物的血小板解聚用途、解聚试剂及解聚方法 - Google Patents

一种组合物的血小板解聚用途、解聚试剂及解聚方法 Download PDF

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WO2022016343A1
WO2022016343A1 PCT/CN2020/103133 CN2020103133W WO2022016343A1 WO 2022016343 A1 WO2022016343 A1 WO 2022016343A1 CN 2020103133 W CN2020103133 W CN 2020103133W WO 2022016343 A1 WO2022016343 A1 WO 2022016343A1
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substituted
group
unsubstituted
alkyl
alkaryl
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PCT/CN2020/103133
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French (fr)
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高飞
张子千
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深圳迈瑞生物医疗电子股份有限公司
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Priority to PCT/CN2020/103133 priority Critical patent/WO2022016343A1/zh
Priority to CN202080104849.7A priority patent/CN116249522A/zh
Publication of WO2022016343A1 publication Critical patent/WO2022016343A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/131—Amines acyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/14—Quaternary ammonium compounds, e.g. edrophonium, choline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/145—Amines having sulfur, e.g. thiurams (>N—C(S)—S—C(S)—N< and >N—C(S)—S—S—C(S)—N<), Sulfinylamines (—N=SO), Sulfonylamines (—N=SO2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/02—Ammonia; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/04—Sulfur, selenium or tellurium; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A61K33/10—Carbonates; Bicarbonates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/18—Iodine; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors

Definitions

  • the invention relates to in vitro blood detection, in particular to the use of a compound capable of eliminating platelet aggregation in platelet depolymerization, a reagent comprising the compound and a depolymerization method.
  • platelet pseudoaggregation can cause erroneous blood cell counts and classifications, leading to erroneous diagnosis and treatment of patients.
  • HIP heparin-induced in vivo platelet aggregation
  • DIC disseminated intravascular coagulation
  • EDTA-PTCP ethylenediaminetetraacetic acid-dependent pseudothrombocytopenia
  • ICSH International Committee for Standardization of Hematology
  • Antiplatelet antibodies that recognize the adhesion receptor glycoprotein IIb-IIIa (GpIIb-IIIa) on the platelet membrane when blood is mixed with EDTA anticoagulant in vitro, causing the expression of platelet aggregation-activating antigens such as granules Membrane protein 140 (GMP140, alias CD62P or P-selectin), type III lysosomal glycoprotein (Gp55, alias CD63) and thrombin-sensitive protein, etc., then activate tyrosine kinase, leading to platelet aggregation.
  • GMP140 granules Membrane protein 140
  • type III lysosomal glycoprotein Gp55, alias CD63
  • thrombin-sensitive protein etc.
  • Pseudo-aggregation of platelets not only leads to errors in platelet-related clinical parameters, but also affects the accuracy of clinical parameters of other blood cells, especially leukocytes, and brings adverse consequences to clinical diagnosis and treatment. Therefore, finding and eliminating platelet aggregation in blood samples is an ongoing problem in in vitro blood testing.
  • additives for example, anticoagulants (such as sodium citrate, sodium heparin, ACD, CTAD, CPT, a mixture of CaCl 2 and sodium heparin, etc.); platelet count diluents (containing sodium azide, calcium azide, fluorine antiplatelet drugs (in 10 minutes after blood collection); aminoglycoside antibiotics (such as amikacin and kanamycin, added within 1 hour after blood collection, but only effective for some samples) .
  • anticoagulants such as sodium citrate, sodium heparin, ACD, CTAD, CPT, a mixture of CaCl 2 and sodium heparin, etc.
  • platelet count diluents containing sodium azide, calcium azide, fluorine antiplatelet drugs (in 10 minutes after blood collection); aminoglycoside antibiotics (such as amikacin and kanamycin, added within 1 hour after blood collection, but only effective for some samples) .
  • the present invention intends to provide the use of such substances to prevent and/or eliminate platelet aggregation in blood samples in in vitro blood testing, and a simple and convenient operation for this use, which can be applied to different detection methods and equipment.
  • Reagents that interfere with platelet aggregation The substance and reagent for platelet depolymerization of the present invention can eliminate most of the platelet aggregation, the effect is stable and reliable, does not need repeated confirmation, and has no adverse effect on normal blood testing.
  • a composition for preventing and/or eliminating platelet aggregation in a blood sample in an in vitro blood test wherein the composition comprises at least one ammonium ion-containing compound.
  • the ammonium ion-containing compound contains a compound selected from the group consisting of chloride ion, bromide ion, iodide ion, hydroxide, phosphate, hydrogen phosphate, dihydrogen phosphate, nitrate, hydrogen sulfide, thiocyanate
  • the ammonium salt is selected from ammonium chloride, ammonium bromide, ammonium iodide, ammonium phosphate, ammonium hydrogen phosphate, ammonium dihydrogen phosphate, ammonium nitrate, ammonium thiocyanate, ammonium hydrogen sulfite, ammonium oxalate , at least one of the group consisting of ammonium hydroxide, ammonium hydrogen sulfate and ammonium hydrogen carbonate.
  • the prevention and/or elimination of platelet aggregation in the blood sample is performed in a solution with a pH value of at least 7.0.
  • the pH value is 7.5-11, more preferably 9.5-11.
  • the concentration of the ammonium ion-containing compound in the composition is 1-50 mmol/L, preferably 2-20 mmol/L .
  • the composition further comprises at least one amino group-containing compound containing at least one primary or secondary amino group.
  • the amino group-containing compound contains 1 to 20 groups selected from primary amino groups, secondary amino groups and imino groups.
  • the compound with amino group is selected from the group consisting of compounds represented by formula (I) and salts thereof:
  • R1 and R2 are the same or different, and each independently is a group selected from the following group, provided that R1 and R2 are not H at the same time, and the group is composed of H, -SO 3 H, -NH 2 , -C ( NH)-NH 2 , substituted or unsubstituted C1-16 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-14 alkaryl, substituted or unsubstituted C7-14 aralkane base, -C(O)-Q1 and -C(O)-O-Q2,
  • Q1 is H, -NH 2 , substituted or unsubstituted C1-16 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-14 alkaryl or substituted or unsubstituted C7 -14 Aralkyl;
  • Q2 is H, substituted or unsubstituted C1-16 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-14 alkaryl or substituted or unsubstituted C7-14 aralkyl;
  • the substituted refers to at least one selected from -NP1P2, -SO 3 H, -OH, halogen, -CN, -C(O)-O-P3, -O-C1-16 alkyl, -O -C6-10 aryl, -O-C7-14 alkaryl, -O-C7-14 aralkyl, -C(O)-C1-16 alkyl, -C(O)-C6-10 aryl , -C(O)-C7-14 alkaryl, -C(O)-C7-14 aralkyl and -C(O)-NP1P2 group consisting of groups substituted, wherein the -O- C1-16 alkyl, -O-C6-10 aryl, -O-C7-14 alkaryl, -O-C7-14 aralkyl, -C(O)-C1-16 alkyl, -C( O)-C6-10 aryl, -C(C(
  • P1, P2 and P3 are each independently a group selected from the group consisting of H, C1-16 alkyl, C6-10 aryl, C7-14 alkaryl and C7-14 aralkyl, wherein said C1 -16 alkyl, C6-10 aryl, C7-14 alkaryl and C7-14 aralkyl are respectively unsubstituted or further selected by at least one of -NH 2 , -OH, -SO 3 H, halogen, -CN, -COOH, and -C (O) NH 2 of the group consisting of substituted groups.
  • R1 and R2 are the same or different, and each independently is a group selected from the following group, provided that R1 and R2 are not H at the same time, and the group consists of H, -SO 3 H, -NH 2 , -C(NH)-NH 2 , substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkaryl base, substituted or unsubstituted C7-10 aralkyl, -C(O)-Q1 and -C(O)-O-Q2,
  • Q1 is H, -NH 2 , substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkaryl or substituted or unsubstituted C7 -10 aralkyl groups;
  • Q2 is H, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkaryl or substituted or unsubstituted C7-10 aralkyl;
  • the substituted refers to at least one selected from -NP1P2, -SO 3 H, -OH, -CN, -C(O)-O-P3, -O-C1-10 alkyl, -O-C6 -10 aryl, -O-C7-10 alkaryl, -O-C7-10 aralkyl and -C(O)-NP1P2 substituted with a group in which the -O-C1- 10 alkyl, -O-C6-10 aryl, -O-C7-10 alkaryl and -O-C7-10 aralkyl are respectively unsubstituted or further selected from at least one of -NH 2 , -OH , -SO 3 H, -CN, -COOH and -C(O)NH 2 are substituted by groups in the group consisting of;
  • P1, P2, and P3 are each independently a group selected from the group consisting of H, C1-10 alkyl, C6-10 aryl, C7-10 alkaryl, and C7-10 aralkyl, wherein said C1 -10 alkyl group, C6-10 aryl, C7-10 alkaryl and C7-10 aralkyl groups are unsubstituted or further substituted at least one selected from the group consisting of -NH 2, -OH, -SO 3 H , -CN , -COOH and -C(O)NH 2 are substituted with groups in the group.
  • R1 and R2 are the same or different, and each independently is a group selected from the following group, provided that R1 and R2 are not H at the same time, and the group is composed of H, -SO 3 H, -NH 2 , -C(NH)-NH 2 , substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkaryl group of C7-10 aralkyl, substituted or unsubstituted, -C(O)-Q1 and -C(O)-OH,
  • Q1 is H, -NH 2 , substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkaryl or substituted or unsubstituted C7 -10 aralkyl groups;
  • P1 and P2 are each independently a group selected from the group consisting of H, C1-10 alkyl, C6-10 aryl, C7-10 alkaryl, and C7-10 aralkyl, wherein said C1-10 alkyl group, C6-10 aryl, C7-10 alkaryl and C7-10 aralkyl groups are unsubstituted or further substituted at least one selected from the group consisting of -NH 2, -OH, -SO 3 H , -CN, - substituted with groups in the group consisting of COOH and -C(O)NH 2 .
  • the pKa of the amino group of the compound having an amino group is in the range of 1-16, preferably 4-14.
  • the pKa value of the amino group of the compound having an amino group is lower than the pH value of the solution for preventing and/or eliminating platelet aggregation in the blood sample.
  • the concentration of the compound having an amino group is 1-50 mmol/L, preferably 2-20 mmol/L.
  • the in vitro blood test includes at least one test selected from the following items: platelet, red blood cell test and white blood cell test.
  • red blood cell detection can further distinguish reticulocytes.
  • white blood cell detection three-category, four-category or five-category detection can also be performed.
  • the red blood cells are at least one of mature red blood cells, reticulocytes and nucleated red blood cells.
  • the leukocytes are at least one of neutrophils, eosinophils, basophils, lymphocytes and monocytes.
  • the detection device may comprise an impedance detector and/or an optical detector.
  • the blood sample is peripheral or venous blood from a mammal.
  • an anti-platelet aggregation interference reagent for in vitro blood detection wherein the reagent comprises at least one ammonium-containing ammonium radical with a concentration of 1-50 mmol/L, preferably 2-20 mmol/L ionic compounds.
  • the ammonium ion-containing compound contains a compound selected from the group consisting of chloride, bromide, iodide, hydroxide, phosphate, hydrogen phosphate, dihydrogen phosphate, nitrate, hydrogen sulfide, sulfur Cyanate, sulfate, bisulfate, sulfite, bisulfite, carbonate, bicarbonate, formate, acetate, oxalate, propionate, malonate, citrate and combinations thereof group of anions.
  • the ammonium ion-containing compound is selected from ammonium chloride, ammonium bromide, ammonium iodide, ammonium phosphate, ammonium hydrogen phosphate, ammonium dihydrogen phosphate, ammonium nitrate, ammonium thiocyanate, hydrogen sulfite At least one of the group consisting of ammonium, ammonium oxalate, ammonium hydroxide, ammonium hydrogen sulfate, and ammonium hydrogen carbonate.
  • the pH value of the reagent is at least 7.0, preferably, the pH value is 7.5-11, more preferably 9.5-11.
  • the platelet depolymerization reagent further comprises at least one compound having an amino group at a concentration of 1-50 mmol/L, preferably 2-20 mmol/L, the compound containing at least one primary or secondary amino group.
  • the compound having an amino group contains 1-20 groups selected from primary amino groups, secondary amino groups and imino groups.
  • the compound with amino group is selected from the group consisting of compounds represented by formula (I) and salts thereof:
  • the pKa of the amino group of the compound having an amino group is in the range of 1-16, preferably 4-14.
  • the pKa value of the amino group of the compound having an amino group is smaller than the pH value of the reagent.
  • the reagent further comprises a buffer, an osmotic pressure regulator and optionally at least one selected from the group consisting of surfactants, fluorescent dyes and erythrocyte lysing agents.
  • a method of preventing and/or eliminating platelet aggregation in a sample in an in vitro blood test comprising the step of treating the blood sample with a composition or reagent as defined above.
  • the method further includes the step of detecting the above-treated blood sample with a blood analyzer.
  • the reagents containing the compounds, the above-mentioned uses and the depolymerization of aggregated platelets in the in vitro blood detection method can deaggregate platelets in a short time, without the need for additional water bath temperature control and prolongation of the reaction time. conditions, so there is no need to change the existing detection steps, suitable for any blood analyzer.
  • the reagent has no adverse effect on platelets and other detection reagents, and can achieve a good depolymerization effect in various platelet aggregation conditions, can easily eliminate the false aggregation of platelets in the sample, and obtain accurate blood cell counts. Check parameters.
  • Figure 1 shows a schematic diagram of the mechanism of depolymerization and repolymerization of aggregated platelets by compounds with amino groups
  • FIG. 2 shows a three-dimensional scattergram of particles in a blood sample obtained in an optical detection device, wherein the optical information characteristics of aggregated platelets and deaggregated platelets are respectively shown;
  • Figure 3 shows the depolymerization effect of various ammonium ion-containing compounds on ADP-induced aggregated platelets at different concentrations
  • Figure 4 shows a graph of the depolymerization effect of a compound having an amino group on aggregated platelets under different ambient pH conditions
  • Figure 5 shows a graph of the depolymerization effect of compounds with different amino pKa values on aggregated platelets at pH 9.5;
  • Figure 6 shows a graph of the induction of platelet aggregation in blood samples by ADP at different final concentrations as a function of time
  • Figure 7 shows a graph of the depolymerization effect of compounds with amino groups on platelet aggregation induced by ADP at no concentration as a function of time
  • Figure 8 shows a graph of the induction of platelet aggregation in blood samples by THR at different final concentrations as a function of time
  • Figure 9 shows a graph of the depolymerization effect of compounds with amino groups on platelet aggregation induced by THR at no concentration as a function of time
  • Figure 10 shows a graph of the induction of platelet aggregation in blood samples by COL over time at various final concentrations
  • Figure 11 shows a graph of the depolymerization effect of compounds with amino groups on platelet aggregation induced at no concentration of COL as a function of time
  • Figure 12 shows a graph of the induction of platelet aggregation in blood samples by RIS at different final concentrations as a function of time
  • Figure 13 shows a graph of the depolymerization effect of compounds with amino groups on platelet aggregation induced at no concentration of RIS as a function of time
  • Figure 14 shows the deaggregation effect of 1-acetylammonium and ammonium chloride each and in combination at different concentrations on aggregated platelets
  • Figure 15 shows three-dimensional scattergrams of blood samples detected in an optical detection device using conventional staining solutions and staining solutions containing ammonium chloride for platelets and red blood cells in the sample before and after induction of aggregation;
  • Figure 16 shows three-dimensional scattergrams of platelets and red blood cells in blood samples detected in an optical detection device using conventional diluents and diluents containing ammonium chloride in an optical detection device before and after induction of aggregation;
  • Figure 17 Histograms showing detection of platelets in blood samples using conventional diluents and diluents containing ammonium chloride in an impedance detection device before and after induction of aggregation;
  • Figure 18 shows three-dimensional scattergrams of blood samples before and after induction of aggregation using conventional lysates and lysates containing ammonium chloride in an optical detection device to detect platelets and leukocytes in the samples;
  • Figure 19 shows three-dimensional scatter plots of platelets and red blood cells in blood samples detected in an optical detection device before and after induction of aggregation using conventional lysates and diluents containing combinations with amino compounds and ammonium ion-containing compounds;
  • Figure 20 shows three-dimensional scatter plots of blood samples detected in an optical detection device using conventional staining solutions and staining solutions containing combinations of amino compounds and ammonium ion-containing compounds to detect platelets and erythrocytes in blood samples before and after induction of aggregation;
  • Figure 21 shows three-dimensional scattergrams of platelets and red blood cells in blood samples detected in an optical detection device using conventional diluents and diluents containing combinations with amino compounds and ammonium ion-containing compounds in blood samples before and after induction of aggregation;
  • Figure 22 shows a graph comparing the depolymerization effect of conventional dilution and dilution containing ammonium chloride in clinical for samples with pseudoaggregation of platelets.
  • Deagglomeration of pseudoaggregated platelets can not only obtain the real platelet count in the blood sample, thereby providing a reliable reference for clinical decision-making, but also eliminate the large particles formed by platelet aggregation that cause the classification and counting of other blood cells, especially white blood cells. Impact.
  • platelets when a solution containing ammonium ions is added to a blood sample in which pseudoaggregation of platelets occurs, platelets can be rapidly deaggregated. It can be seen from the following examples that various ammonium salts have generally similar depolymerization effects on aggregated platelets at a certain pH.
  • the ammonium ion-containing compound according to the present invention may contain a compound selected from chloride, bromide, iodide, hydroxide, phosphate, hydrogen phosphate, dihydrogen phosphate, nitrate, hydrogen sulfide, thiocyanate , sulfate, bisulfate, sulfite, bisulfite, oxalate, carbonate, bicarbonate, formate, acetate, propionate, malonate, citrate and combinations thereof in the group consisting of the anion.
  • ammonium ions deaggregate platelets is unclear. It is speculated that it is related to the concentration of ammonium ions in the solution to form hydrated ammonia. However, if ammonia water is used directly, although it also has good depolymerization performance, it is worse than using compounds containing ammonium ions, especially ammonium salts such as ammonium chloride, ammonium phosphate and ammonium halide. Therefore, it is speculated that some kinds of anions in the solution may also play a certain role in promoting platelet depolymerization. However, in the absence of ammonium ions in solution, these anions have no significant depolymerization effect.
  • Exemplary ammonium ion containing compounds can be ammonium chloride, ammonium bromide, ammonium iodide, ammonium phosphate, ammonium hydrogen phosphate, ammonium dihydrogen phosphate, ammonium nitrate, ammonium thiocyanate, ammonium hydrogen sulfite, ammonium oxalate , ammonium hydroxide, ammonium hydrogen sulfate, ammonium hydrogen carbonate, etc., but not limited thereto. More preferred ammonium ion-containing compounds such as ammonium chloride, ammonium bromide, ammonium phosphate, ammonium hydrogen phosphate and the like.
  • compositions for preventing and/or eliminating pseudoaggregation of platelets in a blood sample in an in vitro blood test includes at least one of the aforementioned ammonium ion-containing compounds. Of course, two or more ammonium ion-containing compounds may also be included.
  • compositions mentioned herein comprising at least one ammonium ion-containing compound may be in the form of the compound itself or in the form of a solution, eg soluble in a suitable solvent, for convenient use in the present invention in use.
  • suitable solvents may be water, alcohols (eg, ethanol), aqueous solutions of alcohols, etc., which are commonly used and do not interfere with the detection of blood samples.
  • the at least one ammonium ion-containing compound has a certain concentration in the solution containing the blood sample to depolymerize the platelets.
  • the concentration of the ammonium ion-containing compound in the composition may be in the range of 1 to 50 mmol/L, such as 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50mmol/L.
  • the concentration of the ammonium ion-containing compound is preferably in the range of 2 to 20 mmol/L, and more preferably in the range of 5 to 15 mmol/L.
  • one of the ammonium ion-containing compounds may be used, or two or more of the compounds may be used simultaneously for platelet depolymerization.
  • the total concentration of each compound falls within the above range.
  • the causes of platelet pseudoaggregation vary widely, and the degree of aggregation and difficulty of disaggregation vary widely. Therefore, the ammonium ion-containing compound has a larger concentration range in which it is effective. For easier disaggregation, lower concentrations may be used, whereas for more difficult disaggregation or high levels of aggregation, higher concentrations may be used.
  • the selection of the appropriate concentration can be reasonably determined by those skilled in the art according to the following specific examples in combination with actual needs.
  • the inventors have also unexpectedly found that a class of compounds containing amino groups also have significant depolymerization effects on the pseudoaggregation of platelets caused by different reasons. And when the ammonium ion-containing compound is used in combination with the compound having an amino group, the depolymerization effect on aggregated platelets can be significantly improved.
  • amino refers to primary and/or secondary amino groups unless otherwise specified, and does not include tertiary amino groups.
  • amino group-containing compound refers to a compound containing at least one amino group (primary amino group and/or secondary amino group) unless otherwise specified.
  • the compounds also optionally contain tertiary amino groups and/or imino groups, especially optionally imino groups.
  • Hydrocarbon groups such as alkyl and aryl groups, have little effect on platelet depolymerization as long as they do not affect the certain water solubility of the compound.
  • Hydrophobic groups such as ester groups (-C(O)-O-), carbonyl groups (-C(O)-) can reduce the depolymerization effect to some extent.
  • the compound with amino group has at least one primary amino group or secondary amino group, preferably 1-20 primary amino group, secondary amino group and/or imino group. It is preferable that the compound having an amino group has at least one primary amino group, and it is more preferable to have 1 to 4 primary amino groups.
  • the amino group therein exists in a deprotonated form in the solution.
  • the amino/imine groups in different compounds differ in their ability to bind hydrogen ions in solution. This ability to bind/dissociate hydrogen ions can be represented by the dissociation constant pKa.
  • pKa lg ([R1R2NH] ⁇ [H +] / [R1R2NH 2 +]).
  • the amino group of the compound should have a suitable pKa value.
