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WO2022099659A1 - 白细胞分类试剂、红细胞分析试剂、试剂盒以及分析方法 - Google Patents

白细胞分类试剂、红细胞分析试剂、试剂盒以及分析方法 Download PDF

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WO2022099659A1
WO2022099659A1 PCT/CN2020/128839 CN2020128839W WO2022099659A1 WO 2022099659 A1 WO2022099659 A1 WO 2022099659A1 CN 2020128839 W CN2020128839 W CN 2020128839W WO 2022099659 A1 WO2022099659 A1 WO 2022099659A1
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chemical formula
sample
reagent
red blood
group
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PCT/CN2020/128839
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English (en)
French (fr)
Inventor
樊江莉
陈庚文
夏天平
叶燚
张子千
姚起超
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大连理工大学
深圳迈瑞生物医疗电子股份有限公司
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Application filed by 大连理工大学, 深圳迈瑞生物医疗电子股份有限公司 filed Critical 大连理工大学
Priority to PCT/CN2020/128839 priority Critical patent/WO2022099659A1/zh
Priority to EP21891237.6A priority patent/EP4245812A4/en
Priority to CN202180076309.7A priority patent/CN116888117A/zh
Priority to PCT/CN2021/130478 priority patent/WO2022100717A1/zh
Publication of WO2022099659A1 publication Critical patent/WO2022099659A1/zh
Priority to US18/196,618 priority patent/US20230280336A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5094—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for blood cell populations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/04—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B69/00—Dyes not provided for by a single group of this subclass
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the present invention relates to a reagent for analyzing red and white blood cells and a method for using the same, in particular to a reagent for automatically distinguishing subgroups of white blood cells in blood by an analyzer, a reagent for identifying red blood cells and their types in blood, and a method.
  • red blood cells are divided into three types of cells: red blood cells (RBC), white blood cells (WBC), and platelets (Platelet, PLT). Blood routine examinations often need to detect the classification and number of white blood cells, the number of red blood cells and the number of platelets.
  • Leukocytes in normal peripheral blood can generally be divided into five categories, namely lymphocytes, monocytes, neutrophils, eosinophils and basophils. Analysis of leukocyte populations from blood samples can provide much useful information for the clinical diagnosis of numerous diseases. For example, the proportion and number of various types of white blood cells in the blood may change during the disease, and abnormal white blood cells such as abnormal lymphocytes and myeloid cells may also appear. Thus, classifying and measuring different types of normal and abnormal leukocytes can yield information about disease latency, initiation, and stages of development.
  • Red blood cells in normal peripheral blood include mature red blood cells and a small amount of reticulocytes (RET).
  • Reticulocytes reticulocytes, RET
  • RET reticulocytes
  • Reticulocytes are transitional cells between late immature erythrocytes and mature erythrocytes, slightly larger than mature erythrocytes.
  • Reticulocytes are the stage of nucleated erythrocytes that have just lost their nuclei, and are still immature erythrocytes with basophilic substances such as ribosomes and ribonucleic acid remaining in their cytoplasm. After wrapping, blue or blue-green branches can be seen in the cytoplasm or even reticulated structures, hence the name reticulocytes.
  • the current research suggests that reticulocytes, as an important stage in the red blood cell maturation process, should also be paid attention to.
  • the fluorescence analysis methods of the existing analyzers all use different dyes to realize the analysis of red and white blood cells, which is more complicated for the design of the instrument and the liquid circuit. Therefore, there is a need for a new dye that can be used for the classification and counting of red blood cells and white blood cells, respectively, to simplify the structure of the hematology analyzer.
  • a first aspect of the present application provides a leukocyte sorting reagent, the reagent comprising a fluorescent dye having the formula F:
  • X is selected from the group consisting of C(CH 3 ) 2 , O, S and Se;
  • R 1 and R 2 are each independently selected from the group consisting of H, C 1 -C 18 alkyl, phenyl, OR 6 and halogen;
  • R 3 and R 4 are each independently selected from the group consisting of C 1 -C 18 alkyl, C 1 -C 18 carboxy, C 1 -C 18 hydroxy, C 1 -C 18 NR 5 R 6 , benzyl and substituted benzyl group, wherein the substituent of the substituted benzyl group is selected from C 1 -C 18 alkyl, CN, COOH, NH 2 , NO 2 , OH, SH, C 1 -C 6 alkoxy, C 1 -C 6 alkane the group consisting of amino, C 1 -C 6 amido, halogen and C 1 -C 6 haloalkyl;
  • R 5 and R 6 are each independently selected from the group consisting of H and C 1 -C 18 alkyl;
  • Y - is a negative ion.
  • the fluorescent dye with formula F in the present application belongs to cyanine compounds, has good permeability of living cells, can enter cells to stain nucleic acids without destroying the cell membrane, and is used in blood analysis to stain leukocytes to achieve Classification and/or counting of white blood cells.
  • the excitation light of the dye is blue-green light with a smaller wavelength, which can identify tiny particles and improve the detection capability of small particles.
  • a second aspect of the present application provides a leukocyte analysis kit, which includes the leukocyte classification reagent described in the first aspect.
  • the white blood cell analysis kit of the present application can classify and/or count white blood cells in blood analysis; classify white blood cells into lymphocytes, monocytes, neutrophils, and eosinophils, and can classify the classified Cells are counted.
  • a third aspect of the present application provides a method for classifying leukocytes, the method comprising: mixing a blood sample, a fluorescent dye having formula F, and an erythrocyte lysing agent to form a mixture; wherein, formula F is
  • Leukocytes are classified and/or counted according to the scattered light properties and fluorescence properties.
  • a fourth aspect of the present application provides a red blood cell analysis reagent, the reagent comprises a fluorescent dye having formula F; wherein, formula F is
  • Red blood cell analysis reagents are used in blood analysis to stain red blood cells to obtain parameters of red blood cells.
  • a fifth aspect of the present application provides a red blood cell analysis kit, the kit comprising the red blood cell analysis reagent according to the fourth aspect.
  • the white blood cell analysis kit of the present application can classify and/or count red blood cells in blood analysis; red blood cell classification includes: distinguishing red blood cells into mature red blood cells and/or reticulocytes; the red blood cell counting includes: mature red blood cells and/or reticulocytes. or reticulocyte count.
  • a sixth aspect of the present application provides a method for analyzing red blood cells, comprising: mixing a blood sample, a fluorescent dye having formula F, and a spheroidizing reagent to form a mixture; wherein, formula F is
  • Red blood cell classification and/or counts are obtained based on the scattered light properties and fluorescence properties.
  • the white blood cell sorting reagent and the red blood cell analysis reagent in the present application both contain a fluorescent dye with formula F, which can stain the nucleic acid of white blood cells and red blood cells, so in blood analysis, it can accurately achieve the classification of white blood cells in the sample and/or Counting, and accurate classification and/or enumeration of sample red blood cells, especially identification and/or enumeration of RET.
  • the types of dyes used in blood cell analysis can be simplified.
  • Figure 1 shows a side scatter light-fluorescence scatter plot using a compound of formula I as a fluorescent dye for sorting leukocytes
  • Figure 2 shows a fluorescence-forward scattered light scatter plot for the detection of erythrocytes and platelets using a compound of formula I as a fluorescent dye;
  • Figure 3 shows a side-scattered light-fluorescence scatter plot using a compound of formula II as a fluorescent dye for sorting leukocytes
  • Figure 4 shows a fluorescence-forward scattered light scatter plot for the detection of red blood cells and platelets using a compound of formula II as a fluorescent dye
  • Figure 5 shows a side-scattered light-fluorescence scatter plot using a compound of formula III as a fluorescent dye for sorting leukocytes
  • Figure 6 shows a fluorescence-forward scattered light scatter plot for the detection of erythrocytes and platelets using a compound of formula III as a fluorescent dye
  • Figure 7 shows a side-scattered light-fluorescence scatter plot using a compound of formula IV as a fluorescent dye for sorting leukocytes
  • Figure 8 shows a fluorescence-forward scattered light scatter plot for the detection of red blood cells and platelets using a compound of formula IV as a fluorescent dye
  • Figure 9 shows a side scatter light-fluorescence scatter plot using a compound of formula V as a fluorescent dye for sorting leukocytes
  • Figure 10 shows a fluorescence-forward scattered light scatter plot for the detection of red blood cells and platelets using a compound of formula V as a fluorescent dye
  • Figure 11 shows a side scatter light-fluorescence scatter plot using a compound of formula VI as a fluorescent dye for sorting leukocytes
  • Figure 12 shows a fluorescence-forward scattered light scatter plot for the detection of red blood cells and platelets using a compound of formula VI as a fluorescent dye
  • Figure 13 shows a side scatter light-fluorescence scatter plot using a compound of formula VII as a fluorescent dye for sorting leukocytes
  • Figure 14 shows a fluorescence-forward scattered light scatter plot for the detection of red blood cells and platelets using a compound of formula VII as a fluorescent dye
  • Figure 15 shows a side scatter light-fluorescence scatter plot using a compound of formula VIII as a fluorescent dye for sorting leukocytes
  • Figure 16 shows a fluorescence-forward scattered light scatter plot for the detection of red blood cells and platelets using a compound of formula VIII as a fluorescent dye
  • Figure 17 shows a side scatter-fluorescence scatter plot using a compound of formula IX as a fluorescent dye for sorting leukocytes
  • Figure 18 shows a fluorescence-forward scattered light scatter plot for the detection of red blood cells and platelets using a compound of formula IX as a fluorescent dye
  • Figure 19 shows a side scatter light-fluorescence scatter plot using a compound of formula X as a fluorescent dye for sorting leukocytes
  • Figure 20 shows a fluorescence-forward scattered light scatter plot for the detection of red blood cells and platelets using a compound of formula X as a fluorescent dye
  • Figure 21 shows a side scatter light-fluorescence scatter plot using a compound of formula XI as a fluorescent dye for sorting leukocytes
  • Figure 22 shows a fluorescence-forward scattered light scatter plot for the detection of red blood cells and platelets using a compound of formula XI as a fluorescent dye
  • Figure 23 shows a side scatter-fluorescence scatterplot for sorting leukocytes using a compound of formula XII as a fluorescent dye
  • Figure 24 shows a fluorescence-forward scattered light scatter plot for the detection of red blood cells and platelets using a compound of formula XII as a fluorescent dye
  • Figure 25 shows a side scatter light-fluorescence scatter plot using a compound of formula XIII as a fluorescent dye for sorting leukocytes
  • Figure 26 shows a fluorescence-forward scattered light scatter plot for the detection of red blood cells and platelets using a compound of formula XIII as a fluorescent dye.