  • the pKa of the amino group mentioned herein refers to the first dissociation constant, that is, the pKa of the amino group or imine group of the first dissociated hydrogen ion.
  • amino group of the compound should have a pKa value of 1-16, preferably 1-14, more preferably 4-14.
  • the depolymerization ability is improved.
  • the depolymerization principle of the above-mentioned compounds on aggregated platelets it can be understood that when the pH gradually increases, the balance of the combination and dissociation of amino groups in the compounds with hydrogen ions in solution moves toward the direction of dissociation, and more amino groups are deprotonated. Therefore, more amino groups are bound to the phosphatidylserine membrane of platelets through hydrogen bonds, thereby blocking the influx of calcium and playing a role in depolymerization. This combination further promotes the deprotonation of more amino groups of the compound, so that the depolymerization can be completed in a relatively short time without raising the temperature.
  • the present inventors found that the above-mentioned compound having an amino group is affected by pH when depolymerizing aggregated platelets, but the pH range where the ammonium ion-containing compound can exert platelet depolymerization effect, that is, 7.0 or higher, preferably 7.5-11 , especially preferably in the range of 9.5 to 11, many also have a good depolymerization effect.
  • the pH range where the ammonium ion-containing compound can exert platelet depolymerization effect that is, 7.0 or higher, preferably 7.5-11 , especially preferably in the range of 9.5 to 11, many also have a good depolymerization effect.
  • the pH is also high, it is easier to conform to the law that the depolymerization effect is strengthened when the pKa of the amino group in the above compounds is less than or equal to the pH value of the solution. Therefore, a suitable kind of compound having an amino group can be selected to obtain the desired depolymerization effect.
  • the effective concentration of a compound with an amino group capable of depolymerizing pseudoaggregated platelets is related to the number and type of amino groups contained in the compound, as well as the hydrophilicity of the compound and other factors.
  • the concentration of the compound with amino group for platelet depolymerization is in the range of 1-50 mmol/L, for example: 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50 mmol/L.
  • the concentration is in the range of 2-20 mmol/L, more preferably in the range of 5-15 mmol/L.
  • Such a compound having an amino group may be a compound having a structure represented by formula (I), or may be a salt thereof.
  • Formula (I) is:
  • R1 and R2 are the same or different, and each independently is a group selected from the following group, provided that R1 and R2 are not H at the same time, and the group is composed of H, -SO 3 H, -NH 2 , -C ( NH)-NH 2 , substituted or unsubstituted C1-16 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-14 alkaryl, substituted or unsubstituted C7-14 aralkane base, -C(O)-Q1 and -C(O)-O-Q2,
  • Q1 is H, -NH 2 , substituted or unsubstituted C1-16 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-14 alkaryl or substituted or unsubstituted C7 -14 Aralkyl;
  • Q2 is H, substituted or unsubstituted C1-16 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-14 alkaryl or substituted or unsubstituted C7-14 aralkyl;
  • the substituted refers to at least one selected from -NP1P2, -SO 3 H, -OH, halogen, -CN, -C(O)-O-P3, -O-C1-16 alkyl, -O -C6-10 aryl, -O-C7-14 alkaryl, -O-C7-14 aralkyl, -C(O)-C1-16 alkyl, -C(O)-C6-10 aryl , -C(O)-C7-14 alkaryl, -C(O)-C7-14 aralkyl and -C(O)-NP1P2 group consisting of groups substituted, wherein the -O- C1-16 alkyl, -O-C6-10 aryl, -O-C7-14 alkaryl, -O-C7-14 aralkyl, -C(O)-C1-16 alkyl, -C( O)-C6-10 aryl, -C(C(
  • P1, P2 and P3 are each independently a group selected from the group consisting of H, C1-16 alkyl, C6-10 aryl, C7-14 alkaryl and C7-14 aralkyl, wherein said C1 -16 alkyl, C6-10 aryl, C7-14 alkaryl and C7-14 aralkyl are respectively unsubstituted or further selected by at least one of -NH 2 , -OH, -SO 3 H, halogen, -CN, -COOH, and -C (O) NH 2 of the group consisting of substituted groups.
  • R1 and R2 are the same or different, each independently a group selected from the following group, provided that R1 and R2 are not H at the same time, the said The group consists of H, -SO 3 H, -NH 2 , -C(NH)-NH 2 , substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7 -10 alkaryl, substituted or unsubstituted C7-10 aralkyl, -C(O)-Q1 and -C(O)-O-Q2,
  • Q1 is H, -NH 2 , substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkaryl or substituted or unsubstituted C7 -10 aralkyl groups;
  • Q2 is H, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkaryl or substituted or unsubstituted C7-10 aralkyl;
  • the substituted refers to at least one selected from -NP1P2, -SO 3 H, -OH, -CN, -C(O)-O-P3, -O-C1-10 alkyl, -O-C6 -10 aryl, -O-C7-10 alkaryl, -O-C7-10 aralkyl and -C(O)-NP1P2 substituted with a group in which the -O-C1- 10 alkyl, -O-C6-10 aryl, -O-C7-10 alkaryl and -O-C7-10 aralkyl are respectively unsubstituted or further selected from at least one of -NH 2 , -OH , -SO 3 H, -CN, -COOH and -C(O)NH 2 are substituted by groups in the group consisting of;
  • P1, P2, and P3 are each independently a group selected from the group consisting of H, C1-10 alkyl, C6-10 aryl, C7-10 alkaryl, and C7-10 aralkyl, wherein said C1 -10 alkyl group, C6-10 aryl, C7-10 alkaryl and C7-10 aralkyl groups are unsubstituted or further substituted at least one selected from the group consisting of -NH 2, -OH, -SO 3 H , -CN , -COOH and -C(O)NH 2 are substituted with groups in the group.
  • R1 and R2 are the same or different, and each independently is a group selected from the following group, provided that R1 and R2 are not H at the same time, and the group consists of H, - SO 3 H, -NH 2 , -C(NH)-NH 2 , substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkaryl , the group of substituted or unsubstituted C7-10 aralkyl, -C(O)-Q1 and -C(O)-OH,
  • Q1 is H, -NH 2 , substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted C7-10 alkaryl or substituted or unsubstituted C7 -10 aralkyl groups;
  • P1 and P2 are each independently a group selected from the group consisting of H, C1-10 alkyl, C6-10 aryl, C7-10 alkaryl, and C7-10 aralkyl, wherein said C1-10 alkyl group, C6-10 aryl, C7-10 alkaryl and C7-10 aralkyl groups are unsubstituted or further substituted at least one selected from the group consisting of -NH 2, -OH, -SO 3 H , -CN, - substituted with groups in the group consisting of COOH and -C(O)NH 2 .
  • R1 and R2 are the same or different, and each independently is a group selected from the following group, provided that R1 and R2 are not H at the same time, and the group is composed of H, - C (NH) -NH 2, substituted or unsubstituted C1-6 alkyl group, a substituted or unsubstituted phenyl group, a substituted or unsubstituted benzyl, substituted or unsubstituted phenethyl group, and -C (O) - Composition of Q1, wherein Q1 is as defined above.
  • Q1 is substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted benzyl or substituted or unsubstituted benzene ethyl;
  • substituted refers to being substituted by at least one group selected from the group consisting of -NP1P2, -SO 3 H, -OH and -C(O)-NP1P2,
  • P1, P2 and P3 are each independently a group selected from the group consisting of H, C1-6 alkyl, phenyl, benzyl and phenethyl, wherein said C1-6 alkyl, phenyl, benzene methyl and phenethyl groups are unsubstituted or further substituted at least one selected from the group consisting of -NH 2, -OH, -SO 3 H , -COOH groups, and -C (O) NH 2 consisting of substituted groups.
  • the C1-16 alkyl group described herein may be a straight-chain or branched chain alkyl group, preferably a C1-14 or C1-12 alkyl group, more preferably a C1-10 alkyl group, more preferably a C1-6 alkyl group.
  • the C6-10 aryl group described herein can be phenyl or naphthyl.
  • the C7-14 alkaryl groups described herein may be mono- or polyalkyl substituted aryl groups.
  • Preferred are C7-10 alkaryl groups, such as tolyl, ethylphenyl, propylphenyl, butylphenyl, xylyl, methylethylphenyl, diethylphenyl, etc., but are not limited thereto.
  • the C7-14 aralkyl groups described herein may be phenylalkyl groups.
  • C7-10 aralkyl groups such as benzyl, phenethyl, phenpropyl, phenbutyl and the like are preferred, but not limited thereto.
  • Substituted as described herein refers to substitution with at least one of the defined substituents.
  • the amino substituent -NP1P2 preferably according to the invention can be polyamino substitution, up to 20 amino groups. For example, 1 to 18, such as 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, etc.
  • the amino group is a primary and/or secondary amino group, more preferably a primary amino group.
  • the hydroxy substituent -OH it is the more preferred substituent.
  • the substitution number of hydroxyl groups can be 1 to 6, such as 2, 3, 4, 5, and 6.
  • a sulfonic acid group, a carboxyl group, and a nitrile group are also preferable.
  • Halogen as used herein generally refers to -F, -Cl, -Br, -I, preferably -F, -Cl, -Br.
  • the preferred compounds with amino groups are guanidines, alcoholamines (especially polyhydric alcoholamines, such as 1,3-diamino-2-propanol), amino acids (preferably lysine, glutamic acid) aminoamide, glycine), short peptides (such as dipeptides), and polyamino-substituted branched or linear alkanes (such as 2-(aminomethyl)propane-1,3-diamine, 1,3-propanediamine Wait).
  • alcoholamines especially polyhydric alcoholamines, such as 1,3-diamino-2-propanol
  • amino acids preferably lysine, glutamic acid
  • aminoamide preferably lysine, glutamic acid aminoamide
  • glycine amino acids
  • short peptides such as dipeptides
  • polyamino-substituted branched or linear alkanes such as 2-(aminomethyl)propane-1,3-diamine, 1,3-prop
  • Examples of compounds of formula (I) that prevent and/or eliminate pseudoaggregation of platelets can be: sulfonic acids, such as sulfamic acid, sulfamic acid, taurine, sulfanilic acid, 2,5-disulfonic acid Aminobenzenesulfonic acid; amino acids such as glutamic acid, glutamine, arginine, lysine, alanine, glycine, N-tris(hydroxymethyl)methylglycine, 4-aminobutyric acid; short Peptides, such as dipeptides, such as dimerized lysine, dimerized glycine; alcohol/phenolamines (especially polyhydroxy-substituted amines), such as ethanolamine, triethanolamine, 3-amino-1-propanol, 4- Amino-1-butanol, 4-hydroxybenzylamine, tyramine (4-hydroxyphenethylamine), 1,3-diamino-2-propanol, 3-
  • More preferred compounds of formula (I) may be hydroxy-substituted amines, alkylamines, benzenesulfonic acid amines, amino acids and short peptides, and guanidines.
  • Salts of the compounds having amino groups are also within the scope of the present invention.
  • salts of compounds containing groups such as acidic sulfonic acid groups and carboxyl groups.
  • the salts can be alkali metal (such as potassium, sodium) salts, alkaline earth metal (such as magnesium) salts, ammonium cation salts, etc.; also can be internal salts. Salts of ammonium cations are particularly preferred due to the effect of the above-mentioned ammonium cations on platelet depolymerization.
  • the ammonium ion-containing compound when the ammonium ion-containing compound is combined with the amino group-containing compound, a good depolymerization effect can be obtained at a lower concentration. That is, when the total concentration of the two is equivalent to the concentration when either is used alone, a better effect can be obtained than when the ammonium ion-containing compound or the amino group-containing compound is used alone.
  • the combination of the two has a synergistic effect on platelet depolymerization. Therefore, when the two are used in combination, the total concentration of the two can be in the range of 2-50 mmol/L, such as 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50 mmol/L.
  • the composition may include 1-20 mmol/L of the amino-containing compound and 1-20 mmol/L of the ammonium ion-containing compound, more preferably, the composition may include 1-10 mmol/L of the amino-containing compound Compounds and 1-10 mmol/L ammonium ion-containing compounds, even including 1-5 mmol/L amino-containing compounds and 1-5 mmol/L ammonium ion-containing compounds.
  • the use ratio of the two is not particularly limited.
  • the molar ratio of the amino group-containing compound and the ammonium ion-containing compound may be in the range of 1:10 to 10:1, preferably in the range of 1:4 to 4:1 to be 1:4, 1:4 2, 1:1, 2:1, 4:1, etc. Most preferably, the molar ratio of the two is 1:1.
  • compositions of the present invention have no damaging effect on erythrocytes within the above concentration range, and the erythrocyte counts are substantially unchanged with and without the composition.
  • the composition can not only obtain an excellent platelet depolymerization effect, but also have no adverse effect on blood cells, especially red blood cells. .
  • the in vitro blood test refers to the detection of blood cells, such as platelets, red blood cells and white blood cells, in a blood sample.
  • the detection can be microscopic examination or instrument detection, preferably instrument detection.
  • the detection may be, for example, using a blood analyzer to detect various cells in the blood, and obtain corresponding detection information.
  • the application of the present invention does not have any limitation on the blood analyzer, and any instrument used for in vitro blood detection can be used for the application of the present invention.
  • the application of the present invention is not limited to detectors, such as impedance detectors and optical detectors.
  • multiple aggregated platelets are recognized as one particle in the optical channel of the hematology analyzer, so the aggregated platelets are larger in volume than a single platelet, have stronger fluorescence intensity than a single platelet, and a smaller number than unaggregated platelets. less platelets.
  • the aggregated platelets show that the platelet forward scattered light intensity information (FS) is too large. , reflecting the large particle size and high fluorescence intensity (FL).
  • FS platelet forward scattered light intensity information
  • FL high fluorescence intensity
  • the aggregated platelets are similarly treated as a particle, producing an electrical signal.
  • the platelet count is low because the particles become larger after aggregation and cannot be identified as platelets.
  • aggregated platelets can also affect white blood cell and red blood cell counts and changes in histograms. This effect was evident in early three-part hematology analyzers.
  • the aggregation of platelets can be prevented, and the aggregated platelets can also be deaggregated, so that accurate platelet counts can be obtained.
  • the leukocyte detection also includes the classification and/or enumeration of, for example, nucleated cells, basophils, eosinophils, neutrophils and/or lymphocytes.
  • detection information includes raw signals (eg, electrical pulses, light intensity, etc.), processed information such as histograms, scattergrams, etc., and finally reported classification and/or count information.
  • red blood cells in blood is much larger than other types of cells, in optical detection, although platelet aggregation has an effect on red blood cells, the effect is small and has little effect on the counting result.
  • the addition of amines and ammonium salts may cause red blood cell lysis.
  • the compounds containing ammonium ions and further compounds with amino groups will not adversely affect the sample environment (such as pH, ionic strength, etc.) within the concentration range defined above, Nor does it cause damage to blood cells. Therefore, the detection of red blood cells after platelet disaggregation also has no effect.
  • the use of the present invention can further classify and separately count mature red blood cells, reticulocytes and nucleated red blood cells.
  • the use of the present invention has effects on platelet aggregation caused by different reasons, so the sample used for the use is not particularly limited.
  • the sample can be from peripheral blood, such as from peripheral blood or venous blood.
  • the method of obtaining the sample is also not particularly limited, such as blood collection by finger stick or venous blood collection with an anticoagulant tube.
  • Anticoagulant tubes coated with conventional anticoagulants such as EDTA can be used for freshly collected blood, since the composition of the present invention also has a good effect on the pseudoaggregation of platelets induced by EDTA.
  • the blood sample can be subjected to any conventional processing to obtain the sample to be tested.
  • the treatment can be, for example, dilution treatment, dyeing treatment, lysis treatment, and the like.
  • the composition can be added to the sample as a treatment agent alone (eg, as a platelet deaggregating agent), or can be added to the sample together with the treatment agent in any step of processing the sample. middle.
  • the composition of the present invention can be formed as a single treatment agent; also by including the amino group-containing compound and optional ammonium ion-containing compound in the reagent for dilution, dyeing or lysis treatment, A diluent, stain or lysing agent with platelet disaggregation effect is formed so as to simultaneously prevent/eliminate platelet disaggregation in any one step of treatment without interfering with the treatment.
  • the composition does not require additional incubation (eg, low temperature or heat) in response to platelet depolymerization. About 30 seconds after the treatment agent containing the composition is mixed with the sample, the platelet depolymerization can be completed, and the sample can be detected.
  • the sample may be from a mammal, preferably a primate, more preferably a human.
  • the present invention also provides a method for preventing and/or eliminating platelet aggregation in a sample in an in vitro blood test, the method comprising a processing step for preventing/eliminating platelet aggregation interference on the blood sample.
  • the blood sample is processed under the above-mentioned conditions using a composition or reagent comprising the above-mentioned ammonium ion-containing compound and optionally a compound having an amino group.
  • the present invention provides an in vitro blood detection method that is resistant to platelet aggregation interference.
  • the method may comprise treating the blood sample to prevent and/or eliminate platelet aggregation in the blood sample with a depolymerizing agent comprising at least one compound selected from the group consisting of ammonium ion-containing and optional amino-containing compounds , and use a blood analyzer to detect the blood sample treated with the depolymerizing agent, so as to obtain at least the detection information of the blood cells after eliminating the interference of blood cell aggregation.
  • a depolymerizing agent comprising at least one compound selected from the group consisting of ammonium ion-containing and optional amino-containing compounds
  • the blood cells can be platelets, red blood cells and/or white blood cells.
  • the detection information can be a pulse signal for the particles in the sample, or an optical signal.
  • the blood analyzer may process the detection information to further obtain detection parameters of blood cells such as histograms, scatter plots, classification and/or counts.
  • the blood analyzer may comprise an impedance detector.
  • the method may comprise the steps of: detecting the blood sample treated with the depolymerizing agent with an impedance detector in a hematology analyzer to obtain electrical signals of particles in the blood sample, and further to obtain platelets and/or red blood cells detection parameters.
  • the impedance detector is still used for detection
  • the blood sample can be further treated with a red blood cell lysing agent in addition to the treatment with a depolymerizing agent before the detection
  • the method includes the following steps: The blood sample treated with the depolymerizing agent and the red blood cell lysing agent is detected with an impedance detector in a hematology analyzer to obtain electrical signals of particles in the blood sample, and further obtain white blood cell detection parameters.
  • Three classifications and counts of white blood cells can be obtained by an impedance detector.
  • the hematology analyzer includes an optical detector
  • the method includes detecting the blood sample treated with the depolymerizing agent with an optical detector in the hematology analyzer to obtain the concentration of the blood sample in the blood sample.
  • at least one scattered light intensity signal of the particle and further obtain the detection parameter of the platelet; preferably, the at least one scattered light intensity signal is a forward scattered light intensity signal. More preferably, the detection parameters of platelets can be obtained from the forward and side scattered light intensity signals.
  • the blood sample is further dyed with a fluorescent dye before the detection, and the method includes: treating the depolymerizing agent and the fluorescent dye
  • the blood sample is detected by an optical detector in the blood analyzer, and the fluorescence intensity signal and at least one scattered light intensity signal of the particles in the blood sample are obtained to obtain the detection parameters of platelets and/or red blood cells.
  • the at least one scattered light intensity signal is a forward scattered light intensity signal.
  • the detection parameters of platelets and/or red blood cells can also be obtained using the fluorescence intensity signal, the forward scattered light intensity signal and the side scattered light intensity signal
  • the blood sample is further dyed with a fluorescent dye and hemolyzed with an erythrocyte lysing agent
  • the method comprises: treating the depolymerizing agent, the fluorescent dye
  • the blood sample treated with erythrocyte lysing agent is detected by an optical detector in a blood analyzer, and at least two optical signals of particles in the blood sample are obtained to obtain detection information of white blood cells.
  • the at least two light signals are selected from the group consisting of fluorescence intensity signals, forward scattered light intensity signals and side scattered light intensity signals.
  • white blood cells for example, four categories (ie, eosinophils, neutrophils, monocytes, and lymphocytes) can be obtained by using the DIFF detection channel of the Mindray blood test instrument, or further use such as in the Mindray blood test instrument.
  • the WNB channel obtains the detection information of basophils.
  • the depolymerizing agent, the fluorescent dye and the erythrocyte lysing agent can be independent reagents, and can also be arbitrarily combined into mixed reagents.
  • the depolymerizing agent can be formed as a diluent, a staining agent, a lysing agent, or a dye-lysing agent, etc., which has the effect of platelet deaggregation.
  • the present invention also provides an anti-platelet aggregation interference reagent for in vitro blood detection.
  • the concentration, pH and other processing conditions of the above-mentioned purposes are generally also applicable to the anti-platelet aggregation interference of the present invention. reagents.
  • the reagent may include 1-50 mmol/L of at least one compound containing ammonium ion, and may further include 1-50 mmol/L of at least one compound having amino group.
  • concentration as defined in the present invention is the total concentration of two or more ammonium ion-containing compounds. Likewise, the stated concentration is also the total concentration of two or more compounds having amino groups.
  • the at least one ammonium ion-containing compound is as defined above and will not be repeated here.
  • the concentration thereof is also preferably 2 to 20 mmol/L, and more preferably 5 to 10 mmol/L.
  • At least one compound having an amino group is also as defined above, while its concentration is preferably 2 to 20 mmol/L, more preferably 5 to 10 mmol/L.
  • the reagent contains both the ammonium ion-containing compound and the amino group-containing compound, and the concentration ranges of both can be in the range of 1-20 mmol/L, 1-10 mmol/L, or even 1-5 mmol/L. varies within the range of L.
  • the molar ratio of the two can be in any ratio, for example, it can be varied in the range of ammonium ion-containing compound: compound with amino group of 1:10 to 10:1, preferably 1:4 to 4:1.
  • the pH of the reagent is not particularly limited, and is related to the use requirements of the reagent.
  • the pH of the reagent is at least 7.0, preferably 7.5-11, more preferably 9.5-11.0.
  • the pH of the reagents can be adjusted by conventional buffers.
  • the present invention has no particular limitation on the kind of buffer used for the reagent. For example, citrate buffer pair, phosphate buffer pair, borate buffer pair, phthalate buffer pair, 3-(N-morpholino)ethanesulfonic acid buffer pair, 4-hydroxyethylpiperazineethanesulfonic acid buffer pair can be used Equivalent, but not limited to this.
  • the reagent should have a certain osmotic pressure.
  • the osmotic pressure can be changed according to the actual requirements of the reagent.
  • it can be in the range of 180-240mOsm/L, preferably 200mOsm/L; Preferably 90mOsm/L.
  • the agent may include an osmotic pressure regulator.
  • the agent of the present invention is not particularly limited to the osmotic pressure regulator, for example, it can be inorganic salts, such as sodium chloride, potassium chloride, etc.; sugars, such as glucose, mannose, fructose, maltose, etc., but not limited thereto.
  • the agent may also include a surfactant.
  • the reagents may include surfactants with different functions.
  • a surfactant that spheroidizes red blood cells may be included in the reagent as a diluent.
  • Such surfactants are, for example, N-alkylbetaine, N,N-dimethyloctadecylamine sodium hydrochloride, sodium dodecylbenzenesulfonate, etc., but are not limited thereto.
  • the reagent may include a surfactant that changes the permeability of the cell membrane, so that the nucleic acid dye can penetrate the cell membrane and enter the interior of the cell to combine with the nucleic acid.
  • surfactants are, for example, phenoxyethanol, sodium dioctadecylamine hydrochloride, sodium N-methylamide carboxylate, etc., but are not limited thereto.
  • the agent may also include other necessary components as required, such as preservatives, antibacterial agents, cell membrane protective agents, chelating agents, and the like. Those skilled in the art can choose to join as needed.
  • the reagent may act as a diluent for the blood sample.
  • it can be a diluent for impedance detection, or it can be a diluent for optical detection.
  • the agent may also be a dye.
  • dyes are stored separately in the form of organic solutions in platelet detectors because they are more soluble in organic solvents. If necessary, dyes can also be added to the above-mentioned diluents to prepare a dye with platelet deaggregation function.
  • the dyes in the stain can be nucleic acid dyes. Nucleic acid-specific dyes such as asymmetric cyanine, thiazole orange TO, oxazole orange YO, acridine orange AO, etc., are specific, such as PI, DAPI, Hoechst series (such as Hoechst33258, Hoechst33342), etc. limited to this.
  • nucleic acid dyes are asymmetric cyanine dyes such as SYBR Green.
  • the pH value of optimal dyeing of this type of dye is roughly the same as the pH value of the optimal effect of the ammonium ion-containing compound and the compound having an amino group in the present invention.
  • the stain can also be a mitochondrial stain (eg Janus Green B, MitoLite Red, Rhodamine 123, Mitotracker Green, Mitotracker Deep Red, Mitotracker Red, etc.) or a membrane stain (eg DiA, DiD, DiI, DiO, DiR, DiS, FDA, Alexa Fluor 488, Super Fluor 488, etc.).
  • Such a stain formulation may be:
  • the reagent of the present invention can also be a lysing agent, which is used in the detection of leukocytes.
  • the lysing agent used in the reagent can be any conventional red blood cell lysing agent.
  • the compounds with amino groups, especially ammonium ion-containing compounds, in the reagents of the present invention have no adverse effect on erythrocytes within the defined concentration range, so the types and concentrations of the lysing agents in the reagents, and The usage is the same as in the prior art.
  • red blood cell lysing agents examples include quaternary ammonium salts (such as dimethylbenzyl hydrocarbyl ammonium chloride, dodecyl trimethyl ammonium chloride, dimethyl benzyl alkyl ammonium chloride, etc.), but are not limited to this.
  • quaternary ammonium salts such as dimethylbenzyl hydrocarbyl ammonium chloride, dodecyl trimethyl ammonium chloride, dimethyl benzyl alkyl ammonium chloride, etc.
  • the reagents of the present invention comprising the ammonium ion-containing compound and the compound having an amino group which may be further included have a low concentration of the active ingredient in preventing/eliminating the pseudoaggregation of platelets, and no additional reaction is required conditions (such as cooling or heating), and it is not necessary to prolong the reaction time, the depolymerization of platelets can be completed in 30s, so the compound has no adverse effect on the detection of blood, and has no effect on the detection steps and conditions. It can be used in existing Eliminate the interference of platelet aggregation under the equipment and detection method.
  • This example measures the effect of ammonium cation-containing compounds on the depolymerization of aggregated platelets.
  • the diluent is composed as follows: citric acid (0.5g/L), surfactant phenoxyethanol (0.1g/L), bacteriostatic agent (6g/L), sodium chloride (3g/L), EDTA (0.1g/L) g/L).
  • citric acid 0.5g/L
  • surfactant phenoxyethanol 0.1g/L
  • bacteriostatic agent 6g/L
  • sodium chloride 3g/L
  • EDTA 0.1g/L g/L
  • the above-mentioned ammonium ion-containing compound (final concentration: 0.01 mol/L) was added to the diluted solution to prepare a treatment solution. Adjust the pH of the treatment solution to 9.5 and the osmotic pressure to 200mOsm/L.
  • the venous blood of healthy subjects was collected into a blood anticoagulation tube for use. Take two 1ml blood, one of which is added with the above diluent, and after mixing, the platelet count is measured in a blood cell analyzer (Minray BC-6000Plus), which is recorded as: PLT_O (unpolymerized); the other is added with platelet aggregation inducer ADP (final concentration is 0.01mmol/L), and mix well. After 5 minutes, platelets will form aggregation. At this time, after adding the above diluent, use the blood cell analyzer to detect the blood containing aggregated platelets to obtain the platelet count, which is recorded as: PLT_O (not deaggregated).
  • PLT_O disaggregation
  • disaggregation rate referred to herein is calculated by the following formula:
  • Depolymerization rate PLT_O (depolymerized)/PLT_O (unpolymerized); or
  • depolymerization rate PLT_O (not depolymerized)/PLT_O (not depolymerized).
  • This example investigates the effect of ambient pH on the depolymerization effect of ammonium ion-containing compounds on aggregated platelets.
  • 0.01 mol/L ammonium chloride was added to the diluent to prepare the treatment solution, and the pH of the treatment solution was adjusted to 4.0, 5.5, 6.5, 7.5, 8.5, 9.5 and 11.0.
  • the depolymerization rate of platelet depolymerization in the samples is shown in Table 1 below.
  • the difference is that the compounds in Table 2 above are used to replace the ammonium ion-containing compounds respectively, and the pH of the diluent is adjusted to 14 and the osmotic pressure to 200 mOsm/L to prepare a treatment solution.
  • This example compares the effects of different numbers of amino groups on structurally similar compounds on platelet depolymerization. According to the same method in Example 3, the compounds containing different amino groups were tested respectively, and the platelet deaggregation rate and pKa value of each compound were obtained as shown in Table 4.
  • This example compares the effects of different amino pKa of structurally similar compounds on platelet depolymerization under the same pH environment.
  • the method is basically the same as Example 3, except that different types of compounds are adjusted to a more suitable pH value, and compounds containing different amino groups are tested respectively, and the platelet depolymerization rate and pKa value of each compound are obtained as shown in Table 5. Show.
  • Example 5 From Example 5, it can be seen that there is a certain relationship between the pH of the environment and the pKa of the amino group of the amino compound.
  • the compound 1-acetylguanidine with a pKa of 8.33 was selected in this example. According to the method similar to Example 1, only A set of tests was carried out by changing the pH of the environment between 7.0 and 10. A graph of pH versus depolymerization rate was obtained, as shown in Figure 4.
  • the pH of the environment has a strong correlation with the pka of the substance in platelet disaggregation.
  • the pH increased, the depolymerization ability of the amino group-containing substances was enhanced, and the depolymerization rate increased from 35.23% to 97.32%.
  • the pH is above 9.5, the depolymerization rate is basically the same.
  • the environmental pH is greater than the amino pKa of the compound to a certain extent, continuing to increase the pH has little effect on the depolymerization effect. This is consistent with the previously speculated depolymerization mechanism. That is, at a suitable pH, most of the amino groups of the compound exist in solution in a deprotonated form, thereby promoting or even accelerating depolymerization.
  • Arginine has more amino/imine groups, but the depolymerization effect is not very good, which may be related to its ability to form cyclic lactam, which leads to the weakening of the role of amino groups.
  • the hydroxyl group seems to be more beneficial to enhance the depolymerization effect of the amino group in the compound.
  • tris(hydroxymethyl acetate) aminomethane contains only one secondary amino group, but the depolymerization rate is still higher than that of glycine.
  • Tris with primary amino group has better effect on platelet depolymerization.
  • the hydroxyl group is favorable for the compound to be close to the cell membrane of platelets, while the primary amino group has better effect on platelet depolymerization than the secondary amino group.
  • the higher depolymerization rate is also related to the lower pKa (8.1) of these two compounds.
  • the tertiary amino group has almost no depolymerization effect. It is also understood from the above-mentioned mechanism that it is difficult for the amino group to function without a hydrogen atom capable of forming a hydrogen bond.
  • Example 8 Depolymerization effect of amino group-containing compounds on different platelet aggregation models
  • Aggregation of platelets can be induced by a variety of substances, leading to blood coagulation.
  • the substances that induce platelet aggregation in the blood coagulation detection protocol were selected, and various models of platelet aggregation were established. These include adenosine diphosphate (ADP), thrombin (THR), collagen (COL) and antibioticithin (RIS).
  • ADP adenosine diphosphate
  • THR thrombin
  • COL collagen
  • RIS adenosine diphosphate
  • RIS adenosine diphosphate
  • THR thrombin
  • COL collagen
  • RIS adenosine diphosphate
  • the amino group-containing compound can also induce different aggregation and aggregation in a certain concentration and time range. degree of platelet efficient disaggregation.
  • FIG. 6 shows the extent to which platelet aggregation was induced using different ADP concentrations (0.01-1 mM) versus time. Specifically, 1 ml of venous blood was taken, and ADP was added to make the final concentrations 0.01 mM, 0.25 mM, 0.5 mM and 1 mM, respectively. Blood containing aggregated platelets is tested with a blood cell analyzer. The blood cell analyzer obtains the characteristics of various cell particles in the blood, including the number, size, etc., through impedance channels and optical channels. After the normal blood is collected into the blood anticoagulation tube, the number of platelet particles detected by the impedance channel obtained by the blood cell analyzer is recorded as (PLT_I).
  • PLT_I (after aggregation).
  • PLT_I (after aggregation)
  • Such amino group-containing compounds were not added to the diluent of the impedance channel, so the impedance channel reflects the degree of platelet aggregation in the aggregation model.
  • the count before platelet aggregation is PLT_I (unaggregated)
  • the count after platelet aggregation is PLT_I (aggregated)
  • the aggregation rate PLT_I (aggregated)/PLT_I (unaggregated).
  • the number of platelet particles detected by the optical channel obtained by the blood cell analyzer was recorded as (PLT_O).
  • PLT_O The number of platelet particles detected by the optical channel obtained by the blood cell analyzer was recorded as (PLT_O).
  • the count before platelet aggregation is PLT_O (unaggregated), and the count after platelet aggregation is PLT_O (not disaggregated).
  • PLT_O (not depolymerized) ⁇ PLT_O (depolymerized) ⁇ PLT_O (not polymerized)
  • depolymerization rate PLT_O (depolymerized)/PLT_O (not polymerized).
  • FIG. 7 there is shown a graph of the effect of amino-containing compounds on platelet disaggregation over time for blood samples induced with different ADP concentrations.
  • the amino group-containing compound (1-acetylguanidine) was added to the diluent, and each blood sample added with different concentrations of the aggregation-inducing agent was treated according to the method similar to Example 3. Measurements were performed at 5-minute intervals to obtain the time-dependent change in the depolymerization rate.
  • composition of the diluent is: citric acid (0.5g/L), surfactant phenoxyethanol (0.1g/L), bacteriostatic agent (6g/L), sodium chloride (3g/L), EDTA (0.1g) /L) and 1-acetylguanidine (10 mmol/L), the pH was 9.5, and the osmotic pressure was 200 mOsm/L.
  • the same compound has a significant depolymerization effect on different platelet aggregation degrees at the same concentration, and the depolymerization effect reaches the highest immediately after adding the amino group-containing compound, and decreases with time. decreased, but basically leveled off after 10 minutes.
  • the lower the concentration of ADP the more stable the effect.
  • the blood cell analyzer can quickly complete the detection of the sample with only a small amount of sample, it can be within 5 minutes after adding the treatment solution containing the amino-containing compound, preferably within 2 minutes, more preferably immediately after 30s. Start detection.
  • the degree of aggregation can be increased, thereby increasing the difficulty of depolymerization.
  • Figures 8 and 9 are graphs of assays for the induction of platelet aggregation with different concentrations of THR. Use basically the same method as above, except replace ADP with THR.
  • the concentrations of THR were 0.1U, 0.15U, 0.2U, 0.23U, 0.25U, 0.28U, 0.30U and 0.35U, respectively. It can be seen that the effect of platelet aggregation is weak at low concentration of THR, and the platelet aggregation rate increases rapidly after 1 minute at high concentration.
  • Figures 10 and 11 are graphs of assays for the induction of platelet aggregation with different concentrations of COL. Use basically the same method as above, except replace ADP with COL.
  • the concentrations of COL were 0.05 mM, 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM and 20 mM, respectively. It can be seen that COL can rapidly aggregate platelets to nearly 100% at different concentrations, and the lower the concentration, the faster the aggregation.
  • the depolymerization effect of 1-acetylguanidine except for the samples with the lowest COL concentration, reached the highest immediately for other COL concentrations, but began to decrease significantly after about 4-8 minutes. This is also due to the conversion of soluble fibrinogen to insoluble fibrinogen after the addition of COL, resulting in blood coagulation.
  • Figures 12 and 13 are graphs of assays of platelet aggregation induced with different concentrations of RIS. Use basically the same method as above, except replace ADP with RIS.
  • the concentrations of RIS were 0.15 mM, 0.375 mM, 0.75 mM, 1.5 mM and 3 mM, respectively. It can be seen that the induction effect of RIS on platelet aggregation takes a long time to play out, and after 15-20 minutes, the degree of platelet aggregation begins to reach the highest level. At the same time, the depolymerization effect of 1-acetylguanidine reached the highest immediately for each RIS concentration, and was relatively stable within 10 minutes. The rapid drop in the depolymerization rate after 15 minutes was also due to blood coagulation in the sample.
  • the amino group-containing compound can have a good depolymerization effect on a variety of platelet aggregation conditions within a certain concentration and time range, and all can take effect quickly, which is convenient for immediate detection without additional reaction conditions (such as heating or longer reaction times).
  • this example determines the effect of the combination of the ammonium ion-containing compound and the amino group-containing compound on platelet depolymerization. Effect.
  • the difference is that the ammonium ion-containing compounds are replaced with ammonium chloride, 1-acetylguanidine and ammonium chloride with concentrations of 1, 2, 5, 10, 20 and 50 mmol/L respectively.
  • the two have synergy in platelet depolymerization. Therefore, when the two are used in combination, they are preferably used at lower concentrations (eg, as low as 1-20 mM, 1-10 mM, or even 1-5 mM, respectively).
  • Test Example 1 Detection of platelets and red blood cells by compounds containing ammonium ions in staining solution
  • This test example provides a staining solution for a blood cell analyzer, which can be used to exclude platelet aggregation from interfering with the detection of red blood cells and platelets.
  • This test example and the following test examples were all measured with a blood cell analyzer (BC-6000Plus) from Shenzhen Mindray Biomedical Co., Ltd. Use the following staining solution formulations:
  • ammonium chloride was added to make the final concentration 0.01mol/L.
  • control dyeing solution and dyeing solution A were prepared with distilled water at 25°C, the pH was adjusted to 9, and the osmotic pressure was 200 mOsm/L.
  • the temperature can be maintained at about 42 °C, using Fluorescence intensity information (FL) of blood sample cells after treatment was measured by lateral fluorescence at a measurement angle of 90°, and side scattered light intensity information of treated blood sample cells was measured by side scattered light at a measurement angle of 90° ( SS), the forward scattered light intensity information (FS) of the processed blood sample cells was measured by the forward scattered light at a measurement angle of 2°-5° to obtain a three-dimensional scattergram.
  • FL Fluorescence intensity information
  • SS side scattered light intensity information
  • FS forward scattered light intensity information
  • the fluorescence intensity (FL) of platelets in the 3D scattergram decreases
  • the forward scattering intensity information (FS) decreases
  • the volume of aggregated platelets is larger than that of individual platelets, and the fluorescence intensity of aggregated platelets is also enhanced.
  • the proportion of reticulocytes is calculated by calculating the percentage of the number of particles in the distribution area of reticulocytes to the number of particles in the mature red blood cell and reticulocyte area on the three-dimensional scattergram.
  • the reticulocytes have stronger fluorescent signals than normal red blood cells.
  • the total erythrocyte count measured in the unaggregated sample was 4.46 ⁇ 10 12 , of which reticulocytes accounted for 1.28% of the total erythrocytes, and the sample with stain A had a total erythrocyte count of 4.43 ⁇ 10 after deaggregation. 12.
  • the total red blood cell count was not disturbed by aggregated platelets.
  • the platelet counts measured in the unaggregated sample were: 274 ⁇ 10 9 ; in the unaggregated sample: 239 ⁇ 10 9 ; in the disaggregated sample: 42 ⁇ 10 9 .
  • Test Example 2 Detection of platelets and red blood cells by ammonium ion-containing compounds in diluents
  • This test example provides a diluent for a blood cell analyzer, which can be used to exclude platelet aggregation from interfering with the detection of red blood cells and platelets.
  • a diluent for a blood cell analyzer which can be used to exclude platelet aggregation from interfering with the detection of red blood cells and platelets.
  • ammonium chloride was added to make the final concentration 0.01mol/L.
  • control diluent and diluent A were prepared with distilled water at 25°C, the pH was adjusted to 9.5, and the osmotic pressure was 200mOsm/L.
  • test was carried out in the same manner as in Test Example 1, except that the samples were treated with the above control diluent and diluent A, and further added with a dye (SYBR Green, 50 mg/L) and a surfactant (N-alkyl betaine) , 0.01g/L) to stain nucleated cells and spheroidize blood cells to obtain 4 groups of scattergrams as shown in Figure 16.
  • a dye SYBR Green, 50 mg/L
  • a surfactant N-alkyl betaine
  • the measured total red blood cell count of the unaggregated sample was 5.72 x 10 12 , of which the proportion of reticulocytes in the total red blood cells was 1.02%, and the total red blood cell count after deaggregation was measured for the sample using Diluent A was 5.69 ⁇ 10 12 , the total red blood cell count was not disturbed by aggregated platelets.
  • the counts of platelets measured in the unaggregated sample were: 198 ⁇ 10 9 ; in the aggregated sample: 42 ⁇ 10 9 ; in the disaggregated sample: 190 ⁇ 10 9 .
  • Test Example 3 Detecting platelets with ammonium ion-containing compounds in diluent by impedance detection
  • This test example is basically the same as Test Example 2, except that the blood cell analyzer uses the impedance channel of Shenzhen Mindray Biomedical Co., Ltd. BC-6000Plus for detection, and the obtained histogram is shown in Figure 17.
  • the horizontal axis of the platelet histogram is the volume of platelets (unit fL), and the vertical axis is the number of platelets.
  • Test Example 4 The detection of leukocytes in lysate by compounds containing ammonium ions
  • This test example provides a lysis solution for a blood cell analyzer, which can be used to exclude platelet aggregation from interfering with the detection of red blood cells and platelets.
  • ammonium chloride was added to make the final concentration 0.01mol/L.
  • control lysate and lysate A were prepared with distilled water at 25°C, the pH was adjusted to 9, and the osmotic pressure was 90 mOsm/L.
  • Test Example 1 takes 1 ml of human venous blood samples collected in anticoagulation tubes, divide the samples into two groups, and use the hematology analyzer for measurement, in which the control lysate and lysate A were used, respectively, in 20 ⁇ l
  • the samples were mixed in a ratio of 1 ml of lysate, and then 20 ⁇ l of DNA stain (Hoechst33342) was added.
  • the temperature could be kept at about 42 °C, and the fluorescence intensity information of the treated blood sample cells ( FL), the side scattered light intensity information (SS) of the treated blood sample cells was measured using the side scattered light at a measurement angle of 90°, and the processed blood was measured using the forward scattered light at a measurement angle of 2°-5°.
  • the forward scattered light intensity information (FS) of the sample cells was obtained as a three-dimensional scattergram. Take another 1 ml of the above blood sample, add the platelet aggregation inducer ADP (final concentration is 0.01 mmol/L), and mix well. The platelets formed aggregates after 5 min. At this time, the samples were divided into two groups, which were also detected by a blood cell analyzer, treated with control lysate and lysate A respectively, and DNA dye was added, and a three-dimensional scattergram was obtained in the same way. As above, the four sets of scattergrams obtained from the measurement of this sample are shown in FIG. 18 .
  • the white blood cell count in the unaggregated sample was measured to be 7.49 ⁇ 10 9 , including 4.74 ⁇ 10 9 neutrophils, 2.16 ⁇ 10 9 lymphocytes, 0.42 ⁇ 10 9 monocytes, and eosinophils 0.14 ⁇ 10 9 .
  • the white blood cell count after depolymerization was 8.72 ⁇ 10 9
  • the differential count was 5.16 ⁇ 10 9 neutrophils, 2.57 ⁇ 10 9 lymphocytes, 0.61 ⁇ 10 9 monocytes, and eosinophils 0.32 ⁇ 10 9
  • the white blood cell count after disaggregation measured with Lysate A sample was 7.58 ⁇ 10 9 , including 4.79 ⁇ 10 9 neutrophils, 2.21 ⁇ 10 9 lymphocytes, 0.39 ⁇ 10 9 monocytes, and eosinophils 0.15 ⁇ 10 9 . It can be seen that the count and classification of white blood cells are not affected by platelet aggregation after using lysing agent A, and the count is more accurate.