  • blood or blood sample as used herein refers to a sample of bodily fluids comprising blood cells, eg, peripheral blood, bone marrow fluid, and the like.
  • alkyl may be understood in its broadest sense to mean any linear, branched or cyclic alkyl substituent.
  • C 1-18 alkyl as used herein generally refers to a saturated hydrocarbon group having 1 to 18 carbon atoms in configuration, C 1-18 alkyl includes, but is not limited to, C 1-12 alkyl, C 1 -6 alkyl, etc.
  • alkyl generally refers to an unsubstituted alkyl.
  • alkyl includes the substituents methyl (Me), ethyl (Et), n-propyl (nPr), isopropyl (iPr), cyclopropyl, n-butyl (nBu), isobutyl (iBu), sec-butyl (sBu), tert-butyl (tBu), cyclobutyl, 2-methylbutyl, n-pentyl, sec-pentyl, tert-pentyl, 2-pentyl, neopentyl, Cyclopentyl, n-hexyl, sec-hexyl, tert-hexyl, 2-hexyl, 3-hexyl, neohexyl, cyclohexyl, 1-methylcyclopentyl, 2-methylpentyl, n-heptyl, 2-heptyl , 3-heptyl, 4-heptyl, cycloheptyl, cyclo
  • carboxy includes any linear, branched or cyclic carboxy substituent.
  • C 1-18 carboxyl as used herein generally refers to a carboxyl substituted group having 1 to 18 carbon atoms in configuration, and C 1-18 carboxyl includes, but is not limited to, C 1-12 carboxyl, C 1-6 carboxyl, etc. .
  • carboxyl refers to a group having a carboxyl group attached to an alkyl group as previously defined.
  • carboxyl exemplarily includes methylcarboxy, ethylcarboxy, propylcarboxy, butylcarboxy, pentylcarboxy, hexylcarboxy, heptylcarboxy, and octylcarboxy, isomers thereof, and the like.
  • hydroxy includes any linear, branched or cyclic hydroxy substituent.
  • C 1-18 hydroxy as used herein generally refers to a hydroxy-substituted group having 1 to 18 carbon atoms in configuration, C 1-18 hydroxy including but not limited to C 1-12 hydroxy, C 1-6 hydroxy, etc. .
  • the term hydroxy refers to a group having a hydroxy group attached to an alkyl group as previously defined.
  • the term hydroxy exemplarily includes hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, hydroxyheptyl, and hydroxyoctyl, isomers thereof, and the like.
  • alkoxy includes any linear, branched or cyclic alkoxy substituent.
  • alkoxy refers to an alkoxy group attached to an alkyl group as previously defined.
  • the term alkoxy exemplarily includes methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy and 2-methyl Butoxy, etc.
  • halogen and “halo” may be understood in the broadest sense to mean preferably fluorine, chlorine, bromine or iodine.
  • blue-green light wavelengths are approximately in the range of 400-560 nm, if not more specifically defined in a particular context.
  • the fluorescent dyes of the present application belong to cyanine compounds, have good permeability to living cells, and can enter cells for specific staining of nucleic acids (RNA, DNA) without destroying the cell membrane.
  • the dye is a blue-green light-excited fluorescent dye, which can be excited by blue or green lasers emitted by devices that provide blue or green region lasers, such as blue or green semiconductor lasers. Equipment such as hematology analyzers or flow cytometers as light sources.
  • the fluorescent dye has the structure shown in formula F:
  • X is selected from the group consisting of C(CH 3 ) 2 , O, S and Se;
  • R 1 and R 2 are each independently selected from the group consisting of H, C 1 -C 18 alkyl, phenyl, OR 6 and halogen;
  • R 3 and R 4 are each independently selected from the group consisting of C 1 -C 18 alkyl, C 1 -C 18 carboxy, C 1 -C 18 hydroxy, C 1 -C 18 NR 5 R 6 , benzyl and substituted benzyl group, wherein the substituent of the substituted benzyl group is selected from C 1 -C 18 alkyl, CN, COOH, NH 2 , NO 2 , OH, SH, C 1 -C 6 alkoxy, C 1 -C 6 alkane the group consisting of amino, C 1 -C 6 amido, halogen and C 1 -C 6 haloalkyl;
  • R 5 and R 6 are each independently selected from the group consisting of H and C 1 -C 18 alkyl;
  • Y - is a negative ion.
  • X in formula F is selected from the group consisting of C( CH3 ) 2 and S.
  • R 1 and the R 2 in formula F are each independently selected from the group consisting of H, C 1 -C 12 alkyl, phenyl, OR 6 and halogen.
  • R 1 and the R 2 in formula F are each independently selected from the group consisting of H, C 1 -C 6 alkyl, phenyl, OR 6 and halogen.
  • R 1 in Formula F is selected from the group consisting of H, C 1 -C 6 alkyl, phenyl, and halogen.
  • R2 in formula F is H.
  • R 3 and R 4 in formula F are each independently selected from C 1 -C 12 alkyl, C 1 -C 12 carboxyl, C 1 -C 12 hydroxyl, C 1 -C 12 NR 5 R 6 , benzyl and the group consisting of substituted benzyl, wherein the substituent of the substituted benzyl is selected from the group consisting of C 1 -C 12 alkyl, CN, COOH, NH 2 , NO 2 , OH, SH, C 1 -C 6 alkane The group consisting of oxy, C 1 -C 6 alkylamino, C 1 -C 6 amido, halogen and C 1 -C 6 haloalkyl.
  • R 3 and R 4 in formula F are each independently selected from C 1 -C 6 alkyl, C 1 -C 6 carboxyl, C 1 -C 6 hydroxyl, C 1 -C 6 NR 5 R 6 , benzyl and the group consisting of substituted benzyl, wherein the substituent of the substituted benzyl is selected from the group consisting of C 1 -C 6 alkyl, CN, COOH, NH 2 , NO 2 , OH, SH, C 1 -C 6 alkane The group consisting of oxy, C 1 -C 6 alkylamino, C 1 -C 6 amido, halogen and C 1 -C 6 haloalkyl.
  • R 3 in formula F is selected from the group consisting of C 1 -C 6 alkyl, C 1 -C 6 hydroxyl, C 1 -C 6 carboxyl, C 1 -C 6 NR 5 R 6 and benzyl .
  • R 4 in Formula F is selected from the group consisting of C 1 -C 6 alkyl, C 1 -C 6 hydroxy, C 1 -C 6 carboxy, and benzyl.
  • R 5 and R 6 in formula F are each independently selected from the group consisting of H and C 1 -C 12 alkyl.
  • R 5 and R 6 in Formula F are each independently selected from the group consisting of H and C 1 -C 6 alkyl.
  • R 5 and R 6 in formula F are each independently C 1 -C 6 alkyl.
  • Y - in formula F is selected from the group consisting of halide anion, ClO 4 - , PF 6 - , BF 4 - , CH 3 COO - or OTs - .
  • the fluorescent dye comprises one of chemical formula I, chemical formula II, chemical formula III, chemical formula IV, chemical formula V, chemical formula VI, chemical formula VII, chemical formula VIII, chemical formula IX, chemical formula X, chemical formula XI, chemical formula XII and chemical formula XIII the structure shown,
  • the fluorescent dye of the present application can be dissolved in glycerol, ethylene glycol, ethylene glycol and stored as a storage solution, and can also be dissolved in other non-aqueous solvents.
  • the present invention preferably uses ethylene glycol as a solvent, and optionally contains 5-10% methanol as a diluent component.
  • the present invention can also optionally include a nonionic surfactant as a dispersant, so that the fluorescent dye can maintain a certain solubility without aggregation.
  • nonionic surfactants are, for example, polyoxyethylene glycol (POE), polypropylene glycol (POP), polyoxyethylene glycol-polypropylene glycol (POE-POP), Brij series nonionic Surfactant.
  • the present invention preferably uses Brij 35, Brij56, etc. as the dispersant of the fluorescent dye, and the concentration is 0.02-2%.
  • the fluorescent dyes of the present application can be formulated with erythrocyte lysing or spheronizing reagents to form a one-component reagent.
  • the erythrocyte lysing agent used may comprise a cationic surfactant, a nonionic surfactant, an anionic surfactant, or any combination thereof, and a buffer, which maintains the pH of the assay system at 5-11.
  • Cationic surfactants nonionic surfactants, anionic surfactants, or any combination thereof can be employed as red blood cell lysing agents. They can dissolve red blood cells in blood samples, and properly destroy the membrane structure of each subgroup of leukocytes, so that each subgroup of leukocytes in the blood shrinks to an appropriate size, and promotes different aggregation of the internal structure of leukocytes, resulting in differences in scattered light characteristics.
  • Cationic surfactants can be octyltrimethylammonium bromide (OTAB), decyltrimethylammonium bromide (DTAB), dodecyltrimethylammonium chloride (LTAC), tetradecyltrimethylammonium Ammonium bromide (CTAB), tetradecyl trimethyl ammonium chloride (CTAC), etc., preferably LTAC, and the concentration used is 300-800 mg/L.
  • Nonionic surfactants can be polyoxyethylene type nonionic surfactants, such as long-chain fatty alcohol polyoxyethylene, alkylphenol polyoxyethylene ether, fatty acid polyoxyethylene ester, fatty amine polyoxyethylene ether, etc., preferably non-ionic surfactants.
  • the ionic surfactant is polyoxyethylene-2,3-dodecyl ether (Brij35), and the concentration used is 1-5g/L.
  • Anionic surfactants are selected from dodecylbenzene sulfonic acid, sodium fatty alcohol acyl sulfate, sodium ethoxylated fatty acid methyl ester sulfonate, sodium secondary alkyl sulfonate, alcohol ether carboxylate, and the concentration is 0.1-2g /L.
  • the reagents of the present application may contain buffers that maintain pH and appropriately dilute the blood sample.
  • the buffer keeps the pH in a constant range, thereby stabilizing the staining effect of each subpopulation of leukocytes, and the buffer is used at a concentration in the range of 0.01-200 mM.
  • the types of buffers are not particularly limited as long as they are suitable for the purpose of the present invention, such as carboxylates, phosphates, citrates, Tris-HCl, MOPS, and other organic buffers at appropriate concentrations.
  • the suitable pH in the reagent of the present invention varies with the specific fluorescent dye selected, and is generally in the range of pH 5.0-11.0, and the preferred pH is 8.0-10.0.
  • the erythrocyte lysing agent of the present invention may also optionally contain an appropriate proportion of alcohol, such as methanol, to promote the coagulation of the internal structure of leukocytes.
  • alcohol such as methanol
  • the present invention also provides a kit for distinguishing and counting leukocytes in blood, the kit comprising a leukocyte sorting reagent comprising a fluorescent dye having the structure shown in formula F:
  • X is selected from the group consisting of C(CH 3 ) 2 , O, S and Se;
  • R 1 and R 2 are each independently selected from the group consisting of H, C 1 -C 18 alkyl, phenyl, OR 6 and halogen;
  • R 3 and R 4 are each independently selected from the group consisting of C 1 -C 18 alkyl, C 1 -C 18 carboxy, C 1 -C 18 hydroxy, C 1 -C 18 NR 5 R 6 , benzyl and substituted benzyl group, wherein the substituent of the substituted benzyl group is selected from C 1 -C 18 alkyl, CN, COOH, NH 2 , NO 2 , OH, SH, C 1 -C 6 alkoxy, C 1 -C 6 alkane the group consisting of amino, C 1 -C 6 amido, halogen and C 1 -C 6 haloalkyl;
  • R 5 and R 6 are each independently selected from the group consisting of H and C 1 -C 18 alkyl;
  • Y - is a negative ion.
  • the fluorescent dye has a structure selected from the group consisting of formula I, formula II, formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, formula X, formula XI, formula XII and formula XIII,
  • the leukocyte sorting reagent further comprises an erythrocyte lysing agent as described above.
  • the fluorescent dye is present as a stock solution, kept in a separate container.
  • the concentration of the fluorescent dye is: 0.1-1000mg/L, preferably 20-500mg/L, more preferably 50-200mg/L.
  • the fluorescent dye is formulated together with the above-mentioned erythrocyte lysing agent as a mixed reagent solution.
  • the kit preferably contains a fluorescent dye suitable for storage in compartments that are hermetically maintained in at least one container.
  • the kit may also contain other leukocyte sorting reagents required to sort leukocytes in blood and instructions for methods of measuring leukocytes.
  • the kit may also contain a control sample or series of control samples that can be assayed and compared to the test sample.
  • the components of the kit for classifying and/or enumerating the various types of leukocytes in the blood can be packaged in a single container, the different containers all in separate packages along with instructions.
  • the spheronizing agent used may contain amphoteric surfactants, osmotic pressure regulators, buffers, and optionally cationic surfactants and/or preservatives.
  • the surfactant in the spheroidization reagent swells the blood cells and slightly damages the cell membrane to allow the dye to enter the cells.
  • Concentration range of amphoteric surfactant 100-2000 mg/L, preferably 100-500 mg/L.
  • the amphoteric surfactant can be one or more of alkyl betaine, sulfobetaine, cocamidopropyl betaine and the like.
  • Cationic surfactants can be octyltrimethylammonium bromide (OTAB), decyltrimethylammonium bromide (DTAB), dodecyltrimethylammonium chloride (LTAC), tetradecyltrimethylammonium Ammonium bromide (CTAB), tetradecyl trimethyl ammonium chloride (CTAC), etc., the use concentration is 0-50mg/L.
  • the osmotic pressure regulator can be: sodium chloride, sodium sulfate and other common salts, and the osmotic pressure range is: 200mOsm/kg-380mOsm/kg.
  • the types of buffers are not particularly limited as long as they are suitable for the purpose of the present invention, such as carboxylates, phosphates, citrates, Tris-HCl, MOPS, and other organic buffers at appropriate concentrations.
  • the suitable pH in the reagent of the present invention varies with the specific fluorescent dye selected, and is generally in the range of pH 5.0-11.0, and the preferred pH is 8.0-10.0.
  • the present invention also provides a kit for being able to distinguish and count red blood cells in blood, especially reticulocytes, comprising a red blood cell analysis reagent comprising the formula Fluorescent dyes of the structure shown in F:
  • X is selected from the group consisting of C(CH 3 ) 2 , O, S and Se;
  • R 1 and R 2 are each independently selected from the group consisting of H, C 1 -C 18 alkyl, phenyl, OR 6 and halogen;
  • R 3 and R 4 are each independently selected from the group consisting of C 1 -C 18 alkyl, C 1 -C 18 carboxy, C 1 -C 18 hydroxy, C 1 -C 18 NR 5 R 6 , benzyl and substituted benzyl group, wherein the substituent of the substituted benzyl group is selected from C 1 -C 18 alkyl, CN, COOH, NH 2 , NO 2 , OH, SH, C 1 -C 6 alkoxy, C 1 -C 6 alkane the group consisting of amino, C 1 -C 6 amido, halogen and C 1 -C 6 haloalkyl;
  • R 5 and R 6 are each independently selected from the group consisting of H and C 1 -C 18 alkyl;
  • Y - is a negative ion.
  • the fluorescent dye has a structure selected from the group consisting of formula I, formula II, formula III, formula IV, formula V, formula VI, formula VII, formula VIII, formula IX, formula X, formula XI, formula XII and formula XIII,
  • the red blood cell analysis reagent further comprises the spheroidization reagent described above.
  • the fluorescent dye is present as a stock solution, kept in a separate container.
  • the concentration of the fluorescent dye is: 0.1-1000mg/L, preferably 20-500mg/L, more preferably 50-200mg/L.
  • the fluorescent dye is formulated together with the above-mentioned spheronization reagent as a mixed reagent solution.
  • the kit preferably contains a fluorescent dye suitable for storage in compartments that are hermetically maintained in at least one container.
  • the kit may also contain other red blood cell sorting reagents required to sort red blood cells in blood and instructions for methods of measuring red blood cells.
  • the kit may also contain a control sample or series of control samples that can be assayed and compared to the test sample.
  • the components of the kit for classifying and/or counting various types of red blood cells in the blood can be packaged in a single container, the different containers being all in separate packages along with instructions red blood cells.
  • the present application also provides methods of classifying and/or counting leukocytes in blood. Briefly, in the method of the present application, by mixing a blood sample with a leukocyte sorting reagent, then measuring at least one scattered light property and at least one fluorescent property of the sample, and classifying and classifying the sample according to the scattered light property and the fluorescent property. / or count.
  • the blood sample used in the method of the present application may be whole blood or blood component.
  • the blood sample can be mixed with an erythrocyte lysing agent first, so that the erythrocytes are lysed and the leukocytes of each subgroup shrink to different degrees. This step simultaneously forms pores in the cell membrane of the leukocytes to be assayed, which are small enough to allow the fluorescent dye molecules to pass through the cell membrane.
  • the fluorescent dye stock solution is then added to allow the leukocytes to be fluorescently labeled.
  • the total volume of the blood sample and the reagent should ensure sufficient cell concentration to pass through the measuring cell of the instrument.
  • the reagent composition of the present application dilutes the blood sample to 1:10, 1:50, or 1:100 or any value in any of the above ranges, so long as the dilution meets the requirements of actual use. Such adjustments are within the scope of those skilled in the art.
  • the blood sample can also be mixed simultaneously with the erythrocyte lysing agent and the fluorescent dye.
  • the sample mixture can be incubated in the incubation cell for a time not exceeding 60 seconds, preferably 30 seconds, 24 seconds; the incubation temperature can be any suitable temperature, such as 40°C.
  • White blood cells can be detected and analyzed by a blood analyzer or flow cytometer.
  • the dissolved, diluted and stained blood sample is introduced into the flow chamber of the measuring instrument, and the light emitted by the light source illuminates the particles in the flow chamber to generate optical information.
  • the optical information includes at least one scattered light and at least one fluorescence.
  • Scattered light in this application refers to scattered light that can be detected by a commercially available hematology analyzer or similar flow cytometer.
  • Such scattered light includes, but is not limited to, side scattered light, forward low angle scattered light (accepted light angle of about 0-5 degrees), and forward high angle scattered light (accepted light angle of about 5-20 degrees).
  • Scattered light having such an angle reflects information on the size or internal structure of leukocytes, and thus serves as the scattered light in the present invention.
  • Side scattered light is preferred.
  • Fluorescent dyes that bind to nucleic acids in cells emit fluorescence. Fluorescence properties are parameters that reflect the amount of intracellular fluorescent dye in a blood sample. Due to the different intracellular metabolic activities of different subgroups, resulting in differences in nucleic acid content, the fluorescence characteristics of leukocytes in different subgroups are different in some aspects.
  • the appropriate excitation wavelength is selected depending on the specific dye used, and the emission at the corresponding wavelength is monitored.
  • a green or blue wavelength region laser emitted by a green or blue semiconductor laser can be used as the detection light source.
  • the green or blue wavelength light source there is no particular limitation on the green or blue wavelength light source, as long as it can emit green light (for example, light with a wavelength of about 501-560 nm) or blue light (for example, about a wavelength of about 400-500 nm) near the excitation wavelength of the selected fluorescent dye. of light).
  • the light source may be configured to emit light having a wavelength of about 450 nm or 520 nm. This type of laser is relatively low cost relative to other lasers, which reduces the cost of the equipment.
  • the leukocytes of each subgroup were identified by the characteristics of scattered light and fluorescence, and the relative classification and count of each cluster scattered point were performed to calculate the percentage of leukocytes of each subgroup.
  • Scattered light reflects the degree of granularity inside the cell.
  • the granularity inside the cell is roughly as follows: eosinophils have two-lobed nuclei and many acid dye-stained brittle particles inside; neutrophils have nuclei (lobed or rod-shaped) and have internal There are more granules; monocytes have less granules in the single macronucleus; LYM cells have almost no granules in the single macronucleus. Therefore, under the same conditions, the order of scattered light intensity characteristics of various leukocytes is EO (eosinophils)>NEUT (neutrophils)>MONO (monocytes)>LYM (lymphocytes).
  • the present application also provides methods of classifying and/or counting red blood cells in blood. Briefly, in the method of the present application, by mixing a blood sample with a red blood cell analysis reagent, then measuring at least one scattered light characteristic and at least one fluorescence characteristic of the sample, and classifying and classifying the sample according to the scattered light characteristic and the fluorescence characteristic / or count.
  • the blood sample used in the method of the present application may be whole blood or blood component.
  • the blood sample can be first mixed with a spheroidizing reagent, which causes red and white blood cells to swell and slightly damage the cell membrane, creating pores in the red and white blood cell membranes that are large enough to allow the fluorescent dye molecules to pass through the cell membrane.