  • Test Example 5 Combination of amino group-containing compound and ammonium ion-containing compound for white blood cell detection
  • This test example is tested according to the method of Test Example 4, the difference is that the lysate C is prepared to replace the lysate A, that is, on the basis of the control lysate, an equimolar amount of 1-acetylguanidine + NH 4 Cl is added to make the final The concentration is 0.01mol/L.
  • control lysate and lysate C were prepared with distilled water at 25°C, the pH was adjusted to 9, and the osmotic pressure was 90 mOsm/L.
  • the white blood cell count measured in the unaggregated sample was 6.19 ⁇ 10 9 , of which the differential count was 3.87 ⁇ 10 9 for neutrophils, 1.91 ⁇ 10 9 for lymphocytes, 0.22 ⁇ 10 9 for monocytes, and 0.22 ⁇ 10 9 for monocytes. Acid granulocytes 0.08 ⁇ 10 9 .
  • the white blood cell count after depolymerization was 6.13 ⁇ 10 9 , the differential count was 3.91 ⁇ 10 9 neutrophils, 1.88 ⁇ 10 9 lymphocytes, 0.21 ⁇ 10 9 monocytes, and eosinophils 0.07 ⁇ 10 9 .
  • the white blood cell count after disaggregation was 6.97 ⁇ 10 9
  • the differential count was 4.21 ⁇ 10 9 neutrophils, 2.21 ⁇ 10 9 lymphocytes, 0.34 ⁇ 10 9 monocytes, and eosinophils 0.17 ⁇ 10 9 . It can be seen that the white blood cell count and classification are not affected by platelet aggregation after using Lysis Agent C, and the count is more accurate.
  • Test Example 6 Detection of platelets and erythrocytes by staining solution comprising a combination of amino-containing compound and ammonium ion-containing compound
  • This test example was basically carried out in the same manner as in Test Example 1, except that the control dyeing solution was prepared according to the following composition, and dyeing solution C was used instead of dyeing solution B.
  • Test Example 2 The test was carried out in the same manner as in Test Example 1, except that ADP at a final concentration of 1 mol/L (100 times that in Test Example 1) was used to induce platelet aggregation in the sample. A three-dimensional scatter plot is obtained, as shown in Figure 20.
  • the total erythrocyte count in the unaggregated sample was measured to be 6.14 ⁇ 10 12 , in which the proportion of reticulocytes in the total erythrocytes was 1.34%, and the total erythrocyte count in the sample after platelet aggregation and disaggregation was induced was 6.11 ⁇ 10 12 , the total red blood cell count was not disturbed by aggregated platelets.
  • Measured platelet counts in unaggregated samples were: 219 ⁇ 10 9 ; counts in aggregated samples: 17 ⁇ 10 9 ; counts in deaggregated samples: 182 ⁇ 10 9 .
  • Test Example 7 Detection of platelets and erythrocytes using diluent containing amino-containing compound in combination with ammonium ion-containing compound
  • This test example was carried out in the same manner as in Test Example 2, except that the diluent C was replaced by the diluent B.
  • Diluent C is based on the control diluent, adding 1-acetylguanidine to make the final concentration 0.01mol/L, and adding ammonium chloride to make the final concentration 0.01mol/L. Adjust the pH of each dilution to 9.5 and the osmotic pressure to 200mOsm/L.
  • Test Example 21 The test was carried out in the same manner as in Test Example 1, except that ADP at a final concentration of 1 mol/L (100 times that in Test Example) was used to induce platelet aggregation in the sample. A 3D scatter plot is obtained, as shown in Figure 21.
  • the total erythrocyte count in the unaggregated sample was measured to be 5.12 ⁇ 10 12 , the proportion of reticulocytes in the total erythrocyte was 1.21%, and the total erythrocyte count in the sample after platelet aggregation and disaggregation was induced was 5.08 ⁇ 10 12 , the total red blood cell count was not disturbed by aggregated platelets.
  • the counts of platelets measured in unaggregated samples were: 278 ⁇ 10 9 ; in aggregated samples: 18 ⁇ 10 9 ; in deaggregated samples: 248 ⁇ 10 9 .
  • Test Example 8 Clinical Depolymerization Effect of Compounds Containing Ammonium Ion on Pseudo Platelet Aggregation
  • This test case is to achieve deaggregation of pseudoaggregated platelets in the clinic.
  • the detection was performed using the same diluent as Diluent A of Test Example 2.
  • the formula of the diluent is: citric acid (0.5g/L), surfactant phenoxyethanol (0.1g/L), bacteriostatic agent (6g/L), sodium chloride (3g/L), NH 4 Cl (0.01mol/L). Adjust the pH of the diluent to 9.5 and the osmotic pressure to 200mOsm/L.
  • the specific steps are: collect blood from outpatients and inpatients, use EDTA ⁇ K2 for anticoagulation, PLT-I platelet count is significantly reduced, blood smear microscopy finds platelet aggregation, and 17 specimens that meet the diagnostic criteria of EDTA-PTCP are in the experimental group , including 9 males and 8 females, with an average age of 51 years.
  • the blood of patients with platelet pseudo-aggregation is drawn. After blood is drawn without anticoagulant, it is immediately detected on a blood cell analyzer within 1 minute (the platelets do not aggregate at this time).
  • the platelet count measured by this method is the true value of platelets PLT_O (true value).
  • the patient's blood was drawn with an EDTA anticoagulant tube, and after 2 hours (the platelets of the pseudoaggregated patient would aggregate), the diluent containing the depolymerized substance 1-acetylguanidine was tested on the same blood cell analyzer.
  • the platelet count value is the platelet disaggregation value PLT_O (disaggregation).
  • the patient's blood was drawn with an EDTA anticoagulant tube, and after 2 hours (the platelets of the pseudoaggregated patient would aggregate), the same blood cell analyzer was used to detect the diluent without the depolymerized substance NH 4 Cl.
  • the obtained platelet count value is the non-disaggregated value of platelets PLT_O (non-disaggregated).
  • the depolymerization effect is calculated by the formula:
  • Depolymerization rate PLT_O (depolymerization)/PLT_O (true value); or
  • Depolymerization rate (control) PLT_O (not depolymerized)/PLT_O (true value).
  • the detection results of each sample are shown in FIG. 22 .
  • the pseudoaggregated platelets can be depolymerized to different degrees.
  • the depolymerization effect still reached 70% after the patient's blood was placed for 2 hours in 13 cases.
  • the depolymerization effect was all below 20%, and the depolymerization diluent containing 10 mM ammonium chloride had a significant depolymerization effect.

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Abstract

一种组合物在体外血液检测中防止和/或消除血液样品中血小板聚集的用途，其中，所述组合物包括至少一种含铵根离子的化合物。一种包括所述化合物的用于体外血液检测的抗血小板聚集干扰的试剂，以及在体外血液检测中防止和/或消除样品中血小板聚集的方法。所述化合物对多种血小板情聚集的情况均有解聚作用，在短时间内即可使血小板解聚，无需水浴控温和延长反应时间等额外条件，可方便地消除样品中血小板聚集，获得准确的血细胞的检测参数。

Description

一种组合物的血小板解聚用途、解聚试剂及解聚方法 技术领域
本发明涉及体外血液检测，特别涉及能消除血小板聚集的化合物在血小板解聚中的用途、包括该化合物的试剂及解聚方法。
背景技术
在血液细胞的分析中，血小板假性聚集会引起错误的血液细胞计数及分类，进而导致病人的错误诊断和治疗。我们很容易混淆血小板假性聚集与某些危及生命的疾病，比如肝素诱导的体内血小板聚集(HIP),弥漫性血管内凝血(DIC),或者产生错误的治疗决策，比如错误的用药，给患者进行不恰当的血小板输注，甚至脾切除手术。血小板假性聚集的原因繁多且复杂，常见原因包括：乙二胺四乙酸依赖性假性血小板减少(EDTA-PTCP)、多重抗凝剂依赖的血小板聚集、血小板卫星现象、高胆固醇和高三酰甘油血症、低温环境引起的血小板冷聚集、采血不顺和抗凝管的材质引起的聚集等。关于血小板假性聚集机理的研究不多，多数集中在EDTA-PTCP。EDTA是国际血液学标准化委员会(ICSH)认定的临床上广泛使用的抗凝剂，EDTA引起的血小板假性聚集最早于1969年首次报道，其主要原因可能是EDTA-PTCP患者的血液中存在依赖EDTA的抗血小板抗体，当血液在体外与EDTA抗凝剂混合时，这些抗体可以识别血小板膜上的黏连受体糖蛋白Ⅱb-Ⅲa(GpⅡb-Ⅲa)，引起血小板聚集激活抗原的表达，比如颗粒膜蛋白140(GMP140，别名CD62P或P-选择素),Ⅲ型溶酶体糖蛋白(Gp55，别名CD63)以及凝血酶敏感蛋白等，进而激活酪氨酸激酶，导致血小板聚集。
血小板假性聚集不仅导致血小板相关临床参数错误，还会影响到其他血细胞，特别是白细胞的临床参数的准确性，给临床诊断和治疗带来不利后果。因此，发现并消除血液样品中的血小板聚集，是体外血液检测中一直期望解决的问题。
目前，临床上已采取一些解决方案来消除或减少血小板假性聚集。例如，将血小板假性聚集的血液样品加热到37摄氏度，同时震荡一段时间，或者在血液样品中加入添加剂来阻止血小板聚集。这些添加剂比如，抗凝剂(如：柠檬酸钠、肝素钠、ACD、CTAD、CPT、CaCl 2和肝素钠的混合物等)；血小板计数稀释液(含有叠氮化钠、叠氮化钙、氟化钠等混合物)；抗血小板药物(在取血后10分钟内加入)；氨基糖苷类抗生素(如阿米卡星和卡那霉素，采血后1小时内加入，但仅对部分样品有效)。
这些防止离体血液样品中血小板聚集的方法和试剂，要么造成检测步骤复杂，要么解聚血小板的效果不够理想或者仅对部分原因造成的聚集有效果，往往需要重新采血并配制特定的解聚剂。在实践中，加入添加剂后还需要镜检观察血小板是否解聚，以确定解聚效果是否可以进行血液分析。必要时需要在冰浴或水浴中延长作用时间，以保证血小板的解聚效果。
因此，在体外血液检测中仍需要操作简单、普适性好的血小板解聚方法和试剂。
发明内容
针对上述体外血液检测中血小板假性聚集的问题，本发明人意外发现在血液样品中加入含有铵根阳离子的溶液获得了意想不到的防止血小板聚集以及对已发生血小板聚集的样品良好的解聚效果。因此，本发明意在提供此类物质在体外血液检测中预防和/或消除血液样品中血小板聚集的用途，以及用于该用途的一种操作简单、方便，能适用不同检测方式和设备的抗血小板聚集干扰的试剂。本发明的用于血小板解聚的物质和试剂能够消除多数血小板聚集的情况，效果稳定可靠，不需反复确认，且对正常的血液检测没有不利影响。
因此，本发明的第一方面，提供一种组合物在体外血液检测中防止和/或消除血液样品中血小板聚集的用途，其中，所述组合物包括至少一种含铵根离子的化合物。
根据具体实施方式，所述含铵根离子的化合物含有选自由氯离子、溴离子、碘离子、氢氧根、磷酸根、磷酸氢根、磷酸二氢根、硝酸根、硫氢根、硫氰酸根、硫酸根、硫酸氢根、亚硫酸根、亚硫酸氢根、碳酸根、碳酸氢根、甲酸根、乙酸根、草酸根、丙酸根、丙二酸根和柠檬酸根及其组合所组成的组中的阴离子。
更具体地，所述铵盐选自由氯化铵、溴化铵、碘化铵、磷酸铵、磷酸氢铵、磷酸二氢铵、硝酸铵、硫氰酸铵、亚硫酸酸氢铵、草酸铵、氢氧化铵、硫酸氢铵和碳酸氢铵所组成的组中的至少一种。
本发明中，所述防止和/或消除血液样品中血小板聚集在pH值至少为7.0的溶液中进行。优选地，所述pH值为7.5～11，更优选为9.5～11。
根据本发明，在进行所述防止和/或消除血液样品中血小板聚集的溶液中，所述组合物中所述含铵根离子的化合物的浓度为1～50mmol/L，优选2～20mmol/L。
根据一种实施方式，所述组合物进一步包括至少一种含氨基的化合物，所述含氨基的化合物含有至少一个伯氨基或仲氨基。优选地，所述含氨基的化合物含有1～20个选 自伯氨基、仲氨基和亚胺基的基团。
根据一种具体的实施方式，所述具有氨基的化合物选自由式(I)所示的化合物及其盐所组成的组：
R1-NH-R2   (I)
其中，R1和R2相同或不同，各自独立地为选自以下组中的基团，前提是R1和R2不同时为H，所述组由H、-SO 3H、-NH 2、-C(NH)-NH 2、取代或未取代的C1-16烷基、取代或未取代的C6-10芳基、取代或未取代的C7-14烷芳基、取代或未取代的C7-14芳烷基、-C(O)-Q1和-C(O)-O-Q2组成，
其中，Q1为H、-NH 2、取代或未取代的C1-16烷基、取代或未取代的C6-10芳基、取代或未取代的C7-14烷芳基或取代或未取代的C7-14芳烷基；
Q2为H、取代或未取代的C1-16烷基、取代或未取代的C6-10芳基、取代或未取代的C7-14烷芳基或取代或未取代的C7-14芳烷基；
其中，所述取代的是指被至少一个选自由-NP1P2、-SO 3H、-OH、卤素、-CN、-C(O)-O-P3、-O-C1-16烷基、-O-C6-10芳基、-O-C7-14烷芳基、-O-C7-14芳烷基、-C(O)-C1-16烷基、-C(O)-C6-10芳基、-C(O)-C7-14烷芳基、-C(O)-C7-14芳烷基和-C(O)-NP1P2组成的组中的基团所取代，其中所述-O-C1-16烷基、-O-C6-10芳基、-O-C7-14烷芳基、-O-C7-14芳烷基、-C(O)-C1-16烷基、-C(O)-C6-10芳基、-C(O)-C7-14烷芳基和-C(O)-C7-14芳烷基分别是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、卤素、-CN、-COOH和-C(O)NH 2组成的组中的基团所取代，