  • the fluorescent dye stock solution is then added so that red and white blood cells are fluorescently labeled. Since mature erythrocytes, reticulocytes, and leukocytes have different nucleic acid contents, the fluorescence characteristics are different, and the above three types of cells can be distinguished.
  • the total volume of the blood sample and the reagent of this application should ensure sufficient cell concentration to pass through the measuring cell of the instrument.
  • the reagent composition of the present application dilutes the blood sample to 1:10, 1:50, or 1:100 or any value in any of the above ranges, so long as the dilution meets the requirements of actual use. Such adjustments are within the scope of those skilled in the art.
  • the blood sample can also be mixed with the spheroidization reagent and fluorescent dye simultaneously.
  • the sample mixture can be incubated in the incubation tank for no more than 60 seconds, preferably 30 seconds, 24 seconds; the incubation temperature can be any suitable temperature, such as 40°C.
  • a blood analyzer or flow cytometer can be used to detect and analyze red blood cells.
  • the spheroidized and stained blood sample is introduced into the flow chamber of the measuring instrument, and the light emitted by the light source illuminates the particles in the flow chamber to generate optical information.
  • the optical information includes at least one scattered light and at least one fluorescence.
  • Scattered light in this application refers to scattered light that can be detected by a commercially available hematology analyzer or similar flow cytometer.
  • Such scattered light includes, but is not limited to, side scattered light, forward low angle scattered light (accepted light angle of about 0-5 degrees), and forward high angle scattered light (accepted light angle of about 5-20 degrees).
  • Scattered light with such an angle reflects information on cell size or internal structure, and thus serves as the scattered light in the present invention.
  • Forward scattered light is preferred.
  • a green or blue wavelength region laser emitted by a green or blue semiconductor laser can also be used as the detection light source.
  • Identify mature red blood cells, reticulocytes and platelets by using scattered light characteristics and fluorescence characteristics, and classify and count each cluster scatter to obtain red blood cell (including mature red blood cell and reticulocyte) count, reticulocyte count and platelet count .
  • the forward scattered light reflects the information of the size of the cell.
  • the platelet volume is smaller than that of the red blood cell, and its forward scattered light intensity is relatively weak; the content of nucleic acid and RNA in mature red blood cells and reticulocytes is different, and the content of nucleic acid and RNA in reticulocytes is relatively high, and its fluorescence relatively high strength.
  • Exemplary fluorescent dyes are synthesized in the following manner to prepare Compound A having the structure shown in Formula I,
  • X is S
  • R 1 is H
  • R 2 is H
  • R 3 is benzyl
  • R 4 is benzyl
  • the first step prepares 2-methylthiobenzothiazole (right side of reaction 1) according to following reaction formula I,
  • the resulting mixture was cooled to room temperature, after which it was poured into a large amount of water. Extract 3 times with an appropriate amount of ethyl acetate, and combine the extracted organic phases. The organic phase was washed twice with distilled water, and then dried over anhydrous magnesium sulfate overnight.
  • the reacted mixture was subjected to suction filtration, and then the filter cake was washed three times with 50 mL of toluene to obtain a crude product.
  • the 3rd step prepares 3-benzyl-2-thione benzothiazole (the right side of reaction formula III) according to following reaction formula III,
  • the reacted mixture was suction filtered, and then the filter cake was washed three times with 50 mL of toluene to obtain a crude product.
  • the 4th step prepares 3-benzyl-2-ethylsulfanyl benzothiazole (reaction formula IV right side) according to following reaction formula IV,
  • the 5th step prepares compound A (reaction formula V right side) according to following reaction formula V,
  • X is S
  • R 1 is H
  • R 2 is H
  • R 3 is carboxypentyl
  • R 4 is methyl
  • the reagent system consisting of the following was prepared.
  • Fluorescent dye (with chemical formula I) 50mg
  • the process of testing the sample is carried out automatically in Mindray's self-developed improved BC-6000 blood analyzer), setting the suction volume to 20 microliters, the injection volume of hemolyzing agent solution B to 1 ml, and the injection volume of fluorescent dye storage solution A.
  • the volume was 20 microliters, and the blood sample was anticoagulated blood.
  • erythrocyte lysing agent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40 °C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, and the side scattered light intensity and fluorescence intensity of the test sample are measured to obtain a white blood cell scattergram.
  • the test results of the blood sample (No. 1 blood sample) are shown in Figure 1, indicating that the application can realize the classification and identification of 4 subgroups of white blood cells.
  • the four clusters of particles marked in Figure 1 are lymphocytes, monocytes, medium neutrophils and eosinophils, indicating that the leukocyte classification reagent of the present application can effectively classify each subgroup of leukocytes.
  • the process of testing the sample is carried out automatically in the blood analysis instrument independently developed by Mindray.
  • the blood sample is anticoagulated blood.
  • spheroidization reagent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40°C to form a test sample for measurement.
  • Sample. The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, the forward scattered light intensity and the fluorescence intensity of the test sample are measured, and a scatter diagram containing red blood cells is obtained.
  • the test results of the blood sample (No. 1 blood sample) are shown in Figure 2, indicating that the application can realize the classification and identification of red blood cells and platelets.
  • the three clusters of particles marked in Figure 2 are red blood cells (i.e. mature red blood cells), reticulum Red blood cells (RET) and platelets, indicating that the red blood cell detection reagent of the present application can realize the classification and identification of red blood cells and platelets, especially the identification of RET.
  • test results of the fluorescent dye with chemical formula I on leukocyte grouping and reticulocytes are relatively consistent with the test results of BC-6800.
  • the reagent system consisting of the following was prepared.
  • Fluorescent dye (with formula II) 50mg
  • the process of testing the sample is carried out automatically in the improved BC-6000 blood analysis instrument independently developed by Mindray. is 20 microliters, and the blood sample is anticoagulated blood.
  • erythrocyte lysing agent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40 °C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, and the side scattered light intensity and fluorescence intensity of the test sample are measured to obtain a white blood cell scattergram.
  • the test results of the blood sample (No. 2 blood sample) are shown in Figure 3, indicating that the application can achieve the classification and identification of 4 subgroups of white blood cells.
  • the four clusters of particles marked in Figure 3 are lymphocytes, monocytes, medium neutrophils and eosinophils, indicating that the leukocyte classification reagent of the present application can effectively classify each subgroup of leukocytes.
  • the process of testing the sample is carried out automatically in the blood analysis instrument independently developed by Mindray.
  • the blood sample is anticoagulated blood.
  • spheroidization reagent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40°C to form a test sample for measurement.
  • Sample. The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, the forward scattered light intensity and the fluorescence intensity of the test sample are measured, and a scatter diagram containing red blood cells is obtained.
  • the test results of the blood sample (No. 2 blood sample) are shown in Figure 4, indicating that the application can realize the classification and identification of red blood cells and platelets.
  • the three clusters of particles marked in Figure 4 are red blood cells (i.e. mature red blood cells), reticulum Red blood cells (RET) and platelets, indicating that the red blood cell detection reagent of the present application can realize the classification and identification of red blood cells and platelets, especially the identification of RET.
  • the reagent system consisting of the following was prepared.
  • Fluorescent dye (with formula III) 50mg
  • the process of testing the sample is carried out automatically in the improved BC-6000 blood analysis instrument independently developed by Mindray. is 20 microliters, and the blood sample is anticoagulated blood.
  • erythrocyte lysing agent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40 °C to form a test sample for measurement.
  • Sample The blood analysis instrument is equipped with a blue laser, the excitation wavelength is 450 nm, and the side scattered light intensity and fluorescence intensity of the test sample are measured to obtain a white blood cell scattergram.
  • the test results of the blood sample (No. 3 blood sample) are shown in Figure 5, indicating that the application can realize the classification and identification of 4 subgroups of leukocytes.
  • the four clusters of particles marked in Figure 5 are lymphocytes, monocytes, medium neutrophils and eosinophils, indicating that the leukocyte classification reagent of the present application can effectively classify each subgroup of leukocytes.
  • the process of testing the sample is carried out automatically in the blood analysis instrument independently developed by Mindray.
  • the blood sample is anticoagulated blood.
  • spheroidization reagent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40°C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, the forward scattered light intensity and the fluorescence intensity of the test sample are measured, and a scatter diagram containing red blood cells is obtained.
  • the test results of the blood sample (blood sample No. 3) are shown in Figure 6, indicating that the application can realize the classification and identification of red blood cells and platelets.
  • the three clusters of particles marked in Figure 6 are red blood cells (i.e. mature red blood cells), reticulum Red blood cells (RET) and platelets, indicating that the red blood cell detection reagent of the present application can realize the classification and identification of red blood cells and platelets, especially the identification of RET.
  • test results of the fluorescent dye with chemical formula III on leukocyte grouping and reticulocytes are relatively consistent with the test results of BC-6800.
  • the reagent system consisting of the following was prepared.
  • Fluorescent dye (with formula IV) 100mg
  • the process of testing the sample is carried out automatically in the improved BC-6000 blood analysis instrument independently developed by Mindray. is 20 microliters, and the blood sample is anticoagulated blood.
  • erythrocyte lysing agent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40 °C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, and the side scattered light intensity and fluorescence intensity of the test sample are measured to obtain a white blood cell scattergram.
  • the test results of the blood sample (No. 4 blood sample) are shown in Figure 7, indicating that the application can achieve the classification and identification of 4 subgroups of leukocytes.
  • the four clusters of particles marked in Figure 7 are lymphocytes, monocytes, medium neutrophils and eosinophils, indicating that the leukocyte classification reagent of the present application can effectively classify each subgroup of leukocytes.
  • the process of testing the sample is carried out automatically in the blood analysis instrument independently developed by Mindray.
  • the blood sample is anticoagulated blood.
  • spheroidization reagent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40°C to form a test sample for measurement.
  • Sample. The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, the forward scattered light intensity and the fluorescence intensity of the test sample are measured, and a scatter diagram containing red blood cells is obtained.
  • the test results of the blood sample (No. 4 blood sample) are shown in Figure 8, indicating that the application can realize the classification and identification of red blood cells and platelets.
  • the three clusters of particles marked in Figure 8 are red blood cells (i.e. mature red blood cells), reticulum Red blood cells (RET) and platelets, indicating that the red blood cell detection reagent of the present application can realize the classification and identification of red blood cells and platelets, especially the identification of RET.
  • test results of leukocyte grouping and reticulocytes by the fluorescent dye with chemical formula IV are relatively consistent with the test results of BC-6800.
  • the reagent system consisting of the following was prepared.
  • Fluorescent dye (with chemical formula V) 500mg
  • the process of testing the sample is carried out automatically in the improved BC-6000 blood analysis instrument independently developed by Mindray. is 20 microliters, and the blood sample is anticoagulated blood.
  • erythrocyte lysing agent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40 °C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, and the side scattered light intensity and fluorescence intensity of the test sample are measured to obtain a white blood cell scattergram.
  • the test results of the blood sample (No. 5 blood sample) are shown in Figure 9, indicating that the application can realize the classification and identification of 4 subgroups of leukocytes.
  • the four clusters of particles marked in Figure 9 are lymphocytes, monocytes, medium neutrophils and eosinophils, indicating that the leukocyte classification reagent of the present application can effectively classify each subgroup of leukocytes.
  • the process of testing the sample is carried out automatically in the blood analysis instrument independently developed by Mindray.
  • the blood sample is anticoagulated blood.
  • spheroidization reagent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40°C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, the forward scattered light intensity and the fluorescence intensity of the test sample are measured, and a scatter diagram containing red blood cells is obtained.
  • the test results of the blood sample (No. 5 blood sample) are shown in Figure 10, indicating that the application can realize the classification and identification of red blood cells and platelets.
  • Red blood cells (RET) and platelets indicating that the red blood cell detection reagent of the present application can realize the classification and identification of red blood cells and platelets, especially the identification of RET.
  • test results of the fluorescent dye with chemical formula V on leukocyte grouping and reticulocytes are relatively consistent with the test results of BC-6800.
  • the reagent system consisting of the following was prepared.
  • Fluorescent dye (with formula VI) 50mg
  • the process of testing the sample is carried out automatically in the improved BC-6000 blood analysis instrument independently developed by Mindray. is 20 microliters, and the blood sample is anticoagulated blood.
  • erythrocyte lysing agent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40 °C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, and the side scattered light intensity and fluorescence intensity of the test sample are measured to obtain a white blood cell scattergram.
  • the test results of the blood sample (No. 6 blood sample) are shown in Figure 11, indicating that the application can realize the classification and identification of 4 subgroups of leukocytes.
  • the four clusters of particles marked in Figure 11 are lymphocytes, monocytes, medium neutrophils and eosinophils, indicating that the leukocyte classification reagent of the present application can effectively classify each subgroup of leukocytes.
  • the process of testing the sample is carried out automatically in the blood analysis instrument independently developed by Mindray.
  • the blood sample is anticoagulated blood.
  • spheroidization reagent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40°C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, the forward scattered light intensity and the fluorescence intensity of the test sample are measured, and a scatter diagram containing red blood cells is obtained.
  • the test results of the blood sample (No. 6 blood sample) are shown in Figure 12, indicating that the application can realize the classification and identification of red blood cells and platelets.
  • Red blood cells (RET) and platelets indicating that the red blood cell detection reagent of the present application can realize the classification and identification of red blood cells and platelets, especially the identification of RET.
  • the reagent system consisting of the following was prepared.
  • Fluorescent dye 250mg
  • the process of testing the sample is carried out automatically in the improved BC-6000 blood analysis instrument independently developed by Mindray. is 20 microliters, and the blood sample is anticoagulated blood.
  • erythrocyte lysing agent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40 °C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, and the side scattered light intensity and fluorescence intensity of the test sample are measured to obtain a white blood cell scattergram.
  • the test results of the blood sample (blood sample No. 7) are shown in Figure 13, indicating that the application can achieve the classification and identification of 4 subgroups of white blood cells.
  • the four clusters of particles marked in Figure 13 are lymphocytes, monocytes, medium neutrophils and eosinophils, indicating that the leukocyte classification reagent of the present application can effectively classify each subgroup of leukocytes.
  • the process of testing the sample is carried out automatically in the blood analysis instrument independently developed by Mindray.
  • the blood sample is anticoagulated blood.
  • spheroidization reagent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40°C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, the forward scattered light intensity and the fluorescence intensity of the test sample are measured, and a scatter diagram containing red blood cells is obtained.
  • the test results of the blood sample (blood sample No. 7) are shown in Figure 14, indicating that the application can realize the classification and identification of red blood cells and platelets.
  • Red blood cells (RET) and platelets indicating that the red blood cell detection reagent of the present application can realize the classification and identification of red blood cells and platelets, especially the identification of RET.
  • test results of leukocyte grouping and reticulocytes by the fluorescent dye with chemical formula VII are relatively consistent with the test results of BC-6800.
  • the reagent system consisting of the following was prepared.
  • Fluorescent dye (with formula VIII) 50mg
  • the process of testing the sample is carried out automatically in the improved BC-6000 blood analysis instrument independently developed by Mindray. is 20 microliters, and the blood sample is anticoagulated blood.
  • erythrocyte lysing agent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40 °C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, and the side scattered light intensity and fluorescence intensity of the test sample are measured to obtain a white blood cell scattergram.
  • the test results of the blood sample (No. 8 blood sample) are shown in Figure 15, indicating that the application can realize the classification and identification of 5 subgroups of white blood cells.
  • the five clusters of particles marked in Figure 15 are lymphocytes, monocytes, medium neutrophils, eosinophils and basophils, indicating that the leukocyte classification reagent of the present application can achieve effective classification of each subgroup of leukocytes.
  • the process of testing the sample is carried out automatically in the blood analysis instrument independently developed by Mindray.
  • the blood sample is anticoagulated blood.
  • spheroidization reagent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40°C to form a test sample for measurement.
  • Sample. The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, the forward scattered light intensity and the fluorescence intensity of the test sample are measured, and a scatter diagram containing red blood cells is obtained.
  • the test results of the blood sample (No. 8 blood sample) are shown in Figure 16, indicating that the application can achieve the classification and identification of red blood cells and platelets.
  • the three clusters of particles marked in Figure 16 are red blood cells (i.e. mature red blood cells), reticulum Red blood cells (RET) and platelets, indicating that the red blood cell detection reagent of the present application can realize the classification and identification of red blood cells and platelets, especially the identification of RET.
  • test results of the fluorescent dye with chemical formula VIII on leukocyte grouping and reticulocytes are relatively consistent with the test results of BC-6800.
  • the reagent system consisting of the following was prepared.
  • Fluorescent dye (with formula IX) 50mg
  • the process of testing the sample is carried out automatically in the improved BC-6000 blood analysis instrument independently developed by Mindray. is 20 microliters, and the blood sample is anticoagulated blood.
  • erythrocyte lysing agent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40 °C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, and the side scattered light intensity and fluorescence intensity of the test sample are measured to obtain a white blood cell scattergram.
  • the test results of the blood sample (blood sample No. 9) are shown in Figure 17, indicating that the application can realize the classification and identification of 4 subgroups of leukocytes.
  • the four clusters of particles marked in Figure 17 are lymphocytes, monocytes, medium neutrophils and eosinophils, indicating that the leukocyte classification reagent of the present application can effectively classify each subgroup of leukocytes.
  • the process of testing the sample is carried out automatically in the blood analysis instrument independently developed by Mindray.
  • the blood sample is anticoagulated blood.
  • spheroidization reagent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40°C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, the forward scattered light intensity and the fluorescence intensity of the test sample are measured, and a scatter diagram containing red blood cells is obtained.
  • the test results of the blood sample (blood sample No. 9) are shown in Figure 18, which shows that the application can realize the classification and identification of red blood cells and platelets.
  • Red blood cells (RET) and platelets indicating that the red blood cell detection reagent of the present application can realize the classification and identification of red blood cells and platelets, especially the identification of RET.
  • test results of the fluorescent dye with chemical formula IX on leukocyte grouping and reticulocytes are relatively consistent with the test results of BC-6800.
  • the reagent system consisting of the following was prepared.
  • Fluorescent dye (with formula X) 50mg
  • the process of testing the sample is carried out automatically in the improved BC-6000 blood analysis instrument independently developed by Mindray. is 20 microliters, and the blood sample is anticoagulated blood.
  • erythrocyte lysing agent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40 °C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, and the side scattered light intensity and fluorescence intensity of the test sample are measured to obtain a white blood cell scattergram.
  • the test results of the blood sample (No. 10 blood sample) are shown in Figure 19, indicating that the application can achieve the classification and identification of 4 subgroups of leukocytes.
  • the four clusters of particles marked in Figure 19 are lymphocytes, monocytes, medium neutrophils and eosinophils, indicating that the leukocyte classification reagent of the present application can effectively classify each subgroup of leukocytes.
  • the process of testing the sample is carried out automatically in the blood analysis instrument independently developed by Mindray.
  • the blood sample is anticoagulated blood.
  • spheroidization reagent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40°C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, the forward scattered light intensity and the fluorescence intensity of the test sample are measured, and a scatter diagram containing red blood cells is obtained.
  • the test results of the blood sample (No. 10 blood sample) are shown in Figure 20, indicating that the application can realize the classification and identification of red blood cells and platelets.
  • Red blood cells (RET) and platelets indicating that the red blood cell detection reagent of the present application can realize the classification and identification of red blood cells and platelets, especially the identification of RET.
  • test results of leukocyte grouping and reticulocytes by the fluorescent dye with chemical formula X are relatively consistent with the test results of BC-6800.
  • the reagent system consisting of the following was prepared.
  • Fluorescent dye (with chemical formula XI) 50mg
  • the process of testing the sample is carried out automatically in the improved BC-6000 blood analysis instrument independently developed by Mindray. is 20 microliters, and the blood sample is anticoagulated blood.
  • erythrocyte lysing agent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40 °C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, and the side scattered light intensity and fluorescence intensity of the test sample are measured to obtain a white blood cell scattergram.
  • the test results of the blood sample (No. 11 blood sample) are shown in Figure 21, indicating that the application can achieve the classification and identification of 4 subgroups of white blood cells.
  • the four clusters of particles marked in Figure 21 are lymphocytes, monocytes, medium neutrophils and eosinophils, indicating that the leukocyte classification reagent of the present application can effectively classify each subgroup of leukocytes.
  • the process of testing the sample is carried out automatically in the blood analysis instrument independently developed by Mindray.
  • the blood sample is anticoagulated blood.
  • spheroidization reagent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40°C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, the forward scattered light intensity and the fluorescence intensity of the test sample are measured, and a scatter diagram containing red blood cells is obtained.
  • the test results of the blood sample (blood sample No. 11) are shown in Figure 22, indicating that the application can realize the classification and identification of red blood cells and platelets.
  • the three clusters of particles marked in Figure 22 are red blood cells (i.e. mature red blood cells), reticulum Red blood cells (RET) and platelets, indicating that the red blood cell detection reagent of the present application can realize the classification and identification of red blood cells and platelets, especially the identification of RET.