P1、P2和P3各自独立地为选自以下组中的基团：H、C1-16烷基、C6-10芳基、C7-14烷芳基和C7-14芳烷基，其中所述C1-16烷基、C6-10芳基、C7-14烷芳基和C7-14芳烷基分别是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、卤素、-CN、-COOH和-C(O)NH 2组成的组中的基团所取代。
更具体地，所述式(I)的化合物中，R1和R2相同或不同，各自独立地为选自以下组中的基团，前提是R1和R2不同时为H，所述组由H、-SO 3H、-NH 2、-C(NH)-NH 2、取代或未取代的C1-10烷基、取代或未取代的C6-10芳基、取代或未取代的C7-10烷芳基、取代或未取代的C7-10芳烷基、-C(O)-Q1和-C(O)-O-Q2组成，
其中，Q1为H、-NH 2、取代或未取代的C1-10烷基、取代或未取代的C6-10芳基、取代或未取代的C7-10烷芳基或取代或未取代的C7-10芳烷基；
Q2为H、取代或未取代的C1-10烷基、取代或未取代的C6-10芳基、取代或未取 代的C7-10烷芳基或取代或未取代的C7-10芳烷基；
其中，所述取代的是指被至少一个选自由-NP1P2、-SO 3H、-OH、-CN、-C(O)-O-P3、-O-C1-10烷基、-O-C6-10芳基、-O-C7-10烷芳基、-O-C7-10芳烷基和-C(O)-NP1P2组成的组中的基团所取代，其中所述-O-C1-10烷基、-O-C6-10芳基、-O-C7-10烷芳基和-O-C7-10芳烷基分别是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、-CN、-COOH和-C(O)NH 2组成的组中的基团所取代；
P1、P2和P3各自独立地为选自以下组中的基团：H、C1-10烷基、C6-10芳基、C7-10烷芳基和C7-10芳烷基，其中所述C1-10烷基、C6-10芳基、C7-10烷芳基和C7-10芳烷基分别是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、-CN、-COOH和-C(O)NH 2组成的组中的基团所取代。
更进一步地，所述式(I)的化合物中，R1和R2相同或不同，各自独立地为选自以下组中的基团，前提是R1和R2不同时为H，所述组由H、-SO 3H、-NH 2、-C(NH)-NH 2、取代或未取代的C1-10烷基、取代或未取代的C6-10芳基、取代或未取代的C7-10烷芳基、取代或未取代的C7-10芳烷基、-C(O)-Q1和-C(O)-O-H的组，
其中，Q1为H、-NH 2、取代或未取代的C1-10烷基、取代或未取代的C6-10芳基、取代或未取代的C7-10烷芳基或取代或未取代的C7-10芳烷基；
其中，所述取代的是指被至少一个选自由-NP1P2、-SO 3H、-OH、-CN、-C(O)-O-H和-C(O)-NP1P2组成的组中的基团所取代；
P1和P2各自独立地为选自以下组中的基团：H、C1-10烷基、C6-10芳基、C7-10烷芳基和C7-10芳烷基，其中所述C1-10烷基、C6-10芳基、C7-10烷芳基和C7-10芳烷基分别是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、-CN、-COOH和-C(O)NH 2组成的组中的基团所取代。
根据本发明，所述具有氨基的化合物具有的氨基的pKa在1～16，优选4～14的范围内。
根据较优的实施方式，所述具有氨基的化合物的氨基的pKa的值小于进行所述防止和/或消除血液样品中血小板聚集的溶液的pH值。
根据本发明，在进行所述防止和/或消除血液样品中血小板聚集的溶液中，所述具有氨基的化合物的浓度为1～50mmol/L，优选2～20mmol/L。
在本发明的用途中，所述体外血液检测包括选自以下项目中的至少一项检测：血小板、红细胞检测和白细胞检测。其中红细胞检测可进一步区分出网织红细胞。白细胞检 测中还可进行三分类、四分类或五分类的检测。更具体地，所述红细胞为成熟红细胞、网织红细胞和有核红细胞中的至少一种。所述白细胞是中性粒细胞、嗜酸性粒细胞、嗜碱性粒细胞、淋巴细胞和单核细胞中的至少一种。
所述检测设备可以包括阻抗检测器和/或光学检测器。
所述血液样品是来自哺乳动物的末梢血或静脉血。
根据本发明的第二方面，提供一种用于体外血液检测的抗血小板聚集干扰的试剂，其中所述试剂包括浓度为1～50mmol/L，优选2～20mmol/L的至少一种含铵根离子的化合物。
根据一种具体的实施方式，含铵根离子的化合物含有选自由氯离子、溴离子、碘离子、氢氧根、磷酸根、磷酸氢根、磷酸二氢根、硝酸根、硫氢根、硫氰酸根、硫酸根、硫酸氢根、亚硫酸根、亚硫酸氢根、碳酸根、碳酸氢根、甲酸根、乙酸根、草酸根、丙酸根、丙二酸根、柠檬酸根及其组合所组成的组中的阴离子。
更具体地，所述含铵根离子的化合物选自由氯化铵、溴化铵、碘化铵、磷酸铵、磷酸氢铵、磷酸二氢铵、硝酸铵、硫氰酸铵、亚硫酸酸氢铵、草酸铵、氢氧化铵、硫酸氢铵和碳酸氢铵所组成的组中的至少一种。
根据本发明，所述试剂的pH值至少为7.0，优选地，所述pH值为7.5～11，更优选为9.5～11。
根据一种实施方式，所述血小板解聚试剂进一步包括浓度为1～50mmol/L，优选2～20mmol/L的至少一种具有氨基的化合物，所述化合物含有至少一个伯氨基或仲氨基。优选地，所述具有氨基的化合物含有1～20个选自伯氨基、仲氨基和亚胺基的基团。
根据一种具体的实施方式，所述具有氨基的化合物选自由式(I)所示的化合物及其盐所组成的组：
R1-NH-R2   (I)
其中式(I)化合物如前文所定义。
根据本发明，所述具有氨基的化合物具有的氨基的pKa在1～16，优选4～14的范围内。
根据较优的实施方式，所述具有氨基的化合物的氨基的pKa的值小于所述试剂的pH值。
根据一种具体的实施方式，所述试剂进一步包括缓冲剂、渗透压调节剂和任选的选自表面活性剂、荧光染料和红细胞裂解剂中的至少一种。
根据本发明的第三方面，还提供一种在体外血液检测中防止和/或消除样品中血小板聚集的方法，所述方法包括用以上所定义的组合物或试剂对血液样品进行处理的步骤。任选地，所述方法还包括对经上述处理的血液样品用血液分析仪进行检测的步骤。
在本发明中，包含所述化合物的试剂、上述用途以及体外血液检测方法中对聚集的血小板的解聚作用，在短时间内即可使血小板解聚，无需水浴控温和延长反应时间等额外条件，因此也无需改变现有的检测步骤，适用于任何血液分析仪。而且，所述试剂对血小板以及其他检测用试剂无不良作用，且可在多种血小板聚集的情况取得较好的解聚效果，可方便地消除样品中血小板的假性聚集，获得准确的血细胞的检测参数。
附图说明
图1示出了具有氨基的化合物对聚集的血小板解聚和复聚的机理示意图；
图2示出了在光学检测设备中获得的血液样品中粒子的三维散点图，其中分别示出了聚集的血小板和解聚的血小板的光学信息特征；
图3示出了不同浓度下各种含铵根离子的化合物对ADP诱导的聚集的血小板的解聚效果；
图4示出了一种具有氨基的化合物在不同的环境pH条件下对聚集的血小板的解聚效果曲线图；
图5示出了在pH为9.5的条件下，具有不同氨基pKa值的化合物对聚集的血小板的解聚效果曲线图；
图6示出了ADP在不同终浓度下对血液样品中血小板聚集的诱导作用随时间变化的曲线图；
图7示出了具有氨基的化合物对以不浓度的ADP诱导的血小板聚集的解聚效果随时间变化的曲线图；
图8示出了THR在不同终浓度下对血液样品中血小板聚集的诱导作用随时间变化的曲线图；
图9示出了具有氨基的化合物对以不浓度的THR诱导的血小板聚集的解聚效果随时间变化的曲线图；
图10示出了COL在不同终浓度下对血液样品中血小板聚集的诱导作用随时间变化的曲线图；
图11示出了具有氨基的化合物对以不浓度的COL诱导的血小板聚集的解聚效果随 时间变化的曲线图；
图12示出了RIS在不同终浓度下对血液样品中血小板聚集的诱导作用随时间变化的曲线图；
图13示出了具有氨基的化合物对以不浓度的RIS诱导的血小板聚集的解聚效果随时间变化的曲线图；
图14示出了1-乙酰基铵和氯化铵各自及组合在不同浓度下对聚集的血小板的解聚效果；
图15示出了血液样品在诱导聚集前后在光学检测设备中使用常规染色液和含有氯化铵的染色液检测所述样品中血小板和红细胞的三维散点图；
图16示出了血液样品在诱导聚集前后在光学检测设备中使用常规稀释液和含有氯化铵的稀释液检测所述样品中血小板和红细胞的三维散点图；
图17、示出了血液样品在诱导聚集前后在阻抗检测设备中使用常规稀释液和含有氯化铵的稀释液检测所述样品中血小板的直方图；
图18、示出了血液样品在诱导聚集前后在光学检测设备中使用常规裂解液和含有氯化铵的裂解液检测所述样品中血小板和白细胞的三维散点图；
图19示出了血液样品在诱导聚集前后在光学检测设备中使用常规裂解液和含有具有氨基化合物和含铵根离子化合物的组合的稀释液检测所述样品中血小板和红细胞的三维散点图；
图20示出了血液样品在诱导聚集前后在光学检测设备中使用常规染色液和含有具有氨基化合物和含铵根离子化合物的组合的染色液检测所述样品中血小板和红细胞的三维散点图；
图21示出了血液样品在诱导聚集前后在光学检测设备中使用常规稀释液和含有具有氨基化合物和含铵根离子化合物的组合的稀释液检测所述样品中血小板和红细胞的三维散点图；
图22示出了常规稀释液和含有氯化铵的稀释液在临床中针对出现血小板假性聚集的样品的解聚效果对比图。
具体实施方式
下面将结合附图和具体实施例，对本发明实施方式中的技术方案进行清楚、完整地描述，显然，所描述的实施方式仅仅是本发明的一部分实施方式，而不是全部的实施方 式。基于本发明中的实施方式，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施方式，都属于本发明保护的范围。
在整个说明书中，除非另有特别说明，本文使用的术语应理解为如本领域中通常所使用的含义。因此，除非另有定义，本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾，本说明书优先。
需要说明的是，在本发明中，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的方法或者产品不仅包括所明确记载的要素，而且还包括没有明确列出的其他要素，或者是还包括为实施方法或者产品所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不消除在包括该要素的方法或者装置中还存在另外的相关要素。
本发明人发现，当溶液中含有铵根阳离子时，可使假性聚集的血小板解聚。进一步的研究发现，铵根阳离子对不同原因引起的血小板假性聚集有显著的解聚作用，而且不需额外加热或冷却，也不需长的作用时间，仅需在待测样品的溶液中加入少量该物质，大约30s之后血小板聚集就可以被消除。因此，本发明提供了该类物质防止和/或消除血液样品中血小板聚集的用途。解聚假性聚集的血小板不但可以获得血样中真实的血小板数量，从而为临床决策提供可靠的参考依据，还可消除血小板聚集而成的较大颗粒对其他血细胞，特别是白细胞的分类和计数造成的影响。
根据本发明，当在发生血小板假聚的血液样品中加入含有铵根离子的溶液时，可快速使血小板解聚。由以下实施例可知，各类铵盐在一定的pH下，对于聚集的血小板均具有大体相近的解聚效果。根据本发明所述含铵根离子的化合物可含有选自由氯离子、溴离子、碘离子、氢氧根、磷酸根、磷酸氢根、磷酸二氢根、硝酸根、硫氢根、硫氰酸根、硫酸根、硫酸氢根、亚硫酸根、亚硫酸氢根、草酸根、碳酸根、碳酸氢根、甲酸根、乙酸根、丙酸根、丙二酸根、柠檬酸根及其组合所组成的组中的阴离子。
不希望受理论的束缚，铵根离子解聚血小板的机理尚不清楚。推测与溶液中，铵根离子形成水合氨的浓度有关。但是如果直接使用氨水，虽然也有较好的解聚性能，却比使用含铵根离子的化合物，特别是诸如氯化铵、磷酸铵和卤化铵等铵盐，要差。因此，推测溶液中一些种类的阴离子可能也对血小板解聚起到一定的促进作用。但是溶液中不存在铵根离子时，这些阴离子却没有明显的解聚作用。
可列举的含铵根离子的化合物可以是氯化铵、溴化铵、碘化铵、磷酸铵、磷酸氢铵、磷酸二氢铵、硝酸铵、硫氰酸铵、亚硫酸氢铵、草酸铵、氢氧化铵、硫酸氢铵、碳酸氢 铵等，但不限于此。较为优选的含铵根离子的化合物如氯化铵、溴化铵、磷酸铵、磷酸氢铵等。
根据本发明提供一种组合物在体外血液检测中防止和/或消除血液样品中血小板假性聚集中的用途。所述组合物包括至少一种上述含铵根离子的化合物。当然也可以包括两种或更多种的含铵根离子的化合物。
本文中提及的包括至少一种含铵根离子的化合物的组合物，可以是所述化合物本身的形式，也可以是溶液形式，例如可溶解在适当的溶剂中，以方便地用于本发明的用途中。合适的溶剂可以是水、醇(如乙醇)、醇的水溶液等常用的且对血液样品的检测无干扰的溶剂。
所述的至少一种含铵根离子的化合物在含有血液样品的溶液中以一定浓度发挥对血小板的解聚作用。在本发明的用途中，所述组合物中含铵根离子的化合物的浓度可在1～50mmol/L的范围内，例如1、2、5、10、15、20、25、30、35、40、50mmol/L。优选含铵根离子的化合物的浓度在2～20mmol/L，更优选在5～15mmol/L的范围内。
本发明人发现，铵根离子在为9.5以上时具有最佳效果。因此，所述含铵根离子的化合物进行所述解聚时，环境pH最好在7.0以上，优选7.5～11.0，更优选9.5～11.0。
根据本发明，可以使用一种所述含铵根离子的化合物，也可同时使用两种或更多种所述化合物进行血小板解聚。当使用两种以上所述化合物时，则各化合物的总浓度符合以上范围。如前所述，血小板假性聚集的原因多种多样，聚集程度和解聚难度也有很大差异。因此，所述含铵根离子的化合物起效的浓度范围较大。对于较易解聚的情况，可使用较低的浓度，相反较难解聚或聚集程度高的情况，可使用较高的浓度。合适浓度的选取，本领技术人员根据以下具体实施例结合实际需要可以合理确定。
进一步地，本发明人还意外发现一类含有氨基的化合物同样对不同原因引起的血小板假性聚集有显著的解聚作用。并且当含铵根离子化合物与具有氨基的化合物组合使用时，可显著提高对聚集的血小板的解聚效果。
本文所述“氨基”除非特别指明的情况下指伯氨基和/或仲氨基，而不包括叔氨基。
本文所述“含氨基的化合物”除非特别指明，指至少含有一个氨基(伯氨基和/或仲氨基)的化合物。该化合物还可选地含有叔氨基和/或亚胺基，尤其是还可选地含有亚胺基。
不希望受理论的束缚，所述具有氨基和/或亚胺基的化合物对假性聚集的血小板的解聚原理推测为，化合物中的氨基/亚胺基在溶液中结合到血小板的磷脂酰丝氨酸层膜，并与磷脂酰丝氨酸层膜上的羰酯基团(-O-C(O)-)通过氢键结合(参见图1，其中图示了具 有氨基化合物的解聚原理)。这样结合到磷脂酰丝氨酸层膜的化合物的氨基会封闭钙离子通道，阻止钙离子的流入。而已知血小板的聚集需要钙离子流入的参与，因此这类化合物通过氨基阻止钙离子的流入使聚集的血小板解聚。当pH逐渐降低时，所述化合物上的氨基质子化。虽然不清楚具体原因，但推测化合物上质子化的氨基不再和磷脂酰丝氨酸层膜上的羰酯基团通过氢键结合，钙离子重新流入血细胞内，血小板又产生聚集。理论上，除叔氨基外，N原子上连接至少一个氢原子的伯氨基、仲氨基或亚胺基均具有一定的血小板解聚功能。
本发明人进一步的研究发现，具有氨基的化合物中，氨基/亚胺基的数量越多，解聚效果就越好。此外，伯氨基、仲氨基和亚胺基单独的解聚效果依次降低。通常来说，亲水性强的化合物解聚效果也高于亲水性弱的化合物。亲水性取代基，如羟基、羧基、酰胺基、磺酸基等取代基对氨基/亚胺基的解聚效果大体没有影响，有的情况(如羟基、多羟基)甚至利于解聚。这可能与这些基团增加了化合物的水溶性以及与磷脂酰丝氨酸层膜的亲和性有关。烃基，如烷基、芳基等基团，只要不影响化合物一定的水溶性，对血小板解聚的影响不大。疏水性基团，例如酯基(-C(O)-O-)、羰基(-C(O)-)在一定程度上会降低解聚效果。
因此，较优选具有诸如羟基、羧基、磺酸基的化合物，如醇胺类、氨基酸类、磺酸类等物质。此外，具有多个氨基和/或亚胺基的物质也是优选的如胍类，短肽等。优选所述具有氨基化合物至少具有一个伯氨基或仲氨基，优选具有1-20个伯氨基、仲氨基和/或亚胺基。优选具有氨基化合物至少具有一个伯胺基，更优选具有1～4个伯胺基。
根据以上推测的机理，具有氨基的化合物解聚时其中的氨基以去质子化的形式存在于溶液中。不同化合物中的氨基/亚胺基结合溶液中氢离子的能力不同。这种结合/解离氢离子的能力可以用解离常数pKa表示。
具有氨基化合物在溶液中存在如下平衡式：
Figure PCTCN2020103133-appb-000001
pKa＝lg([R1R2NH]×[H +]/[R1R2NH 2 +])。
要获得合适的血小板解聚效果，所述化合物的氨基应具有合适的pKa值。对于具有多个氨基/亚胺基的化合物来说，本文所说的氨基的pKa是指第一解离常数，即第一个解离氢离子的氨基或亚胺基的pKa。
通常所述化合物的氨基应具有1～16的pKa值，优选1～14，更优选4～14。
此外，对于同一种化合物来说，该化合物中氨基的pKa值小于溶液的pH值时，解聚能力得到提升。根据上述化合物对聚集的血小板的解聚原理，可以理解，当pH逐渐升高，化合物中的氨基在溶液中与氢离子结合和解离的平衡向解离的方向移动，更多的氨基去质子化，因而更多氨基通过氢键结合到血小板的磷脂酰丝氨酸层膜，从而阻断钙的流入，起到解聚的作用。而这样的结合进一步推动更多的化合物氨基的去质子化，从而在较短时间内，不需升高温度，就可完成解聚。
本发明人发现，上述具有氨基的化合物对聚集的血小板进行解聚时受到pH的影响，但是在含铵根离子的化合物能够发挥血小板解聚作用的pH范围内，即7.0以上，优选7.5～11，特别优选为9.5～11的范围内时，多数也具有较好的解聚作用。这是因为pH也高时，也就越容易符合上述化合物中氨基的pKa小于等于溶液的pH值时解聚作用加强的规律。因此可选择合适的具有氨基的化合物的种类，以获得所需的解聚效果。
具有氨基的化合物能够解聚假性聚集的血小板的起效浓度与所述化合物中含有的氨基数量和种类有关，也与化合物的亲水性等因素相关。通常来说，具有氨基的化合物进行血小板解聚的浓度在1～50mmol/L的范围，例如：1、2、5、10、15、20、25、30、35、40、50mmol/L内。根据优选的实施方式，所述浓度在2～20mmol/L的范围内，更优选的在5～15mmol/L的范围内。
这样的具有氨基的化合物可为具有式(I)所示的结构的化合物，也可为其盐。式(I)为：
R1-NH-R2    (I)
其中，R1和R2相同或不同，各自独立地为选自以下组中的基团，前提是R1和R2不同时为H，所述组由H、-SO 3H、-NH 2、-C(NH)-NH 2、取代或未取代的C1-16烷基、取代或未取代的C6-10芳基、取代或未取代的C7-14烷芳基、取代或未取代的C7-14芳烷基、-C(O)-Q1和-C(O)-O-Q2组成，
其中，Q1为H、-NH 2、取代或未取代的C1-16烷基、取代或未取代的C6-10芳基、取代或未取代的C7-14烷芳基或取代或未取代的C7-14芳烷基；
Q2为H、取代或未取代的C1-16烷基、取代或未取代的C6-10芳基、取代或未取代的C7-14烷芳基或取代或未取代的C7-14芳烷基；
其中，所述取代的是指被至少一个选自由-NP1P2、-SO 3H、-OH、卤素、-CN、-C(O)-O-P3、-O-C1-16烷基、-O-C6-10芳基、-O-C7-14烷芳基、-O-C7-14芳烷基、-C(O)-C1-16烷基、-C(O)-C6-10芳基、-C(O)-C7-14烷芳基、-C(O)-C7-14芳烷基和 -C(O)-NP1P2组成的组中的基团所取代，其中所述-O-C1-16烷基、-O-C6-10芳基、-O-C7-14烷芳基、-O-C7-14芳烷基、-C(O)-C1-16烷基、-C(O)-C6-10芳基、-C(O)-C7-14烷芳基和-C(O)-C7-14芳烷基分别是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、卤素、-CN、-COOH和-C(O)NH 2组成的组中的基团所取代，
P1、P2和P3各自独立地为选自以下组中的基团：H、C1-16烷基、C6-10芳基、C7-14烷芳基和C7-14芳烷基，其中所述C1-16烷基、C6-10芳基、C7-14烷芳基和C7-14芳烷基分别是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、卤素、-CN、-COOH和-C(O)NH 2组成的组中的基团所取代。
根据一种实施方式，其中，所述式(I)的化合物中，R1和R2相同或不同，各自独立地为选自以下组中的基团，前提是R1和R2不同时为H，所述组由H、-SO 3H、-NH 2、-C(NH)-NH 2、取代或未取代的C1-10烷基、取代或未取代的C6-10芳基、取代或未取代的C7-10烷芳基、取代或未取代的C7-10芳烷基、-C(O)-Q1和-C(O)-O-Q2组成，
其中，Q1为H、-NH 2、取代或未取代的C1-10烷基、取代或未取代的C6-10芳基、取代或未取代的C7-10烷芳基或取代或未取代的C7-10芳烷基；
Q2为H、取代或未取代的C1-10烷基、取代或未取代的C6-10芳基、取代或未取代的C7-10烷芳基或取代或未取代的C7-10芳烷基；
其中，所述取代的是指被至少一个选自由-NP1P2、-SO 3H、-OH、-CN、-C(O)-O-P3、-O-C1-10烷基、-O-C6-10芳基、-O-C7-10烷芳基、-O-C7-10芳烷基和-C(O)-NP1P2组成的组中的基团所取代，其中所述-O-C1-10烷基、-O-C6-10芳基、-O-C7-10烷芳基和-O-C7-10芳烷基分别是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、-CN、-COOH和-C(O)NH 2组成的组中的基团所取代；
P1、P2和P3各自独立地为选自以下组中的基团：H、C1-10烷基、C6-10芳基、C7-10烷芳基和C7-10芳烷基，其中所述C1-10烷基、C6-10芳基、C7-10烷芳基和C7-10芳烷基分别是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、-CN、-COOH和-C(O)NH 2组成的组中的基团所取代。
优选地，所述式(I)的化合物中，R1和R2相同或不同，各自独立地为选自以下组中的基团，前提是R1和R2不同时为H，所述组由H、-SO 3H、-NH 2、-C(NH)-NH 2、取代或未取代的C1-10烷基、取代或未取代的C6-10芳基、取代或未取代的C7-10烷芳基、取代或未取代的C7-10芳烷基、-C(O)-Q1和-C(O)-O-H的组，
其中，Q1为H、-NH 2、取代或未取代的C1-10烷基、取代或未取代的C6-10芳基、 取代或未取代的C7-10烷芳基或取代或未取代的C7-10芳烷基；
其中，所述取代的是指被至少一个选自由-NP1P2、-SO 3H、-OH、-CN、-C(O)-O-H和-C(O)-NP1P2组成的组中的基团所取代；
P1和P2各自独立地为选自以下组中的基团：H、C1-10烷基、C6-10芳基、C7-10烷芳基和C7-10芳烷基，其中所述C1-10烷基、C6-10芳基、C7-10烷芳基和C7-10芳烷基分别是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、-CN、-COOH和-C(O)NH 2组成的组中的基团所取代。
进一步地，所述式(I)的化合物中，R1和R2相同或不同，各自独立地为选自以下组中的基团，前提是R1和R2不同时为H，所述组由H、-C(NH)-NH 2、取代或未取代的C1-6烷基、取代或未取代的苯基、取代或未取代的苯甲基、取代或未取代的苯乙基和-C(O)-Q1组成，其中Q1如上定义。
更进一步地，所述式(I)的化合物中，Q1为取代或未取代的C1-6烷基、取代或未取代的苯基、取代或未取代的苯甲基或取代或未取代的苯乙基；
其中，所述取代的是指被至少一个选自由-NP1P2、-SO 3H、-OH和-C(O)-NP1P2组成的组中的基团所取代，
P1、P2和P3各自独立地为选自以下组中的基团：H、C1-6烷基、苯基、苯甲基和苯乙基，其中所述C1-6烷基、苯基、苯甲基和苯乙基是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、-COOH和-C(O)NH 2组成的组中的基团所取代。
本文所述C1-16的烷基可以是直链或支链的烷基，优选C1-14、C1-12的烷基，进一步优选C1-10的烷基，更优选C1-6的烷基。可以列举的有甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基、异戊基、新戊基、正己基、正辛基、十二烷基、十六烷基等，但不限于此。
本文所述C6-10芳基可以是苯基或萘基。
本文所述C7-14烷芳基可以单烷基或多烷基取代的芳基。优选C7-10的烷芳基，诸如甲苯基、乙苯基、丙苯基、丁苯基、二甲苯基、甲乙苯基、二乙苯基等，但不限于此。
本文所述C7-14芳烷基可以苯基烷基。优选C7-10的芳烷基，诸如苄基、苯乙基、苯丙基、苯丁基等，但不限于此。
本文所述取代的是指被所限定的取代基中的至少一个取代基所取代。对于氨基取代基-NP1P2，根据本发明优选可为多氨基取代，可多至20个氨基。例如1～18个，如2、3、4、5、6、8、10、12、14、16个等。优选所述氨基为伯氨基和/或仲氨基，更优选为伯氨 基。对于羟基取代基-OH，是较优选的取代基。羟基的取代数目可以是1～6个，如2、3、4、5、6个。此外，磺酸基、羧基、腈基也较为优选。
本文所述卤素通常指-F、-Cl、-Br、-I，优选-F、-Cl、-Br。
根据以下实施例，较优的具有氨基的化合物是胍类、醇胺类(特别是多羟基醇胺类，如1,3-二氨基-2-丙醇)，氨基酸(优选赖氨酸、谷氨酰胺、甘氨酸)、短肽(如二肽)以及多氨基取代的多支链或直链烷烃(如2-(氨基甲基)丙烷-1,3-二胺、1,3-丙二胺等)。
可列举的防止和/或消除血小板假性聚集的式(I)化合物可为：磺酸类，如氨基磺酸、氨基甲磺酸、牛磺酸、对氨基苯磺酸、2,5-二氨基苯磺酸；氨基酸类，如谷氨酸、谷氨酰胺、精氨酸、赖氨酸、丙氨酸、甘氨酸、N-三(羟甲基)甲基甘氨酸、4-氨基丁酸；短肽，如二肽，如二聚赖氨酸、二聚甘氨酸；醇/酚胺类(特别是多羟基取代的胺类)，如乙醇胺、三乙醇胺、3-氨基-1-丙醇、4-氨基-1-丁醇、4-羟基苄胺、酪胺(4-羟基苯乙基胺)、1,3-二氨基-2-丙醇、3-氨基-1,2-丙二醇、三羟甲基氨基甲烷、对羟基苯胺、3,5-二羟基苯胺、4-氨基苯酚；芳胺类，如间苯二胺、间苯三胺；烷基胺类，如乙胺、正丙胺、正丁胺、叔丁胺、1,3-丙二胺、2-(氨基甲基)丙烷-1,3-二胺；氨基腈类，如氨基乙腈、三氨基丙腈；酰胺类，如甲酰胺、乙酰胺、碳酰胺、天冬酰胺甲酯、2-(甲氨基)丁二酰胺、2-氨基乙酰胺、2-氨基丙酰胺、谷氨酰胺；胍类，如胍、双胍、氨基胍、甲基胍、{[氨基(亚氨基)甲基]胺}-乙酸、1-乙酰基胍等，但不限于此。
较优选的式(I)化合物可为羟基取代的胺类、烷基胺类、苯磺酸胺类、氨基酸和短肽、以及胍类。
所述具有氨基的化合物的盐也在本发明的范围内。特别是含有酸性的磺酸基、羧基等基团的化合物的盐。所述盐可以是碱金属(如钾、钠)的盐、碱土金属(如镁)的盐、铵根阳离子的盐等；也可以是内盐。由于上述铵根阳离子对血小板解聚的作用，铵根阳离子的盐是特别优选的。
在本发明的用途中，当含铵根离子的化合物与所述含氨基的化合物组合时，可在较低浓度下可获得良好的解聚效果。即，二者的总浓度与单独使用任何一种时的浓度相当时，可获得比单独使用含铵根离子的化合物或含氨基的化合物更好的效果。二者组合使用对血小板解聚具有协同的作用。因此，当二者组合使用时，二者的总浓度可在2～50mmol/L的范围内，例如1、2、5、10、15、20、25、30、35、40、50mmol/L。优选地，所述组合物可包括1～20mmol/L的含氨基的化合物和1～20mmol/L的含铵根离子的化合物，更优选所述组合物可包括1～10mmol/L的含氨基的化合物和1～10mmol/L的含铵根 离子的化合物，甚至包括1～5mmol/L的含氨基的化合物和1～5mmol/L的含铵根离子的化合物。二者的使用比例没有特别限制。作为例子，具有氨基的化合物和含铵根离子的化合物的摩尔比可在1:10到10:1的范围内，优选在1:4到4:1的范围内以是1:4、1:2、1:1、2:1、4:1等。最优选，二者的摩尔比为1:1。
从以下的实施例可以看出，当组合使用含氨基的化合物和含铵根离子的化合物时，取得了显著提升的解聚效果。
这种组合对于体外血液检测的应用是特别有利的，因为用于血小板解聚的物质浓度得到显著降低。周知，季铵盐类表明活性剂对细胞膜，特别是红细胞的细胞膜是有破坏作用的，因而通常被用作红细胞裂解剂。本发明人观察到，本发明的组合物在上述浓度范围内对红细胞没有破坏作用，在使用所组合物和不使用所述组合物的情况下，红细胞计数基本不变。特别地，在上述具有氨基的化合物和含铵根离子的化合物组合使用的优选浓度下，所述组合物不但可以获得优异的血小板解聚效果，而且对血细胞无任何不良作用，特别对红细胞没有影响。
根据本发明的用途，所述体外血液检测是指对血液样品中的血细胞的检测，诸如血小板、红细胞和白细胞等。本发明用途中，所述检测可以是镜检，也可以是用仪器检测，优选用仪器检测。具体地，所述检测可以是例如使用血液分析仪对各种血液中的细胞进行检测，并获得相应的检测信息。本发明的用途对于血液分析仪没有任何限制，任何用于体外血液检测的仪器均可用于本发明的用途。
本发明的用途对于检测器也没有限制，诸如阻抗检测器、光学检测器均可使用。
以光学检测器为例，多个聚集的血小板在血液分析仪的光学通道中被识别为一个粒子，因此这种聚集的血小板的体积比单个血小板大，荧光强度比单个血小板强，数量较未聚集的血小板少。反应在检测信息中，参见图2所示的在迈瑞血细胞分析仪中测得的三维散点图中可以看到，聚集的血小板聚的样品显示出血小板前向散射光强度信息(FS)偏大，反映出粒子体积偏大，另外荧光强度(FL)高。部分聚集的血小板的体积达到白细胞的体积，还会影响到白细胞的计数。解聚后，血小板在光学通道中，粒子信号识别的体积会减小，荧光强度有所减弱，数量增加。进一步参见图2，从三维散点图可以看到，解聚后血小板的粒子群的反映出前向散射光(FL)对应的血小板体积明显减小，粒子数量增加，荧光强度降低。
对于阻抗检测器，类似地，聚集的血小板也会被作为一个粒子，产生一个电信号。由于聚集后粒子体积变大，而不能被识别为血小板，而导致血小板计数偏低。此外，聚 集的血小板还会影响白细胞和红细胞的计数及直方图的变化。这种影响在早期的三分类血液分析仪中体现得较为明显。
根据本发明的用途，通过在样品中加入上述组合物，可防止血小板聚集的情况，也可使已经聚集的血小板解聚，因而可获得准确的血小板计数。
此外，由于不再存在聚集的血小板，还可同时消除对白细胞检测的干扰，因而可获得准确的白细胞的检测信息。所述白细胞检测还包括对例如有核细胞、嗜碱性粒细胞、嗜酸性粒细胞、中性粒细胞和/或淋巴细胞的分类和/或计数。
本文所述“检测信息”包括原始信号(如电脉冲、光强度等)，经处理获得的诸如直方图、散点图等信息，以及最终报告的分类和/或计数的信息。
另外，由于血液中红细胞数量远大于其他类型的细胞，因此在光学法检测中，虽然血小板聚集对红细胞有所影响，但是影响较小，对于计数结果几乎不产生影响。但是胺类物质，以及铵盐的加入有可能会导致红细胞裂解。对于本发明，由以下实施例可知，所述含铵根离子的化合物以及进一步的具有氨基的化合物，在以上定义的浓度范围内不会对样品环境(如pH、离子强度等)产生不利影响，也不会造成对血细胞的破坏。因此，血小板解聚后对红细胞的检测也没有影响。本发明的用途可进一步对成熟红细胞、网织红细胞和有核红细胞分类并分别计数。
本发明的用途对于不同原因导致的血小板聚集均有效果，因此对于用于所述用途的样品没有特别限制。所述样品可以来自外周血，如来自末梢血或静脉血。样品的获得方式也没有特别限制，如指尖针刺取血或用抗凝管静脉取血均可。新鲜采集的血液可使用涂有诸如EDTA等常规抗凝剂的抗凝试管，因为本发明的组合物对于由于EDTA诱导的血小板假性聚集也有很好的效果。
通常来说，取血后样品可进行任何常规处理，以获得待测试样。所述处理可以是例如：稀释处理、染色处理、裂解处理等。本发明的用途中，所述组合物可单独作为一种处理剂(如作为血小板解聚剂)加入样品中，或在对所述样品进行处理的任何一个步骤中随该步处理剂一起加入样品中。因此，本发明的组合物可形成为一种单独的处理剂；也可通过在用于稀释、染色或裂解处理的试剂中包含所述含氨基的化合物和任选的含铵根离子的化合物，形成具有血小板解聚效果的稀释剂、染色剂或裂解剂，从而在任何一步处理中同时发挥防止/消除血小板解聚的作用，而不会妨碍所述处理。所述组合物对血小板解聚的反应不需额外的保温(如低温或加热)。将含所述组合物的处理剂与样品混匀后大约30秒就能完成血小板的解聚，即可对样品进行检测。
根据本发明，所述样品可来自哺乳动物，优选灵长类动物，更优选人。
由此，本发明还提供一种在体外血液检测中防止和/或消除样品中血小板聚集的方法，所述方法包括对血液样品进行预防/消除血小板聚集干扰的处理步骤。所述处理步骤中采用包括上述含铵根离子的化合物和任选的具有氨基的化合物的组合物或试剂在上述条件下处理所述血液样品。
更具体地，本发明提供一种抗血小板聚集干扰的体外血的检测方法。
所述方法可包括用包括至少一种选自由含铵根离子和任选的含氨基的化合物的化合物的解聚剂，对血液样品进行防止和/或消除所述血液样品中的血小板聚集的处理，并对经所述解聚剂处理的血液样品用血液分析仪进行检测，以至少获得消除血细胞聚集干扰后的血细胞的检测信息。
所述血细胞可为血小板、红细胞和/或白细胞。
所述检测信息，根据所用检测器不同，可以是针对样品中粒子的脉冲信号，也可以是光学信号。所述血液分析仪可对所述检测信息进行处理进一步获得诸如直方图、散点图、分类和/或计数等血细胞的检测参数。
根据一种实施方式，所述血液分析仪可包括阻抗检测器。所述方法可包括以下步骤：经所述解聚剂处理的血液样品用血液分析仪中的阻抗检测器进行检测，以获得所述血液样品中粒子的电信号，并进一步获得血小板和/或红细胞的检测参数。
此外，仍利用所述阻抗检测器进行检测，所述血液样品在进行所述检测前除使用解聚剂进行处理外还可进一步用红细胞裂解剂进行溶血处理，且所述方法包括以下步骤：经所述解聚剂和红细胞裂解剂处理的血液样品用血液分析仪中的阻抗检测器进行检测，以获得所述血液样品中粒子的电信号，并进一步获得白细胞检测参数。
由阻抗检测器可获得白细胞的三分类及计数。
根据另一种实施方式，所述血液分析仪包括光学检测器，所述方法包括经所述解聚剂处理的血液样品用血液分析仪中的光学检测器进行检测，以获得所述血液样品中粒子的至少一种散射光强度信号，并进一步获得血小板的检测参数；优选地，所述至少一种散射光强度信号是前向散射光强度信号。更优的，可通过前向和侧向两种散射光强度信号获得血小板的检测参数。
根据又一种实施方式，仍使用所述光学检测器，所述血液样品在进行所述检测前进一步用荧光染料进行染色处理，且所述方法包括：对经所述解聚剂和荧光染料处理的血液样品用血液分析仪中的光学检测器进行检测，获得所述血液样品中粒子的荧光强度信 号和至少一种散射光强度信号，以获得血小板和/或红细胞的检测参数。
优选地，在该实施方式中，进一步包括区分出网织红细胞并获得网织红细胞的检测参数，更优选地，所述至少一种散射光强度信号是前向散射光强度信号。也可以利用荧光强度信号、前向散射光强度信号和侧向散射光强度信号获得血小板和/或红细胞的检测参数
根据再一种实施方式，仍使用所述光学检测器，所述血液样品进一步用荧光染料进行染色处理并用红细胞裂解剂进行溶血处理，且所述方法包括：对经所述解聚剂、荧光染料和红细胞裂解剂处理的血液样品用血液分析仪中的光学检测器进行检测，获得所述血液样品中粒子的至少两种光信号，以获得白细胞的检测信息。
优选地，在该实施方式中，所述所述至少两种光信号选自荧光强度信号、前向散射光强度信号和侧向散射光强度信号。
在白细胞检测中，例如使用迈瑞血液检测仪的DIFF检测通道可获得四分类(即，嗜酸性粒细胞、中性粒细胞、单核细胞和淋巴细胞)，或者进一步使用如迈瑞血液检测仪中的WNB通道获得嗜碱性粒细胞的检测信息。
在上述检测方法中，所述解聚剂、荧光染料以及红细胞裂解剂可为单独的试剂，也任意组合为混合试剂。例如所述解聚剂可形成为具有血小板解聚效果的稀释剂、染色剂、裂解剂或染色-裂解剂等。
因此，本发明还提供一种用于体外血液检测的抗血小板聚集干扰的试剂。
由于在体外血液检测中，用于处理血液样品的试剂体积远大于血液样品本身的体积，因此前文所述的用途的浓度、pH等处理条件大体同样适用于本发明的所述抗血小板聚集干扰的试剂。
根据本发明，所述试剂可包括1～50mmol/L的至少一种所述含铵根离子的化合物，还可进一步包括1～50mmol/L的至少一种所述具有氨基的化合物。本发明中所定义的浓度对于两种或更多种含铵根离子的化合物来说是其总浓度。同样的，所述浓度对于两种或更多种具有氨基的化合物来说也是其总浓度。所述至少一种含铵根离子的化合物如上文中所定义，在此不再赘述。其浓度也同样优选2～20mmol/L，更优选5～10mmol/L。
进一步含有的至少一种具有氨基的化合物也如上文所定义，同时其浓度优选2～20mmol/L，更优选5～10mmol/L。根据较优的实施方式，所述试剂既含有含铵根离子的化合物也含有具有氨基的化合物，二者的浓度范围均可在1～20mmol/L、1～10mmol/L， 甚至1～5mmol/L的范围内变化。二者的摩尔比可以是任何比例，例如可在含铵根离子的化合物:具有氨基的化合物为1:10至10:1，优选1:4至4:1的范围内变化。
所述试剂的pH没有特别限制，与其试剂使用要求相关。优选地，所述试剂的pH至少为7.0，优选为7.5～11，更优选为9.5～11.0。所述试剂的pH可通过常规的缓冲剂来调节。本发明对于用于所述试剂的缓冲剂的种类没有特别限制。例如可以使用柠檬酸缓冲对、磷酸缓冲对、硼酸缓冲对、邻苯二甲酸盐缓冲对、3-(N-吗啡啉)乙磺酸缓冲对、4-羟乙基哌嗪乙磺酸缓冲对等，但不限于此。
所述试剂应具有一定的渗透压。所述渗透压可根据所述试剂的实际要求变化，例如对于稀释剂，可在180～240mOsm/L的范围内优选200mOsm/L；再如对于裂解剂，可在70～130mOsm/L范围内，优选90mOsm/L。所述试剂可包括渗透压调节剂。本发明的试剂对渗透压调节剂没有特别限制，例如可以是无机盐，如氯化钠、氯化钾等；糖类，如葡萄糖、甘露糖、果糖、麦芽糖等，但不限于此。
所述试剂还可包括表面活性剂。根据检测目的的不同，所述试剂可包括不同功能的表面活性剂。例如对于红细胞检测，作为稀释液的试剂中可包括使红细胞球形化的表面活性剂。这样的表面活性剂例如：N-烷基甜菜碱，N,N-二甲基十八胺盐酸钠，十二烷基苯磺酸钠等，但不限于此。再如对于白细胞检测中，试剂中可包括改变细胞膜透性的表面活性剂，以利于核酸染料透过细胞膜进入到细胞内部与核酸结合。这样的表面活性剂例如：苯氧乙醇，双十八烷基胺盐酸钠，N-甲基酰胺羧酸钠等，但不限于此。
此外，所述试剂还可根据需要包括其他必要的组分，如防腐剂、抗菌剂、细胞膜保护剂、螯合剂等。本领域技术人员可根据需要选择加入。
根据本发明的一种实施方式，所述试剂可以作为血液样品的稀释剂。例如可以是用于阻抗检测的稀释剂，也可以是用于光学检测的稀释剂。可列举的稀释剂的配方，如：
Figure PCTCN2020103133-appb-000002
根据本发明，所述试剂还可以是染色剂。通常，因为染料多易溶于有机溶剂，因此在血小板检测仪中，染料以有机溶液的形式单独贮存。根据需要，也可将染料加入上述 稀释剂中，制成具有血小板解聚功能的染色剂。染色剂中的染料可以是核酸染料。可列举的核酸特异性染料如不对称菁类、噻唑橙TO类、恶唑橙YO类、吖啶橙AO类等，具体的如PI、DAPI、Hoechst系列(如Hoechst33258、Hoechst33342)等，但不限于此。较优选的核酸染料是不对称菁类染料，如SYBR Green。该类染料最佳染色的pH值与本发明中含铵根离子的化合物和具有氨基的化合物的最佳起效的pH值大体一致。根据不同的检测需要，所述染色剂还可以是线粒体染色剂(例如Janus Green B、MitoLite Red、罗丹明123、Mitotracker Green、Mitotracker Deep Red、Mitotracker Red等)或膜染色剂(例如DiA、DiD、DiI、DiO、DiR、DiS、FDA、Alexa Fluor 488、Super Fluor 488等)。
这样的染色剂配方可为：
Figure PCTCN2020103133-appb-000003
此外，本发明的试剂还可以是裂解剂，用于白细胞的检测中。用于所述试剂的裂解剂可以是任何常规的红细胞裂解剂。如前所述，本发明的试剂中的具有氨基的化合物，特别是含铵根离子的化合物，在所定义的浓度范围内对红细胞无不利作用，因此试剂中的裂解剂的种类和浓度，以及使用方式与现有技术中相同。可列举的红细胞裂解剂有季铵盐类(如氯化二甲基苄基烃铵、十二烷基三甲基氯化铵、二甲基苄基烷基氯化铵等)，但不限于此。这样的裂解剂配方可以是：
Figure PCTCN2020103133-appb-000004
再次地，根据前文所述，本发明的包括含铵根离子的化合物以及可进一步包括的具有氨基的化合物的试剂在防止/消除血小板假性聚集中，活性成分的浓度低，不需额外的反应条件(如冷却或加热)，也不需延长反应时间，30s就能完成对血小板的解聚，因此所述化合物对血液的检测没有不良影响，对检测步骤和条件也没有影响，可以在现有的设备和检测方法下消除血小板聚集的干扰。
实施例
实施例1.含铵根离子的化合物对聚集的血小板的解聚作用
本实施例测定了含有铵根阳离子的化合物对聚集的血小板解聚的作用。
将含铵根离子的化合物(氯化铵、溴化铵、碘化铵、磷酸铵、磷酸氢铵、磷酸二氢铵、硫氰酸铵、碳酸氢铵、草酸铵、氨水)以及作为对照的化合物(NaCl)分别加入血细胞分析仪用的稀释液中制得血液样品稀释液。用终浓度0.01mmol/L的血小板聚集诱导剂ADP处理的血液样品获得血小板聚集的血液样品。所述稀释液组成如下：柠檬酸(0.5g/L)，表面活性剂苯氧乙醇(0.1g/L)，抑菌剂(6g/L)，氯化钠(3g/L)，EDTA(0.1g/L)。在该稀释液中分别加入上述含铵根离子的化合物(终浓度0.01mol/L)制备处理液。调整处理液pH至9.5，渗透压至200mOsm/L。
将健康受试者的静脉血采集到血液抗凝管备用。取1ml血液两份，其中一份加入上述稀释液，混匀后在血细胞分析仪(迈瑞BC-6000Plus)中测定血小板计数，记为：PLT_O(未聚)；另一份加入血小板聚集诱导剂ADP(终浓度为0.01mmol/L)，混匀。5分钟后血小板会形成聚集，此时加入上述稀释液后再用所述血细胞分析仪检测含有聚集血小板的血液获得血小板计数，记为：PLT_O(未解聚)。另取1ml血液，加入血小板聚集诱导剂ADP(终浓度为0.01mmol/L)，混匀，5分钟后加入上述处理液，混匀后用所述血细胞分析仪检测经处理后的血液中血小板的计数，记为：PLT_O(解聚)。
一般来说，PLT_O(未解聚)<PLT_O(解聚)<PLT_O(未聚)。
本文提及的术语“解聚率”用以下公式计算：
经解聚样品：解聚率＝PLT_O(解聚)/PLT_O(未聚)；或者
采用上述稀释液对诱导血小板聚集的样品产生的对照解聚效果可用以下公式计算：
未经解聚(对照)样品：解聚率＝PLT_O(未解聚)/PLT_O(未聚)。
测得铵根离子化合物在不同浓度下的解聚率如图3所示。
从图3可以看到，在用0.01mmol/L的ADP诱导血小板聚集的样品中，各含铵根离子的化合物在浓度达到1mmol/L时均产生了一定的解聚作用，并在浓度达到10mmol/L时达到最佳效果。具体地，在10mmol/L时，(NH 4) 2HPO 4为99.25％，NH 4CL为95.70％，(NH 4) 3PO 4为94.87％，NH 4Br为93.48％，NH 4I为92.75％，NH 4HCO 3为88.68％，NH 4C 2O 4为87.67％，NH 4H 2PO 4为86.24％，NH 4SCN为85.78％，NH 4OH为83.05％。当铵根离子化合物换成NaCl时，解聚效果降至18.90％。可以看出，起到血小板解聚作用的是铵根离子，而不是阴离子。而且不同的阴离子对解聚作用有一些影响，但是影响不是很大。
实施例2.环境pH值对含铵根离子的化合物的血小板解聚能力的影响
本实施例研究了环境pH值对含铵根离子的化合物对聚集的血小板的解聚效果的影响。按照实施例1类似的方法，稀释剂中加入0.01mol/L的氯化铵制备处理液，调整处理液的pH为4.0、5.5、6.5、7.5、8.5、9.5和11.0，分别检测氯化铵对样品中血小板解聚的解聚率，如下表1所示。