  • the reagent system consisting of the following was prepared.
  • Fluorescent dye (with chemical formula XII) 1000mg
  • the process of testing the sample is carried out automatically in the improved BC-6000 blood analysis instrument independently developed by Mindray. is 20 microliters, and the blood sample is anticoagulated blood.
  • erythrocyte lysing agent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40 °C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, and the side scattered light intensity and fluorescence intensity of the test sample are measured to obtain a white blood cell scattergram.
  • the test results of the blood sample (No. 12 blood sample) are shown in Figure 23, indicating that the application can realize the classification and identification of 4 subgroups of leukocytes.
  • the four clusters of particles marked in Figure 23 are lymphocytes, monocytes, medium neutrophils and eosinophils, indicating that the leukocyte classification reagent of the present application can effectively classify each subgroup of leukocytes.
  • the process of testing the sample is carried out automatically in the blood analysis instrument independently developed by Mindray.
  • the blood sample is anticoagulated blood.
  • spheroidization reagent 1 ml was mixed with 20 microliters of blood treated with anticoagulant, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40°C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, the forward scattered light intensity and the fluorescence intensity of the test sample are measured, and a scatter diagram containing red blood cells is obtained.
  • the test results of the blood sample (No. 12 blood sample) are shown in Figure 24, indicating that the application can realize the classification and identification of red blood cells and platelets.
  • Red blood cells (RET) and platelets indicating that the red blood cell detection reagent of the present application can realize the classification and identification of red blood cells and platelets, especially the identification of RET.
  • test results of leukocyte grouping and reticulocytes by the fluorescent dye with chemical formula XII are relatively consistent with the test results of BC-6800.
  • the reagent system consisting of the following was prepared.
  • Fluorescent dye (with chemical formula XIII) 50mg
  • the process of testing the sample is carried out automatically in the improved BC-6000 blood analysis instrument independently developed by Mindray. is 20 microliters, and the blood sample is anticoagulated blood.
  • erythrocyte lysing agent 1 ml was mixed with 20 microliters of anticoagulant-treated blood, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40 °C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, and the side scattered light intensity and fluorescence intensity of the test sample are measured to obtain a white blood cell scattergram.
  • the test results of the blood sample (No. 13 blood sample) are shown in Figure 25, indicating that the application can achieve the classification and identification of 4 subgroups of leukocytes.
  • the four clusters of particles marked in Figure 25 are lymphocytes, monocytes, medium neutrophils and eosinophils, indicating that the leukocyte classification reagent of the present application can effectively classify each subgroup of leukocytes.
  • the process of testing the sample is carried out automatically in the blood analysis instrument independently developed by Mindray.
  • the blood sample is anticoagulated blood.
  • spheroidization reagent 1 ml was mixed with 20 microliters of blood treated with anticoagulant, and 20 microliters of fluorescent dye storage solution was immediately added, and the mixture was mixed and incubated for 30 seconds in an incubation pool at 40°C to form a test sample for measurement.
  • Sample The blood analysis instrument is provided with a blue laser, the excitation wavelength is 450 nm, the forward scattered light intensity and the fluorescence intensity of the test sample are measured, and a scatter diagram containing red blood cells is obtained.
  • the test results of the blood sample (blood sample No. 13) are shown in Figure 26, indicating that the application can achieve the classification and identification of red blood cells and platelets.
  • the three clusters of particles marked in Figure 26 are red blood cells (i.e. mature red blood cells), reticulum Red blood cells (RET) and platelets, indicating that the red blood cell detection reagent of the present application can realize the classification and identification of red blood cells and platelets, especially the identification of RET.
  • test results of leukocyte grouping and reticulocytes by the fluorescent dye with chemical formula XIII are relatively consistent with the test results of BC-6800.

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Abstract

一种白细胞分类试剂、试剂盒及分类方法，红细胞分析试剂、试剂盒、及分析方法。白细胞分类试剂和红细胞分析试剂均包含具有通式（F）的荧光染料。该染料可以对白细胞和红细胞的核酸染色，故在血液分析时，能准确实现对样本中白细胞的分类和/或计数，以及准确实现对样本红细胞的分类和/或计数，尤其是对RET的识别和/或计数。此外，还可简化血细胞分析时所用的染料种类。

Description

白细胞分类试剂、红细胞分析试剂、试剂盒以及分析方法
说明书
技术领域
本发明涉及红白细胞分析试剂和其使用方法，具体涉及通过分析仪自动区分血液中白细胞各亚群的试剂、识别血液中红细胞及种类的试剂、和方法。