表1：pH值对氯化铵中铵根离子解聚血小板的影响
pH值 4.0 5.5 6.5 7.5 8.5 9.5 11.0
解聚率 18.62％ 31.70％ 59.61％ 94.05％ 94.62％ 95.70％ 97.72％
由表1可见，环境pH值在7.5以上时，铵根离子对聚集的血小板的解聚作用突然增加到94.05％，并在pH值为7.5～11.0的区间中保持在较高的解聚率。这暗示了在溶液中铵根离子形成了较多的水合氨的情况下，可促进血小板的解聚作用。
实施例3.对比结构类似的含氨基和不含氨基化合物对血小板解聚的能力
本实施例对比了具有相似结构和取代基的含有氨基和不含氨基的化合物对血小板解聚的作用。被试化合物如下表2所示：
表2：被试的含氨基的化合物和结构类似的不含氨基的化合物
Figure PCTCN2020103133-appb-000005
Figure PCTCN2020103133-appb-000006
按照与实施例1类似的方法，区别是用上表2中的化合物分别替代含铵根离子的化合物，并调整稀释液pH至14，渗透压至200mOsm/L，制备处理液。
按上述方法逐一测试含表2各化合物的处理液对ADP诱导的血液处理后，血小板的解聚率，如下表3所示。
表3.含氨基化合物和不含氨基的结构类似化合物对血小板的解聚效果
Figure PCTCN2020103133-appb-000007
由上表3可见，仅含有氨基的化合物对已经聚集的血小板有解聚作用，而其他基团，如磺酸基、羟基、羧基、羰基等对聚集的血小板几乎没有解聚作用(与空白对照NaCl的解聚率相当)。由该实施例可以确定各化合物中对血小板起主要解聚作用的是氨基基团。
实施例4.氨基的数量与血小板解聚的能力的关系
本实施例对比了结构类似化合物上不同氨基数量对血小板解聚的影响。按照实施例3相同的方法对含不同氨基数量的化合物分别进行测试，获得各化合物的血小板解聚率和pKa值如表4所示。
表4.含不同数目氨基的化合物的血小板解聚率及pKa值
Figure PCTCN2020103133-appb-000008
Figure PCTCN2020103133-appb-000009
Figure PCTCN2020103133-appb-000010
由上表4可以看出，结构相似或相同，仅取代氨基的数目不同时，化合物对聚集的血小板的解聚效果随氨基数目的增加而增加，其中短肽、多氨基取代的烷胺、多氨基取代的羟胺和胍类更为优选。
实施例5.相同pH环境下氨基pka与血小板解聚能力的关系
本实施例对比了相同pH环境下，结构类似化合物不同的氨基pKa对血小板解聚的影响。按照实施例3基本相同的方法，区别是对不同类型的化合物调整至较为合适的pH值，对含不同氨基数量的化合物分别进行测试，获得各化合物的血小板解聚率和pKa值如表5所示。
表5.一定的环境pH下具有不同氨基pKa的化合物的血小板解聚率
Figure PCTCN2020103133-appb-000011
Figure PCTCN2020103133-appb-000012
由上表5可以看出，氨基上取代基不同会影响氨基的pKa值不同。
实施例6.环境pH与含氨基的化合物的氨基pKa的关系及其对解聚能力的影响
由实施例5可以看到环境pH与含氨基化合物的氨基pKa之间有一定的关系。为了进一步研究环境pH与含氨基化合物的氨基pKa之间关系以及对血小板解聚能力的影响，本实施例选取了pKa为8.33的化合物1-乙酰基胍，按照与实施例1类似的方法，仅改变环境pH值在7.0～10之间变化，进行了一组测试。得到pH与解聚率的曲线图，如图4所示。
从图4可以看出，环境的pH与物质的pka在血小板解聚中具有强相关性。当pH升高时，含氨基的物质的解聚能力增强，解聚率从35.23％增加到97.32％。而pH达9.5以上时，解聚率基本基本持平。可见，当环境pH大于化合物的氨基pKa到一定程度时，继续提高pH对解聚效果影响不大。这与前述推测的解聚机理是一致的。也就是在合适的pH下，化合物的氨基大部分以去质子化的形式存在于溶液中，从而促进甚至加速了解聚作用。
进一步地，以类似的方法，在pH9.5时，检测了具有不同氨基pKa的化合物对聚集血小板的解聚率，结果显示(图5)。其中，选取了pKa值在8～11之间的一些化合物：N-(三羟甲基)甲基甘氨酸(8.1)、精氨酸(9.0)、谷氨酸(9.6)、甘氨酸(9.6)、对氨基苯磺酸(10.1)和赖氨酸(10.7)。随着氨基pKa8.1升高到10.7，各化合物的解聚效果的趋势是逐渐降低的。
由该实施例可以看到，调整pH值高于氨基pKa值时，有助于提高解聚效果。因此，通过调整环境pH值，可以使含氨基的化合物对聚集血小板的解聚能力提高。反过来说，可以根据检测环境的需要选择合适的含氨基的化合物。
实施例7.含氨基的化合物结构对聚集的血小板的解聚效果
为了考察化合物结构对聚集的血小板解聚的影响，选取了具有代表性结构的化合物，均在pH＝9.5的条件下，按照实施例1的方法进行测试，得到各化合物的解聚率，如下表6所示。
表6.不同化合物对聚集的血小板的解聚效果
Figure PCTCN2020103133-appb-000013
由上表6明显可以看出，氨基与细胞膜的相互作用会受到含氨基的物质本身结构的影响。例如，二聚甘氨酸与甘氨酸相比，pKa相同，并且多了一个仲氨基，但是解聚效果仍略低于甘氨酸。这可归因于分子中多了一个羰基。另外，谷氨酸和甘氨酸都只有一个pKa为9.6的伯胺基，但谷氨酸比甘氨酸多了一个羧基，其解聚率也较低。
从表6可以看出，羰基和羧基对于提升化合物的血小板解聚效果似乎并无帮助，而烷基链对于解聚效果影响不大。
另外，氨基/亚胺基，特别是伯氨基的数量越多，解聚效果越好。精氨酸具有较多氨基/亚胺基，但是解聚效果不是很好，可能与其能够形成环内酰胺，导致氨基的作为被削弱有关。
另外，羟基似乎更有利于增强化合物中氨基的解聚效果。例如，三羟甲基乙酸氨基甲烷仅含有一个仲氨基，但是解聚率却仍高于甘氨酸。具有伯氨基的三羟甲基氨基甲烷对血小板的解聚效果更优。推测羟基有利于化合物靠近血小板的细胞膜，而伯氨基对血小板的解聚效果优于仲氨基。当然，较高的解聚率还与这两个化合物的pKa较低(8.1)也有关系。
另外，叔氨基则几乎没有解聚效果。从上述机理也可了解到，氨基上没有能够形成氢键的氢原子，则难以发挥作用。
实施例8.含氨基的化合物对不同血小板聚集模型的解聚效果
血小板的聚集能够被多种物质诱导，进而导致血液凝固。为了考察含氨基的化合物对于不同因素诱导的血小板聚集的解聚效果，本实施例根据已有的血小板聚集通路，选择血液凝固检测方案中诱导血小板聚集的物质，建立了多种血小板聚集的模型，包括二磷酸腺苷(ADP)、凝血酶(THR)、胶原(COL)和瑞斯西丁素(RIS)。通过本实施例发现，ADP、THR、COL和RIS随着浓度和时间的变化诱导血小板产生不同程度的聚集，同时，含氨基的化合物也能够在一定的浓度和时间范围内对不同诱因、不同聚集程度的血小板有效解聚。
参见图6，其中示出了使用不同ADP浓度(0.01～1mM)诱导血小板聚集的程度与时间的关系。具体地，分别取1ml静脉血，加入ADP使终浓度分别为0.01mM、0.25mM、0.5mM和1mM。用血细胞分析仪检测含有聚集血小板的血液。血细胞分析仪通过阻抗通道和光学通道获得血液中各种细胞粒子的特征，包括数量，大小等。正常的血液采集到血液抗凝管后，通过血细胞分析仪得到阻抗通道检测的血小板粒子数记为(PLT_I)。 当血小板聚集发生时，团聚的血小板的体积远大于单个血小板的体积，因此无法被血小板对应的识别机制正确计数，导致血小板计数下降，记为PLT_I(聚集后)。阻抗通道的稀释液中未加入该类含有氨基的化合物，因此阻抗通道反映了聚集模型中血小板聚集的程度。血小板聚集前的计数为PLT_I(未聚)，血小板聚集后的计数为PLT_I(聚集)，聚集率＝PLT_I(聚集)/PLT_I(未聚)。通过血细胞分析仪得到光学通道检测的血小板粒子数记为(PLT_O)。当血小板聚集发生时，团聚的血小板的体积远大于单个血小板的体积，因此无法被血小板对应的识别机制正确计数，导致PLT_O(未解聚)的下降。在光学通道的稀释液中使用含有该类具有氨基的化合物，实现聚集血小板的解聚，获得解聚后的PLT_O(解聚)。血小板聚集前的计数为PLT_O(未聚)，血小板聚集后的计数为PLT_O(未解聚)。同样，PLT_O(未解聚)<PLT_O(解聚)<PLT_O(未聚)，解聚率＝PLT_O(解聚)/PLT_O(未聚)。通过上述原理，建立了模型，定量检测含有氨基的化合物的解聚效果。
由图6可以看出，加入ADP后，在5分钟的时间内，血小板的聚集基本达到最大。并且ADP的加入量越大，血小板的聚集程度越高。对于ADP终浓度在0.25mM以上时，对血小板聚集的程度影响大体相同。另外，0.01mM浓度的ADP对血小板的聚集程度影响最弱，可使最高约35％的血小板聚集，但是该浓度下血小板解聚也相对缓慢。
进一步参考图7，其中示出了对于采用不同ADP浓度诱导的血液样品，含氨基的化合物对血小板解聚效果随时间的变化曲线。具体地，用含氨基的化合物(1-乙酰基胍)加入稀释液中按照与实施例3相似的方法对加入不同浓度聚集诱导剂的各血液样品进行处理后测定，每个样品加入处理液后间隔5分钟进行一次测定，得到解聚率随时间的变化。稀释液的组成为：柠檬酸(0.5g/L)，表面活性剂苯氧乙醇(0.1g/L)，抑菌剂(6g/L)，氯化钠(3g/L)，EDTA(0.1g/L)和1-乙酰基胍(10mmol/L)，pH为9.5，渗透压为200mOsm/L。
从图7可以看出，相同化合物在相同浓度下，对不同的血小板聚集程度均有显著的解聚效果，且解聚效果在加入含氨基的化合物后立即就达到最高，并随时间推移有所下降，但基本上在10分钟后趋于稳定。另外，ADP的浓度越低，效果越稳定。考虑到血细胞检测仪仅需少量样品就能够能够快速完成对样品的检测，因此可在加入含有所述含氨基的化合物的处理液后5分钟之内，优选2分钟之内，更优选30s后立即开始检测。
因此，通过增加ADP的浓度，可以增加聚集程度，从而增加解聚的难度。
类似地，图8和图9是用不同浓度的THR诱导血小板聚集测试的曲线图。采用与上述基本相同的方法，区别是把ADP换成THR。其中THR的浓度分别为0.1U、0.15U、0.2U、0.23U、0.25U、0.28U、0.30U和0.35U。可以看出，THR低浓度时聚集血小板的效果较弱，高浓度时1分钟后血小板聚集率快速上升。这是因为一定浓度THR加入后，可溶性纤维蛋白原转变为不可溶的纤维蛋白原，造成血液凝固，血小板也被不可溶的纤维蛋白原包裹，而导致聚集率急剧升高。同时，1-乙酰基胍的解聚效果对各个THR浓度均立刻达到最高，但在1分钟后开始明显降低。也与此时解聚剂无法发挥效果有关。但在处理后立即检测的样品中可获得较好的解聚率。
图10和图11是用不同浓度的COL诱导血小板聚集测试的曲线图。采用与上述基本相同的方法，区别是把ADP换成COL。其中COL的浓度分别为0.05mM、0.1mM、0.5mM、1mM、5mM、10mM和20mM。可以看出，COL在不同浓度下可快速使血小板聚集达到接近100％，其中浓度越低，聚集反而越快。同时，1-乙酰基胍的解聚效果，除最低COL浓度的样品外，对其他各个COL浓度均立刻达到最高，但在约4～8分钟后开始明显降低。这同样是因为加入COL后可溶性纤维蛋白原转变为不可溶的纤维蛋白原，造成血液凝固所致。
图12和图13是用不同浓度的RIS诱导血小板聚集测试的曲线图。采用与上述基本相同的方法，区别是把ADP换成RIS。其中RIS的浓度分别为0.15mM、0.375mM、0.75mM、1.5mM和3mM。可以看出，RIS对血小板聚集的诱导作用需要较长时间才能发挥出来，在15～20分钟之后，血小板的聚集程度开始达到最高。同时，1-乙酰基胍的解聚效果对各个RIS浓度均可立刻达到最高，并在10分钟内均比较稳定。15分钟后解聚率的快速下降同样是因为样品中血液发生了凝固。
从以上各个诱导血小板聚集的模型可以看出，不同通路下聚集诱导剂对血小板的聚集程度有不同的影响，但是含氨基的化合物对各诱导剂造成的聚集均可立即起效，但由于可溶性纤维蛋白原转变为不溶性纤维蛋白原造成凝血，使得THR和COL诱导血小板聚集的样品凝血后的解聚效果没有体现。这对于血细胞检测仪进行血小板分类和计数来说，几乎不造成影响。
这说明所述含氨基的化合物可对多种血小板聚集情况在一定浓度和时间范围内均可产生良好的解聚作用，且均能快速起效，便于立即检测，而不需额外的反应条件(如加热或较长反应时间)。
实施例9.含氨基的化合物与含铵根离子的化合物组合使用对血小板解聚的影响
基于含氨基的化合物与含铵根离子的化合物可能以不同的解聚机制对聚集的血小板进行解聚，本实施例测定了含铵根离子的化合物与含氨基的化合物的组合对血小板解聚的效果。按照与实施例1类似的方法，区别是将含铵根离子化合物分别替换为浓度分别为1、2、5、10、20和50mmol/L的氯化铵、1-乙酰基胍、氯化铵和1-乙酰基胍的组合以及作为对照的NaCl，其中氯化铵和1-乙酰基胍的组合中，氯化铵和1-乙酰基胍的浓度相等，总浓度符合以上浓度要求。另外，为了凸显二者组合的效果，本实施例中，采用0.1mmol/L的ADP(即采用实施例1中ADP浓度的10倍)来诱导血小板聚集，在增加解聚难度的情况下研究含氨基的化合物与含铵根离子的化合物解聚血小板的能力。测定的解聚率如图14所示。
结果显示(图14)，在终浓度为0.1mmol/L的ADP诱导的血小板聚集中，随着浓度的升高，NH 4Cl和1-乙酰基胍的解聚效果分别逐渐增强。例如，单独的50mmol/L NH 4Cl解聚效果为66.27％，单独的50mmol/L的1-乙酰基胍解聚效果为62.14％，NH 4Cl和1-乙酰基胍在总浓度50mmol/L共同作用使解聚效果达到95.48％，起到了进一步增强血小板解聚的效果。在其他浓度下也可看到这种增强的作用。暗示二者在血小板解聚作用上存在协同性。因此，二者组合使用时优选以更低的浓度(例如分别低至1～20mM，1～10mM，甚至1～5mM)使用。
以下进一步对本发明的包括具有氨基的化合物的试剂抗血小板解聚干扰在实际检测中的应用情况进行实验。
测试例1.含铵根离子的化合物在染色液中用于血小板和红细胞的检测
本测试例提供一种用于血细胞分析仪的染色液，该染色液可用于排除血小板聚集干扰红细胞和血小板的检测。本测试例以及以下各测试例均采用深圳迈瑞生物医疗股份有限公司的血细胞分析仪(BC-6000Plus)测定。采用以下染色液配方：
对照染色液：
Figure PCTCN2020103133-appb-000014
染色液A：
在对照染色液的基础上，加入氯化铵使终浓度为0.01mol/L。
在25℃用蒸馏水配制对照染色液和染色液A，pH均调整到9，渗透压为200mOsm/L。
取1ml收集在抗凝管中的人静脉血样品，将样品分成两组，用所述血细胞分析仪进行测定，其中分别使用对照染色液和染色液A，以4μl样品加入1ml染色液的比例混合，加入染色剂(SYBR Green，50mg/L)和表面活性剂(N-烷基甜菜碱，0.01g/L)使有核细胞染色，并使血细胞球形化，温度可保持在约42℃，采用测定角度为90°的侧向荧光测定处理后的血液样品细胞的荧光强度信息(FL)，采用测定角度为90°的侧向散射光测定处理后的血液样品细胞的侧向散射光强度信息(SS)，采用测定角度2°-5°的前向散射光测定处理后的血液样品细胞的前向散射光强度信息(FS)得到三维散点图。另取1ml上述血液样品，加入血小板聚集诱导剂ADP(终浓度为0.01mmol/L)，混匀。5min后血小板形成聚集。此时将样品分成两组，用血细胞分析仪检测，分别使用对照染色液和染色液A进行处理，同样的获得三维散点图。如上，该样品测定得到的4组散点图如图15所示。
由图15可见，诱导血小板聚集的血液样品用排除血小板聚集干扰的染色液A处理后，聚集的血小板被解聚，并实现成熟红细胞、网织红细胞和血小板的检测。
如图所示，对诱导血小板聚集的样品使用染色液A，三维散点图中血小板的荧光强度(FL)减小，前向散射强度信息(FS)减小，解聚的三维散点图信息向血小板未聚集的血样的三维散点图信息靠近。在使用对照染色液的散点图中，团聚的血小板的体积大于单个血小板的体积，团聚在一起的血小板的荧光强度也增强。在三维散点图上通过计算网织红细胞分布区域的粒子数占成熟红细胞与网织红细胞区域粒子数的百分比来计算网织红细胞的比例，比正常红细胞具有更强荧光信号的为网织红细胞，未聚集样品测得的总红细胞计数为4.46×10 12，其中网织红细胞在总红细胞中所占的比例为1.28％，使用染色剂A的样品测得解聚后的总红细胞计数为4.43×10 12，总红细胞计数未受聚集血小板的干扰。测得的血小板在未聚集的样品中的计数为：274×10 9；在未聚聚的样品中的计数为：239×10 9；在解聚的样品中的计数为：42×10 9。
同时，通过对血小板计数，测得使用染色液A使诱导聚集的样品中的血小板计数增加，解聚率达到87.16％，使用对照染色液的样品计算得到解聚率为15.36％。
测试例2.含铵根离子化合物在稀释液中用于对血小板和红细胞的检测
本测试例提供一种用于血细胞分析仪的稀释液，该稀释液可用于排除血小板聚集干扰红细胞和血小板的检测。采用以下稀释液配方：
对照稀释液：
Figure PCTCN2020103133-appb-000015
稀释液A：
在对照稀释液的基础上，加入氯化铵使终浓度为0.01mol/L。
在25℃用蒸馏水配制对照稀释液和稀释液A，pH均调整到9.5，渗透压为200mOsm/L。
按照测试例1相似的方法进行检测，区别是分别用上述对照稀释液和稀释液A来处理样品，并进一步加入染色剂(SYBR Green，50mg/L)和表面活性剂(N-烷基甜菜碱，0.01g/L)使有核细胞染色，并使血细胞球形化得到的4组散点图如图16所示。
由图16可见，诱导血小板聚集的血液样品用排除血小板聚集干扰的稀释液A处理后，聚集的血小板被解聚，并实现成熟红细胞、网织红细胞和血小板的检测。
测得未聚集样品测得的总红细胞计数为5.72×10 12，其中网织红细胞在总红细胞中所占的比例为1.02％，使用稀释剂A的样品测得解聚后的总红细胞计数为5.69×10 12，总红细胞计数未受聚集血小板的干扰。测得的血小板在未聚集的样品中的计数为：198×10 9；在聚集的样品中的计数为：42×10 9；在解聚的样品中的计数为：190×10 9。
同时，通过对血小板计数，测得使用染色液A使诱导聚集的样品中的血小板计数增加，解聚率达到95.69％，使用对照染色液的样品计算得到解聚率为20.79％。
测试例3.含铵根离子的化合物在稀释液中用阻抗检测检测血小板
本测试例与测试例2基本相同，仅血细胞分析仪采用深圳迈瑞生物医疗股份有限公司BC-6000Plus的阻抗通道进行检测，获得直方图如图17所示。
如图17所示，血小板直方图的横轴为血小板的体积(单位fL)，纵轴为血小板的数目。当血小板发生聚集时，阻抗通道中血小板的体积变大。在解聚稀释液的作用下，聚集的血小板实现解聚，阻抗通道中血小板的体积变小。
同样按照测试例1中的公式进行计算，获得含有NH 4CL的稀释液A使聚集后的血小板的数目增加，解聚率达到93.17％，不含有NH4CL的对照稀释液解聚率为18.03％。
测试例4.含铵根离子的化合物在裂解液中用于白细胞的检测
本测试例提供一种用于血细胞分析仪的裂解液，该裂解液可用于排除血小板聚集干扰红细胞和血小板的检测。按照以下配方配制裂解液：
对照裂解液：
Figure PCTCN2020103133-appb-000016
裂解液A：
在对照裂解液的基础上，加入氯化铵使终浓度为0.01mol/L。
在25℃用蒸馏水配制对照裂解液和裂解液A，pH均调整到9，渗透压为90mOsm/L。
按照测试例1类似的步骤，取1ml收集在抗凝管中的人静脉血样品，将样品分成两组，用所述血细胞分析仪进行测定，其中分别使用对照裂解液和裂解液A，以20μl样品加入1ml裂解液的比例混合，再加入20μl的DNA染色剂(Hoechst33342)，温度可保持在约42℃，采用测定角度为90°的侧向荧光测定处理后的血液样品细胞的荧光强度信息(FL)，采用测定角度为90°的侧向散射光测定处理后的血液样品细胞的侧向散射光强度信息(SS)，采用测定角度2°-5°的前向散射光测定处理后的血液样品细胞的前向散射光强度信息(FS)得到三维散点图。另取1ml上述血液样品，加入血小板聚集诱导剂ADP(终浓度为0.01mmol/L)，混匀。5min后血小板形成聚集。此时将样品分成两组，同样用血细胞分析仪检测，分别使用对照裂解液和裂解液A进行处理，并加入DNA染料，同样的获得三维散点图。如上，该样品测定得到的4组散点图如图18所示。
在光学通道中，多个聚集的血小板被识别成为一个粒子，部分聚集的血小板的体积达到白细胞的体积，影响到白细胞的计数。的检测。在本测试例中，测得未聚集样品中的白细胞计数为7.49×10 9，其中中性粒细胞4.74×10 9，淋巴细胞2.16×10 9，单核细胞0.42×10 9，嗜酸性粒细胞0.14×10 9。使用对照裂解液的样品测得解聚后的白细胞计数为 8.72×10 9，分类计数中性粒细胞5.16×10 9，淋巴细胞2.57×10 9，单核细胞0.61×10 9，嗜酸性粒细胞0.32×10 9。使用裂解剂A的样品测得解聚后的白细胞计数为数为7.58×10 9，其中中性粒细胞4.79×10 9，淋巴细胞2.21×10 9，单核细胞0.39×10 9，嗜酸性粒细胞0.15×10 9。可见，可见白细胞计数和分类在使用裂解剂A后不受到血小板聚集的影响，计数更为准确。
测试例5.含氨基的化合物和含铵根离子的化合物组合用于白细胞检测
本测试例按照测试例4的方法进行测试，区别是制备裂解液C以代替裂解液A，即，在对照裂解液的基础上，加入等摩尔量的1-乙酰基胍+NH 4Cl使终浓度为0.01mol/L。
在25℃用蒸馏水配制对照裂解液和裂解液C，pH均调整到9，渗透压为90mOsm/L。
按照测试例5类似的步骤进行测试，同样的获得三维散点图如图19所示。
在本测试例中，测得未聚集样品测得的白细胞计数为6.19×10 9，其中分类计数中性粒细胞3.87×10 9，淋巴细胞1.91×10 9，单核细胞0.22×10 9，嗜酸性粒细胞0.08×10 9。使用裂解液C的样品测得解聚后的白细胞计数为6.13×10 9，分类计数中性粒细胞3.91×10 9，淋巴细胞1.88×10 9，单核细胞0.21×10 9，嗜酸性粒细胞0.07×10 9。使用对照裂解液的样品测得解聚后的白细胞计数为6.97×10 9，分类计数中性粒细胞4.21×10 9，淋巴细胞2.21×10 9，单核细胞0.34×10 9，嗜酸性粒细胞0.17×10 9。可见白细胞计数和分类在使用裂解剂C后不受到血小板聚集的影响，计数更为准确。
测试例6.包括含氨基化合物与含铵根离子的化合物组合的染色液用于血小板和红细胞的检测
与测试例1基本相同地进行本测试例，区别是按以下组成配制对照染色液，并以染色液C替代染色液B。
对照染色液：
Figure PCTCN2020103133-appb-000017
染色液C：
在对照染色液的基础上，加入1-乙酰基胍使终浓度为0.01mol/L，并加入氯化铵使终浓度为0.01mol/L。
同样在25℃用蒸馏水配制，pH调整到9，渗透压为200mOsm/L。
按照测试例1相似的方法进行测试，区别是用终浓度为1mol/L的ADP(是测试例1中的100倍)诱导样品中的血小板聚集。获得三维散点图，如图20所示。
测得未聚集样品中的总红细胞计数为6.14×10 12，其中网织红细胞在总红细胞中所占的比例为1.34％，诱导血小板聚集并解聚后的样品中总红细胞计数为6.11×10 12，总红细胞计数未受聚集血小板的干扰。测得的血小板在未聚集的样品中的计数为：219×10 9；在聚集的样品中的计数为：17×10 9；在解聚的样品中的计数为：182×10 9。
同时，通过对血小板计数，测得使用稀释液B使诱导聚集的样品中的血小板计数增加，解聚率达到82.73％，对照染色液测得的解聚率仅为7.61％。
测试例7.包括含氨基化合物与含铵根离子的化合物组合的稀释液用于血小板和红细胞的检测
与测试例2基本相同地进行本测试例，区别是以稀释液C替代稀释液B。稀释液C是在对照稀释液的基础上，加入1-乙酰基胍使终浓度为0.01mol/L，并加入氯化铵使终浓度为0.01mol/L。调整各稀释液pH至9.5，渗透压至200mOsm/L。
按照测试例1相似的方法进行测试，区别是用终浓度为1mol/L的ADP(是测试例中的100倍)诱导样品中的血小板聚集。获得三维散点图，如图21所示。
测得未聚集样品中的总红细胞计数为5.12×10 12，其中网织红细胞在总红细胞中所占的比例为1.21％，诱导血小板聚集并解聚后的样品中总红细胞计数为5.08×10 12，总红细胞计数未受聚集血小板的干扰。测得的血小板在未聚集的样品中的计数为：278×10 9；在聚集的样品中的计数为：18×10 9；在解聚的样品中的计数为：248×10 9。
同时，通过对血小板计数，测得使用稀释液B使诱导聚集的样品中的血小板计数增加，解聚率达到89.20％，对照稀释液测得的解聚率为6.61％。
测试例8临床中含铵根离子的化合物对假性血小板聚集的解聚效果
本测试例是在临床中实现假性聚集血小板的解聚。采用与测试例2的稀释液A相同的稀释液进行检测。该稀释液的配方为：柠檬酸(0.5g/L)，表面活性剂苯氧乙醇(0.1g/L)， 抑菌剂(6g/L)，氯化钠(3g/L)，NH 4Cl(0.01mol/L)。调整稀释液pH至9.5，渗透压至200mOsm/L。
具体步骤为：收集门诊及住院患者血液，以EDTA·K2抗凝，PLT-I血小板计数明显减少，血涂片镜检发现血小板有聚集现象，符合EDTA-PTCP诊断标准的标本17例为实验组，其中男性9例，女性8例，平均年龄51岁。抽取血小板假性聚集患者的血液，无抗凝剂抽血后，1分钟内(此时血小板未发生聚集)立即在血细胞分析仪上检测，用此方法测得血小板计数值为血小板的真值PLT_O(真值)。
用EDTA抗凝管抽取患者血液，在2小时后(假性聚集患者的血小板会聚集)用含有解聚物质1-乙酰基胍的稀释液在同一个血细胞分析仪上检测，用此方法测得的血小板计数值为血小板的解聚值PLT_O(解聚)。同时，用EDTA抗凝管抽取患者血液，在2小时后(假性聚集患者的血小板会聚集)用不含解聚物质NH 4Cl的稀释液的同一个血细胞分析仪的检测，用此方法测得的血小板计数值为血小板的未解聚值PLT_O(未解聚)。通过公式计算解聚效果：
解聚率(解聚)＝PLT_O(解聚)/PLT_O(真值)；或者
解聚率(对照)＝PLT_O(未解聚)/PLT_O(真值)。
各样品的检测结果示于图22中。如图显示，假性聚集血小板均能有不同程度的解聚，在20例样品中，病人血液放置2小时后解聚效果仍达到70％的有13例。对照组中，解聚效果都在20％以下，含10mM的氯化铵的解聚稀释液的解聚效果明显。

Claims (18)

  1. 一种组合物在体外血液检测中防止和/或消除血液样品中血小板聚集的用途，其中，所述组合物包括至少一种含铵根离子的化合物。
  2. 根据权利要求1所述的用途，其中，所述含铵根离子的化合物含有选自由氯离子、溴离子、碘离子、氢氧根、磷酸根、磷酸氢根、磷酸二氢根、硝酸根、硫氢根、硫氰酸根、硫酸根、硫酸氢根、亚硫酸根、亚硫酸氢根、碳酸根、碳酸氢根、甲酸根、乙酸根、草酸根、丙酸根、丙二酸根和柠檬酸根及其组合所组成的组中的阴离子；更优选地，所述铵盐选自由氯化铵、溴化铵、碘化铵、磷酸铵、磷酸氢铵、磷酸二氢铵、硝酸铵、硫氰酸铵、亚硫酸酸氢铵、草酸铵、氢氧化铵、硫酸氢铵和碳酸氢铵所组成的组中的至少一种。
  3. 根据权利要求1所述的用途，其中，所述防止和/或消除血液样品中血小板聚集在pH值至少为7.0的溶液中进行，优选地，所述pH值为7.5～11，更优选为9.5～11。
  4. 根据权利要求1中任一项所述的用途，其中，在进行所述防止和/或消除血液样品中血小板聚集的溶液中，所述组合物中所述含铵根离子的化合物的浓度为1～50mmol/L，优选2～20mmol/L。
  5. 根据权利要求1所述的用途，其中，所述组合物进一步包括至少一种含氨基的化合物，所述化合物含有至少一个伯氨基或仲氨基，优选含有1～20个选自伯氨基、仲氨基和亚胺基的基团。
  6. 根据权利要求5所述的用途，其中，所述具有氨基的化合物选自由式(I)所示的化合物及其盐所组成的组：
    R1-NH-R2  (I)
    其中，R1和R2相同或不同，各自独立地为选自以下组中的基团，前提是R1和R2不同时为H，所述组由H、-SO 3H、-NH 2、-C(NH)-NH 2、取代或未取代的C1-16烷基、取代或未取代的C6-10芳基、取代或未取代的C7-14烷芳基、取代或未取代的C7-14芳烷基、-C(O)-Q1和-C(O)-O-Q2组成，
    其中，Q1为H、-NH 2、取代或未取代的C1-16烷基、取代或未取代的C6-10芳基、取代或未取代的C7-14烷芳基或取代或未取代的C7-14芳烷基；
    Q2为H、取代或未取代的C1-16烷基、取代或未取代的C6-10芳基、取代或未取代的C7-14烷芳基或取代或未取代的C7-14芳烷基；
    其中，所述取代的是指被至少一个选自由-NP1P2、-SO 3H、-OH、卤素、-CN、-C(O)-O-P3、-O-C1-16烷基、-O-C6-10芳基、-O-C7-14烷芳基、-O-C7-14芳烷基、-C(O)-C1-16烷基、-C(O)-C6-10芳基、-C(O)-C7-14烷芳基、-C(O)-C7-14芳烷基和-C(O)-NP1P2组成的组中的基团所取代，其中所述-O-C1-16烷基、-O-C6-10芳基、-O-C7-14烷芳基、-O-C7-14芳烷基、-C(O)-C1-16烷基、-C(O)-C6-10芳基、-C(O)-C7-14烷芳基和-C(O)-C7-14芳烷基分别是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、卤素、-CN、-COOH和-C(O)NH 2组成的组中的基团所取代，
    P1、P2和P3各自独立地为选自以下组中的基团：H、C1-16烷基、C6-10芳基、C7-14烷芳基和C7-14芳烷基，其中所述C1-16烷基、C6-10芳基、C7-14烷芳基和C7-14芳烷基分别是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、卤素、-CN、-COOH和-C(O)NH 2组成的组中的基团所取代；
    优选地，所述式(I)的化合物中，R1和R2相同或不同，各自独立地为选自以下组中的基团，前提是R1和R2不同时为H，所述组由H、-SO 3H、-NH 2、-C(NH)-NH 2、取代或未取代的C1-10烷基、取代或未取代的C6-10芳基、取代或未取代的C7-10烷芳基、取代或未取代的C7-10芳烷基、-C(O)-Q1和-C(O)-O-Q2组成，
    其中，Q1为H、-NH 2、取代或未取代的C1-10烷基、取代或未取代的C6-10芳基、取代或未取代的C7-10烷芳基或取代或未取代的C7-10芳烷基；
    Q2为H、取代或未取代的C1-10烷基、取代或未取代的C6-10芳基、取代或未取代的C7-10烷芳基或取代或未取代的C7-10芳烷基；
    其中，所述取代的是指被至少一个选自由-NP1P2、-SO 3H、-OH、-CN、-C(O)-O-P3、-O-C1-10烷基、-O-C6-10芳基、-O-C7-10烷芳基、-O-C7-10芳烷基和-C(O)-NP1P2组成的组中的基团所取代，其中所述-O-C1-10烷基、-O-C6-10芳基、-O-C7-10烷芳基和-O-C7-10芳烷基分别是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、-CN、-COOH和-C(O)NH 2组成的组中的基团所取代；
    P1、P2和P3各自独立地为选自以下组中的基团：H、C1-10烷基、C6-10芳基、C7-10烷芳基和C7-10芳烷基，其中所述C1-10烷基、C6-10芳基、C7-10烷芳基和C7-10芳烷基分别是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、-CN、-COOH和-C(O)NH 2组成的组中的基团所取代；
    更优选地，所述式(I)的化合物中，R1和R2相同或不同，各自独立地为选自以下组中的基团，前提是R1和R2不同时为H，所述组由H、-SO 3H、-NH 2、-C(NH)-NH 2、 取代或未取代的C1-10烷基、取代或未取代的C6-10芳基、取代或未取代的C7-10烷芳基、取代或未取代的C7-10芳烷基、-C(O)-Q1和-C(O)-O-H的组，
    其中，Q1为H、-NH 2、取代或未取代的C1-10烷基、取代或未取代的C6-10芳基、取代或未取代的C7-10烷芳基或取代或未取代的C7-10芳烷基；
    其中，所述取代的是指被至少一个选自由-NP1P2、-SO 3H、-OH、-CN、-C(O)-O-H和-C(O)-NP1P2组成的组中的基团所取代；
    P1和P2各自独立地为选自以下组中的基团：H、C1-10烷基、C6-10芳基、C7-10烷芳基和C7-10芳烷基，其中所述C1-10烷基、C6-10芳基、C7-10烷芳基和C7-10芳烷基分别是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、-CN、-COOH和-C(O)NH 2组成的组中的基团所取代。
  7. 根据权利要求5所述的用途，其中，所述具有氨基的化合物具有的氨基的pKa在1～16，优选4～14的范围内，更优选所述具有氨基的化合物的氨基的pKa的值小于进行所述防止和/或消除血液样品中血小板聚集的溶液的pH值。
  8. 根据权利要求5所述的用途，其中，在进行所述防止和/或消除血液样品中血小板聚集的溶液中，所述具有氨基的化合物的浓度为1～50mmol/L，优选2～20mmol/L。
  9. 根据权利要求1～8中任一项所述的用途，其中所述体外血液检测包括选自以下项目中的至少一项检测：血小板、红细胞检测和白细胞检测。
  10. 根据权利要求1～8中任一项所述的用途，其中所述血液样品是来自哺乳动物的末梢血或静脉血。
  11. 一种用于体外血液检测的抗血小板聚集干扰的试剂，其中所述试剂包括浓度为1～50mmol/L，优选5～20mmol/L的至少一种含铵根离子的化合物。
  12. 根据权利要求11所述的所述抗血小板聚集干扰的试剂，其中，含铵根离子的化合物含有选自由氯离子、溴离子、碘离子、氢氧根、磷酸根、磷酸氢根、磷酸二氢根、硝酸根、硫氢根、硫氰酸根、硫酸根、硫酸氢根、亚硫酸根、亚硫酸氢根、碳酸根、碳酸氢根、甲酸根、乙酸根、草酸根、丙酸根、丙二酸根、柠檬酸根及其组合所组成的组中的阴离子；更优选地，所述含铵根离子的化合物选自由氯化铵、溴化铵、碘化铵、磷酸铵、磷酸氢铵、磷酸二氢铵、硝酸铵、硫氰酸铵、亚硫酸酸氢铵、草酸铵、氢氧化铵、硫酸氢铵和碳酸氢铵所组成的组中的至少一种。
  13. 根据权利要求11所述的抗血小板聚集干扰的试剂，其中，所述试剂的pH值至少为7.0，优选地，所述pH值为7.5～11，更优选为9.5～11。
  14. 根据权利要求11所述的抗血小板聚集干扰的试剂，其中，所述试剂进一步包括浓度为1～50mmol/L，优选5～20mmol/L的至少一种具有氨基的化合物，所述化合物含有至少一个伯氨基或仲氨基，优选含有1～20个选自伯氨基、仲氨基和亚胺基的基团。
  15. 根据权利要求14所述的抗血小板聚集干扰的试剂，其中，所述具有氨基的化合物选自由式(I)所示的化合物及其盐所组成的组：
    R1-NH-R2    (I)
    其中，R1和R2相同或不同，各自独立地为选自以下组中的基团，前提是R1和R2不同时为H，所述组由H、-SO 3H、-NH 2、-C(NH)-NH 2、取代或未取代的C1-16烷基、取代或未取代的C6-10芳基、取代或未取代的C7-14烷芳基、取代或未取代的C7-14芳烷基、-C(O)-Q1和-C(O)-O-Q2组成，
    其中，Q1为H、-NH 2、取代或未取代的C1-16烷基、取代或未取代的C6-10芳基、取代或未取代的C7-14烷芳基或取代或未取代的C7-14芳烷基；
    Q2为H、取代或未取代的C1-16烷基、取代或未取代的C6-10芳基、取代或未取代的C7-14烷芳基或取代或未取代的C7-14芳烷基；
    其中，所述取代的是指被至少一个选自由-NP1P2、-SO 3H、-OH、卤素、-CN、-C(O)-O-P3、-O-C1-16烷基、-O-C6-10芳基、-O-C7-14烷芳基、-O-C7-14芳烷基、-C(O)-C1-16烷基、-C(O)-C6-10芳基、-C(O)-C7-14烷芳基、-C(O)-C7-14芳烷基和-C(O)-NP1P2组成的组中的基团所取代，其中所述-O-C1-16烷基、-O-C6-10芳基、-O-C7-14烷芳基、-O-C7-14芳烷基、-C(O)-C1-16烷基、-C(O)-C6-10芳基、-C(O)-C7-14烷芳基和-C(O)-C7-14芳烷基分别是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、卤素、-CN、-COOH和-C(O)NH 2组成的组中的基团所取代，
    P1、P2和P3各自独立地为选自以下组中的基团：H、C1-16烷基、C6-10芳基、C7-14烷芳基和C7-14芳烷基，其中所述C1-16烷基、C6-10芳基、C7-14烷芳基和C7-14芳烷基分别是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、卤素、-CN、-COOH和-C(O)NH 2组成的组中的基团所取代；
    优选地，所述式(I)的化合物中，R1和R2相同或不同，各自独立地为选自以下组中的基团，前提是R1和R2不同时为H，所述组由H、-SO 3H、-NH 2、-C(NH)-NH 2、取代或未取代的C1-10烷基、取代或未取代的C6-10芳基、取代或未取代的C7-10烷芳基、取代或未取代的C7-10芳烷基、-C(O)-Q1和-C(O)-O-Q2组成，
    其中，Q1为H、-NH 2、取代或未取代的C1-10烷基、取代或未取代的C6-10芳基、 取代或未取代的C7-10烷芳基或取代或未取代的C7-10芳烷基；
    Q2为H、取代或未取代的C1-10烷基、取代或未取代的C6-10芳基、取代或未取代的C7-10烷芳基或取代或未取代的C7-10芳烷基；
    其中，所述取代的是指被至少一个选自由-NP1P2、-SO 3H、-OH、-CN、-C(O)-O-P3、-O-C1-10烷基、-O-C6-10芳基、-O-C7-10烷芳基、-O-C7-10芳烷基和-C(O)-NP1P2组成的组中的基团所取代，其中所述-O-C1-10烷基、-O-C6-10芳基、-O-C7-10烷芳基和-O-C7-10芳烷基分别是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、-CN、-COOH和-C(O)NH 2组成的组中的基团所取代；
    P1、P2和P3各自独立地为选自以下组中的基团：H、C1-10烷基、C6-10芳基、C7-10烷芳基和C7-10芳烷基，其中所述C1-10烷基、C6-10芳基、C7-10烷芳基和C7-10芳烷基分别是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、-CN、-COOH和-C(O)NH 2组成的组中的基团所取代；
    更优选地，所述式(I)的化合物中，R1和R2相同或不同，各自独立地为选自以下组中的基团，前提是R1和R2不同时为H，所述组由H、-SO 3H、-NH 2、-C(NH)-NH 2、取代或未取代的C1-10烷基、取代或未取代的C6-10芳基、取代或未取代的C7-10烷芳基、取代或未取代的C7-10芳烷基、-C(O)-Q1和-C(O)-O-H的组，
    其中，Q1为H、-NH 2、取代或未取代的C1-10烷基、取代或未取代的C6-10芳基、取代或未取代的C7-10烷芳基或取代或未取代的C7-10芳烷基；
    其中，所述取代的是指被至少一个选自由-NP1P2、-SO 3H、-OH、-CN、-C(O)-O-H和-C(O)-NP1P2组成的组中的基团所取代；
    P1和P2各自独立地为选自以下组中的基团：H、C1-10烷基、C6-10芳基、C7-10烷芳基和C7-10芳烷基，其中所述C1-10烷基、C6-10芳基、C7-10烷芳基和C7-10芳烷基分别是未取代的或进一步被至少一个选自由-NH 2、-OH、-SO 3H、-CN、-COOH和-C(O)NH 2组成的组中的基团所取代。
  16. 根据权利要求14所述的抗血小板聚集干扰的试剂，其中，所述具有氨基的化合物具有的氨基的pKa在1～16，优选4～14的范围内，更优选，所述具有氨基的化合物的氨基的pKa的值小于进行所述试剂的pH值。
  17. 根据权利要求11～16中任一项所述的抗血小板聚集干扰的试剂，其中，所述试剂进一步包括缓冲剂、渗透压调节剂和任选的选自表面活性剂、荧光染料和红细胞裂解剂中的至少一种。
  18. 一种在体外血液检测中防止和/或消除样品中血小板聚集的方法，所述方法包括用根据权利要求1～16中的任一项所定义的组合物或根据权利要求11～17中的任一项所述的试剂对血液样品进行处理的步骤。
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KITAGAWA, S. ; ISHIDA, M. ; KOTANI, K. ; KAMETANI, F.: "Inhibitory effects of long-chain alkyltrimethylammonium ions on aggregation of bovine platelets and the relation of their effects to Ca^2^+ mobilization", BIOCHIMICA ET BIOPHYSICA ACTA, ELSEVIER, AMSTERDAM, NL, vol. 905, no. 1, 27 November 1987 (1987-11-27), AMSTERDAM, NL , pages 75 - 80, XP023504190, ISSN: 0005-2736, DOI: 10.1016/0005-2736(87)90010-1 *
KITAGAWA, S. ; SEKI, H. ; KAMETANI, F.: "Effects of monovalent cations and anions on ADP-induced aggregation of bovine platelets, and mechanism thereof", BIOCHIMICA ET BIOPHYSICA ACTA, ELSEVIER, AMSTERDAM, NL, vol. 840, no. 3, 5 July 1985 (1985-07-05), AMSTERDAM, NL , pages 371 - 376, XP025788851, ISSN: 0304-4165, DOI: 10.1016/0304-4165(85)90217-X *
REHSE KLAUS, KÄMPFE MICHAEL: "Antiaggregatorische und anticoagulante Eigenschaften von Oligoaminen, 10. Mitt: Oligotertiäre Amine und oligoquartäre Ammoniumsalze", ARCHIV DER PHARMAZIE, WILEY VERLAG, WEINHEIM, vol. 322, no. 11, 1 January 1989 (1989-01-01), Weinheim , pages 811 - 815, XP055888141, ISSN: 0365-6233, DOI: 10.1002/ardp.19893221107 *
SHINYA HIROSHI, MATSUO NOBUAKI, TAKEYAMA NAOSHI, TANAKA TAKAYA: "Hyperammonemia inhibits platelet aggregation in rats", THROMBOSIS RESEARCH, ELSEVIER, AMSTERDAM, NL, vol. 81, no. 2, 15 January 1996 (1996-01-15), AMSTERDAM, NL , pages 195 - 201, XP055888137, ISSN: 0049-3848, DOI: 10.1016/0049-3848(95)00236-7 *

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