背景技术
血细胞分为红细胞(Red Blood Cell，RBC)、白细胞(White Blood Cell，WBC)、血小板(Platelet，PLT)等三类细胞，血常规检查时经常需要检测白细胞分类及数量、红细胞数量和血小板数量。
正常外周血中的白细胞通常可分为五类，即淋巴细胞、单核细胞、中性粒细胞、嗜酸性粒细胞和嗜碱性粒细胞。由血液样本分析白细胞群体可为众多疾病的临床诊断提供许多有用的信息。例如，在出现疾病时血液中的各类白细胞的比例和数量可能发生变化，同时还可能出现异常淋巴细胞和幼稚粒细胞等异常白细胞。因此，分类并测定不同类型的正常和异常白细胞可获得有关疾病潜伏、起始和发展阶段等方面的信息。
正常外周血中的红细胞包括成熟红细胞和少量网织红细胞(reticulocyte，RET)。网织红细胞(reticulocyte，RET)是介于晚幼红细胞和成熟红细胞之间的过渡阶段细胞，略大于成熟红细胞。网织红细胞是有核红细胞刚刚失去核的阶段，仍属未完全成熟的红细胞，其胞质中尚有核糖体、核糖核酸等嗜碱性物质残存,经煌焦油蓝或新亚甲蓝活体染包后,胞质中可见蓝或蓝绿色枝点状甚至网织状结构，故名网织红细胞。目前的研究认为，网织红细胞作为红细胞成熟过程中的一个重要阶段也应被重视。
现有分析仪的荧光分析方法，都是通过不同的染料来实现对红白细胞的分析，对于仪器和液路设计比较复杂。因此，需要一种新染料可分别用于红细胞和白细胞的分类和计数，来简化血液分析仪的结构。
发明内容
在发明内容部分中引入了一系列简化形式的概念，这将在具体实施方 式部分中进一步详细说明。本发明的发明内容部分并不意味着要试图限定出所要求保护的技术方案的关键特征和必要技术特征，更不意味着试图确定所要求保护的技术方案的保护范围。
为至少部分地解决上述问题，本申请的第一方面提供了一种白细胞分类试剂，所述试剂包含具有式F的荧光染料：
Figure PCTCN2020128839-appb-000001
其中，X选自由C(CH 3) 2、O、S和Se组成的组；
R 1和R 2各自独立地选自由H、C 1-C 18烷基、苯基、OR 6和卤素组成的组；
R 3和R 4各自独立地选自由C 1-C 18烷基、C 1-C 18羧基、C 1-C 18羟基、C 1-C 18NR 5R 6、苄基和取代苄基组成的组，其中所述取代苄基的取代基选自由C 1-C 18烷基、CN、COOH、NH 2、NO 2、OH、SH、C 1-C 6烷氧基、C 1-C 6烷基氨基、C 1-C 6酰氨基、卤素和C 1-C 6卤代烷基组成的组；
R 5和R 6各自独立地选自由H和C 1-C 18烷基组成的组；
Y -为负离子。
本申请中具有式F的荧光染料，属于菁类化合物，具有良好的活细胞通透性，能够在不破坏细胞膜的情况进入细胞对核酸进行染色，用于血液分析中，对白细胞进行染色，实现对白细胞的分类和/或计数。一个实施例中，所述染料的激发光为波长较小的蓝绿色光，能够识别微小颗粒，提高了对小粒子的检测能力。
本申请的第二方面提供一种白细胞分析试剂盒，其包括上述第一方面所述的白细胞分类试剂。本申请的白细胞分析试剂盒，在血液分析中可对白细胞进行分类和/或计数；将白细胞分类为淋巴细胞、单核细胞、中性粒细胞、和嗜酸性粒细胞，并可对分类后的细胞进行计数。
本申请的第三方面提供一种白细胞分类方法，所述方法包括：将血液样品、具有式F的荧光染料、和红细胞溶解剂混合形成混合物；其中，式F为
Figure PCTCN2020128839-appb-000002
其中，X、R 1、R 2、R 3、R 4和Y -如上述第一方面在所述白细胞分类试剂中所定义；
用光照射所述待测试样中的粒子，测定所述待测试样的至少一种散射光特性和至少一种荧光特性；
根据所述散射光特性和荧光特性对白细胞进行分类和/或计数。
本申请的第四方面提供一种红细胞分析试剂，所述试剂包含具有式F的荧光染料；其中，式F为
Figure PCTCN2020128839-appb-000003
其中，X、R 1、R 2、R 3、R 4和Y -如上述第一方面在所述白细胞分类试剂中所定义。红细胞分析试剂用于血液分析中，可以对红细胞进行染色，获得红细胞的参数。
本申请的第五方面提供一种红细胞分析试剂盒，所述试剂盒包含如第四方面所述的红细胞分析试剂。本申请的白细胞分析试剂盒，在血液分析中可对红细胞进行分类和/或计数；红细胞分类包括：将红细胞区分为成熟红细胞和/或网织红细胞；所述红细胞计数包括：对成熟红细胞和/或网织红细胞的计数。
本申请的第六方面提供一种红细胞分析方法，包括：将血液样品、具有式F的荧光染料、和球形化试剂混合形成混合物；其中，式F为
Figure PCTCN2020128839-appb-000004
其中，X、R 1、R 2、R 3、R 4和Y -如上述第一方面在白细胞分类试剂中所定义；
测定所述混合物的至少一种散射光特性和至少一种荧光特性；
根据所述散射光特性和荧光特性获得红细胞分类和/或计数。
本申请中的白细胞分类试剂和红细胞分析试剂均包含有具有式F的荧光染料，该染料可以对白细胞和红细胞的核酸染色，故在血液分析时，能准确实现对样本中白细胞的分类和/或计数，以及准确实现对样本红细胞的分类和/或计数，尤其是对RET的识别和/或计数。此外，还可简化血细胞分析时所用染料种类。
附图说明
本发明的下列附图在此作为本发明的一部分用于理解本发明。附图中示出了本发明的实施例及其描述，用来解释本发明的原理。
附图中：
图1示出了采用具有化学式I的化合物作为荧光染料用于分类白细胞的侧向散射光-荧光散点图；
图2示出了采用具有化学式I的化合物作为荧光染料用于检测红细胞和血小板的荧光-前向散射光散点图；
图3示出了采用具有化学式II的化合物作为荧光染料用于分类白细胞的侧向散射光-荧光散点图；
图4示出了采用具有化学式II的化合物作为荧光染料用于检测红细胞和血小板的荧光-前向散射光散点图；
图5示出了采用具有化学式III的化合物作为荧光染料用于分类白细胞的侧向散射光-荧光散点图；
图6示出了采用具有化学式III的化合物作为荧光染料用于检测红细胞和血小板的荧光-前向散射光散点图；
图7示出了采用具有化学式IV的化合物作为荧光染料用于分类白细胞的侧向散射光-荧光散点图；
图8示出了采用具有化学式IV的化合物作为荧光染料用于检测红细胞和血小板的荧光-前向散射光散点图；
图9示出了采用具有化学式V的化合物作为荧光染料用于分类白细胞的侧向散射光-荧光散点图；
图10示出了采用具有化学式V的化合物作为荧光染料用于检测红细胞和血小板的荧光-前向散射光散点图；
图11示出了采用具有化学式VI的化合物作为荧光染料用于分类白细胞的侧向散射光-荧光散点图；
图12示出了采用具有化学式VI的化合物作为荧光染料用于检测红细胞和血小板的荧光-前向散射光散点图；
图13示出了采用具有化学式VII的化合物作为荧光染料用于分类白细胞的侧向散射光-荧光散点图；
图14示出了采用具有化学式VII的化合物作为荧光染料用于检测红细胞和血小板的荧光-前向散射光散点图；
图15示出了采用具有化学式VIII的化合物作为荧光染料用于分类白细胞的侧向散射光-荧光散点图；
图16示出了采用具有化学式VIII的化合物作为荧光染料用于检测红细胞和血小板的荧光-前向散射光散点图；
图17示出了采用具有化学式IX的化合物作为荧光染料用于分类白细胞的侧向散射光-荧光散点图；
图18示出了采用具有化学式IX的化合物作为荧光染料用于检测红细胞和血小板的荧光-前向散射光散点图；
图19示出了采用具有化学式X的化合物作为荧光染料用于分类白细胞的侧向散射光-荧光散点图；
图20示出了采用具有化学式X的化合物作为荧光染料用于检测红细胞和血小板的荧光-前向散射光散点图；
图21示出了采用具有化学式XI的化合物作为荧光染料用于分类白细胞的侧向散射光-荧光散点图；
图22示出了采用具有化学式XI的化合物作为荧光染料用于检测红细胞和血小板的荧光-前向散射光散点图；
图23示出了采用具有化学式XII的化合物作为荧光染料用于分类白细胞的侧向散射光-荧光散点图；
图24示出了采用具有化学式XII的化合物作为荧光染料用于检测红细胞和血小板的荧光-前向散射光散点图；
图25示出了采用具有化学式XIII的化合物作为荧光染料用于分类白细胞的侧向散射光-荧光散点图；
图26示出了采用具有化学式XIII的化合物作为荧光染料用于检测红细胞和血小板的荧光-前向散射光散点图。
具体实施方式
在下文的描述中，给出了大量具体的细节以便提供对本发明更为彻底的理解。然而，对于本领域技术人员而言显而易见的是，本发明可以无需一个或多个这些细节而得以实施。在其他的例子中，为了避免与本发明发生混淆，对于本领域公知的一些技术特征未进行描述。
为了彻底理解本发明，将在下列的描述中提出详细的描述。显然，本发明实施方式的施行并不限定于本领域的技术人员所熟悉的特殊细节。本发明的较佳实施例详细描述如下，然而除了这些详细描述外，本发明还可以具有其他实施方式。
应予以注意的是，这里所使用的术语仅是为了描述具体实施例，而非意图限制根据本发明的示例性实施例。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式。此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在所述特征、整体、步骤、操作、元件和/或组分，但不排除存在或附加一个或多个其他特征、整体、步骤、操作、元件、组分和/或它们的组合。
除另有说明外，本文中使用的术语“血液”或“血液样品”是指包含血细胞的体液样品，例如外周血、骨髓液等。
特别地，如本申请自始至终所使用的，术语烷基可以在最广义上理解为任何线性、支化或环状的烷基取代基。如本文使用的术语“C 1-18烷基”一般指在构型中具有1至18个碳原子的饱和烃基团，C 1-18烷基包括但不限于C 1-12烷基、C 1-6烷基等。在未描述为“取代烷基”的情况下，术语“烷基”通常指未取代的烷基。示例性地，术语烷基包括取代基甲基(Me)，乙基(Et)，正丙基(nPr)，异丙基(iPr)，环丙基，正丁基(nBu)，异丁基(iBu)，仲丁基(sBu)，叔丁基(tBu)，环丁基，2-甲基丁基，正戊基，仲戊基，叔戊基，2-戊基，新戊基，环戊基，正己基，仲己基，叔己基，2-己基，3-己基，新己基，环己基，1-甲基环戊基，2-甲基戊基，正庚基，2-庚基，3-庚基，4-庚基，环庚基，1-甲基环己基，正辛基，2-乙基己基， 环辛基，1-双环[2,2,2]辛基，2-双环[2,2,2]-辛基，2-(2,6-二甲基)辛基，3-(3,7-二甲基)辛基，金刚烷基，2,2,2-三氟乙基，1,1-二甲基-正己-1-基，1,1-二甲基-正庚-1-基，1,1-二甲基-正辛-1-基，1,1-二甲基-正癸-1-基，1,1-二甲基-正十二烷-1-基，1,1-二甲基-正十四烷-1-基，1,1-二甲基-正十六烷-1-基，1,1-二甲基-正十八烷-1-基，1,1-二乙基-正己-1-基，1,1-二乙基-正庚-1-基，1,1-二乙基-正辛-1-基，1,1-二乙基-正癸-1-基，1,1-二乙基-正十二烷-1-基，1,1-二乙基-正十四烷-1-基，1,1-二乙基-正十六烷-1-基，1,1-二乙基-正十八烷-1-基，1-(正丙基)-环己-1-基，1-(正丁基)-环己-1-基，1-(正己基)-环己-1-基，1-(正辛基)-环己-1-基和1-(正癸基)-环己-1-基等。
如上文和此处所使用的，术语羧基包括任何线性、支化或环状的羧基取代基。如本文使用的术语C 1-18羧基一般指在构型中具有1至18个碳原子的羧基取代基团，C 1-18羧基包括但不限于C 1-12羧基、C 1-6羧基等。特别的，术语羧基指与如前文定义的烷基连接的具有羧基的基团。术语羧基示例性地包括甲羧基，乙羧基，丙羧基，丁羧基，戊羧基、己羧基、庚羧基和辛羧基，以及它们的同分异构体等。
如上文和此处所使用的，术语羟基包括任何线性、支化或环状的羟基取代基。如本文使用的术语C 1-18羟基一般指在构型中具有1至18个碳原子的羟基取代基团，C 1-18羟基包括但不限于C 1-12羟基、C 1-6羟基等。特别的，术语羟基指与如前文定义的烷基连接的具有羟基的基团。术语羟基示例性地包括羟甲基，羟乙基，羟丙基，羟丁基，羟戊基、羟己基、羟庚基和羟辛基，以及它们的同分异构体等。
如上文和此处所使用的，术语烷氧基包括任何线性、支化或环状的烷氧基取代基。特别的，术语烷氧基指与如前文定义的烷基连接的烷氧基基团。术语烷氧基示例性地包括甲氧基，乙氧基，正丙氧基，异丙氧基，正丁氧基，异丁氧基，仲丁氧基，叔丁氧基和2-甲基丁氧基等。
如上文和此处所使用的，术语“卤素”和“卤代”可以在最广泛的意义上理解为优选氟、氯、溴或碘。
如本文所使用的，如果在特定的上下文中没有更具体地定义，蓝绿色光波长约在400-560nm的范围。
荧光染料
本申请的荧光染料属于菁类化合物，具有良好的活细胞通透性，能够 在不破坏细胞膜的情况进入细胞对核酸(RNA、DNA)进行特异性染色。所述染料为蓝绿光激发荧光染料，能够被蓝色或绿色半导体激光器等提供蓝色或绿色区域激光的器件发出的蓝色或绿色激光所激发，这样本发明试剂可用于采用半导体激光器等廉价设备作为光源的血液分析仪或流式细胞仪等。
在一个实施例中，荧光染料具有式F所示的结构：
Figure PCTCN2020128839-appb-000005
其中，X选自由C(CH 3) 2、O、S和Se组成的组；
R 1和R 2各自独立地选自由H、C 1-C 18烷基、苯基、OR 6和卤素组成的组；
R 3和R 4各自独立地选自由C 1-C 18烷基、C 1-C 18羧基、C 1-C 18羟基、C 1-C 18NR 5R 6、苄基和取代苄基组成的组，其中所述取代苄基的取代基选自由C 1-C 18烷基、CN、COOH、NH 2、NO 2、OH、SH、C 1-C 6烷氧基、C 1-C 6烷基氨基、C 1-C 6酰氨基、卤素和C 1-C 6卤代烷基组成的组；
R 5和R 6各自独立地选自由H和C 1-C 18烷基组成的组；
Y -为负离子。
一个实施例中，式F中的X选自由C(CH 3) 2和S组成的组。
一个实施例中，式F中的R 1和所述R 2各自独立地选自由H、C 1-C 12烷基、苯基、OR 6和卤素组成的组。
一个实施例中，式F中的R 1和所述R 2各自独立地选自由H、C 1-C 6烷基、苯基、OR 6和卤素组成的组。
一个实施例中，式F中的R 1选自由H、C 1-C 6烷基、苯基和卤素组成的组。
一个实施例中，式F中的R 2为H。
一个实施例中，式F中的R 3和R 4各自独立地选自由C 1-C 12烷基、C 1-C 12羧基、C 1-C 12羟基、C 1-C 12NR 5R 6、苄基和取代苄基组成的组，其中所述取代苄基的取代基选自由C 1-C 12烷基、CN、COOH、NH 2、NO 2、OH、SH、C 1-C 6烷氧基、C 1-C 6烷基氨基、C 1-C 6酰氨基、卤素和C 1-C 6卤代烷基组成的组。
一个实施例中，式F中的R 3和R 4各自独立地选自由C 1-C 6烷基、C 1-C 6羧基、C 1-C 6羟基、C 1-C 6NR 5R 6、苄基和取代苄基组成的组，其中所述取代苄基的取代基选自由C 1-C 6烷基、CN、COOH、NH 2、NO 2、OH、SH、C 1-C 6烷氧基、C 1-C 6烷基氨基、C 1-C 6酰氨基、卤素和C 1-C 6卤代烷基组成的组。
一个实施例中，式F中的R 3选自由C 1-C 6烷基、C 1-C 6羟基、C 1-C 6羧基、C 1-C 6NR 5R 6和苄基组成的组。
一个实施例中，式F中的R 4选自由C 1-C 6烷基、C 1-C 6羟基、C 1-C 6羧基、和苄基组成的组。
一个实施例中，式F中的R 5和R 6各自独立地选自由H和C 1-C 12烷基组成的组。
一个实施例中，式F中的R 5和R 6各自独立地选自由H和C 1-C 6烷基组成的组。
一个实施例中，式F中的R 5和R 6各自独立地为C 1-C 6烷基。
一个实施例中，式F中的Y -选自由卤素负离子、ClO 4 -、PF 6 -、BF 4 -、CH 3COO -或OTs -组成的组。
一个实施例中，荧光染料包含化学式I、化学式II、化学式III、化学式IV、化学式V、化学式VI、化学式VII、化学式VIII、化学式IX、化学式X、化学式XI、化学式XII和化学式XIII中的一种所示的结构，
Figure PCTCN2020128839-appb-000006
Figure PCTCN2020128839-appb-000007
本申请的荧光染料可以溶解在甘油、甘醇、乙二醇中并作为储存液保存，也可以溶解在其它非水溶剂中。本发明优选使用乙二醇作为溶剂，并任选含有5-10％的甲醇作为稀释液的成分。此外，为了防止荧光染料聚集，本发明还可任选包含非离子表面活性剂作为分散剂，使荧光染料保持一定的溶解度，不产生聚集。这种非离子表面活性剂例如为聚氧乙烯乙二醇(POE)、聚丙烯乙二醇(POP)、聚氧乙烯乙二醇-聚丙烯乙二醇(POE-POP)、Brij系列非离子表面活性剂。本发明优选使用Brij 35、Brij56等作为荧光染料的分散剂，浓度为0.02-2％。
或者，本申请的荧光染料可与红细胞溶解剂或球形化试剂配制在一起， 形成单组分试剂。
红细胞溶解剂
使用的红细胞溶解剂可包含阳离子表面活性剂、非离子表面活性剂、阴离子表面活性剂或它们的任何组合，以及缓冲剂，其将测定体系的pH保持在5-11。
可采用阳离子表面活性剂、非离子表面活性剂、阴离子表面活性剂或他们的任何组合，作为溶解红细胞的试剂。它们可溶解血液样本中红细胞，并且适当破坏各亚群白细胞的膜结构，使血液中白细胞的各亚群收缩成适当的大小，促进白细胞内部结构产生不同的凝聚，造成散射光特性的差别。阳离子表面活性剂可以为辛基三甲基溴化铵(OTAB)、癸基三甲基溴化铵(DTAB)、十二烷基三甲基氯化铵(LTAC)、十四烷基三甲基溴化铵(CTAB)、十四烷基三甲基氯化铵(CTAC)等，优选为LTAC，使用浓度为300-800mg/L。非离子表面活性剂可采用聚氧乙烯型非离子表面活性剂，如长链脂肪醇聚氧乙烯、烷基酚聚氧乙烯醚、脂肪酸聚氧乙烯酯、脂肪胺聚氧乙烯醚等，优选非离子表面活性剂为聚氧乙烯-2，3-十二烷基醚(Brij35)，使用浓度为1-5g/L。阴离子表面活性剂选自十二烷基苯磺酸、脂肪醇酰硫酸钠、乙氧基化脂肪酸甲酯磺酸钠、仲烷基磺酸钠、醇醚羧酸盐，使用浓度为0.1-2g/L。
本申请的试剂中可含有保持pH并对血液样本进行适当稀释的缓冲液。缓冲液使pH保持在恒定范围内，由此可稳定各亚群白细胞的染色效果，缓冲剂的使用浓度在0.01-200mM范围内。对缓冲液的种类并没有特别限定，只要它们可适用于本发明的目的，例如适当浓度的羧酸盐类、磷酸盐类、柠檬酸盐类、Tris-HCl、MOPS以及其它有机缓冲剂等。本发明试剂中的合适pH因所选择的具体荧光染料不同而有所不同，一般在pH 5.0-11.0范围内，优选的pH是8.0-10.0。
本发明的红细胞溶解剂还可任选包含适当比例的醇，如甲醇，以促进白细胞内部结构的凝聚。
白细胞检测试剂盒
在另一方面，本发明还提供用于能够区分和计数血液中的白细胞的试剂盒，所述所述试剂盒包含白细胞分类试剂，所述白细胞分类试剂包含具有式 F所示结构的荧光染料：
Figure PCTCN2020128839-appb-000008
其中，X选自由C(CH 3) 2、O、S和Se组成的组；
R 1和R 2各自独立地选自由H、C 1-C 18烷基、苯基、OR 6和卤素组成的组；
R 3和R 4各自独立地选自由C 1-C 18烷基、C 1-C 18羧基、C 1-C 18羟基、C 1-C 18NR 5R 6、苄基和取代苄基组成的组，其中所述取代苄基的取代基选自由C 1-C 18烷基、CN、COOH、NH 2、NO 2、OH、SH、C 1-C 6烷氧基、C 1-C 6烷基氨基、C 1-C 6酰氨基、卤素和C 1-C 6卤代烷基组成的组；
R 5和R 6各自独立地选自由H和C 1-C 18烷基组成的组；
Y -为负离子。
其他实施例中，X、R 1、R 2、R 3、R 4和Y -的选择，同前文荧光染料部分的描述所示。
优选，荧光染料具有选自化学式I、化学式II、化学式III、化学式IV、化学式V、化学式VI、化学式VII、化学式VIII、化学式IX、化学式X、化学式XI、化学式XII和化学式XIII的结构，
Figure PCTCN2020128839-appb-000009
Figure PCTCN2020128839-appb-000010
优选所述白细胞分类试剂还包含本文上述的红细胞溶解剂。
在一个实施方案中，所述荧光染料作为储存液形式存在，保存在单独的容器中。其中，所述荧光染料的浓度为：0.1-1000mg/L,优选20-500mg/L，更优选50-200mg/L。
在另一个实施方案中，所述荧光染料与上述红细胞溶解剂一起配制为混合试剂溶液。
此试剂盒优选包含适于密封保持在至少一个容器中的分区存放的荧光染料。所述试剂盒还可包含将血液中白细胞分类所需的其它白细胞分类试剂以及测定白细胞的方法的说明。所述试剂盒还可包含可测定并与测试样品对比的对照样品或一系列对照样品。试剂盒的各组分可封装在单个容器中，不同 容器连同说明一起全部在单独包装中，所述试剂盒用于分类和/或计数血液中的各类白细胞。
球形化试剂
使用的球形化试剂可包含两性表面活性剂，渗透压调节剂，缓冲剂，以及任选的阳离子表面活性剂和/或防腐剂。
球形化试剂中的表面活性剂使血细胞肿胀并轻微损伤细胞膜以便染料进入细胞。两性表面活性剂的浓度范围：100-2000mg/L,优选100-500mg/L。两性表面活性剂可为烷基甜菜碱、磺酸基甜菜碱、椰油酰胺丙基甜菜碱等中一种或多种。阳离子表面活性剂可以为辛基三甲基溴化铵(OTAB)、癸基三甲基溴化铵(DTAB)、十二烷基三甲基氯化铵(LTAC)、十四烷基三甲基溴化铵(CTAB)、十四烷基三甲基氯化铵(CTAC)等，使用浓度为0-50mg/L。渗透压调节剂可为：氯化钠、硫酸钠等常用盐类，渗透压范围为：200mOsm/kg-380mOsm/kg。对缓冲液的种类并没有特别限定，只要它们可适用于本发明的目的，例如适当浓度的羧酸盐类、磷酸盐类、柠檬酸盐类、Tris-HCl、MOPS以及其它有机缓冲剂等。本发明试剂中的合适pH因所选择的具体荧光染料不同而有所不同，一般在pH 5.0-11.0范围内，优选的pH是8.0-10.0。
红细胞检测试剂盒
在另一方面，本发明还提供用于能够区分和计数血液中的红细胞的试剂盒，尤其是对网织红细胞识别和计数，所述试剂盒包含红细胞分析试剂，所述红细胞分析试剂包含具有式F所示结构的荧光染料：
Figure PCTCN2020128839-appb-000011
其中，X选自由C(CH 3) 2、O、S和Se组成的组；
R 1和R 2各自独立地选自由H、C 1-C 18烷基、苯基、OR 6和卤素组成的 组；
R 3和R 4各自独立地选自由C 1-C 18烷基、C 1-C 18羧基、C 1-C 18羟基、C 1-C 18NR 5R 6、苄基和取代苄基组成的组，其中所述取代苄基的取代基选自由C 1-C 18烷基、CN、COOH、NH 2、NO 2、OH、SH、C 1-C 6烷氧基、C 1-C 6烷基氨基、C 1-C 6酰氨基、卤素和C 1-C 6卤代烷基组成的组；
R 5和R 6各自独立地选自由H和C 1-C 18烷基组成的组；
Y -为负离子。
其他实施例中，X、R 1、R 2、R 3、R 4和Y -的选择，同前文荧光染料部分的描述所示。
优选，荧光染料具有选自化学式I、化学式II、化学式III、化学式IV、化学式V、化学式VI、化学式VII、化学式VIII、化学式IX、化学式X、化学式XI、化学式XII和化学式XIII的结构，
Figure PCTCN2020128839-appb-000012
Figure PCTCN2020128839-appb-000013
优选所述红细胞分析试剂还包含本文上述的球形化试剂。
在一个实施方案中，所述荧光染料作为储存液形式存在，保存在单独的容器中。其中，所述荧光染料的浓度为：0.1-1000mg/L,优选20-500mg/L，更优选50-200mg/L。
在另一个实施方案中，所述荧光染料与上述球形化试剂一起配制为混合试剂溶液。
此试剂盒优选包含适于密封保持在至少一个容器中的分区存放的荧光染料。所述试剂盒还可包含将血液中红细胞分类所需的其它红细胞分类试剂以及测定红细胞的方法的说明。所述试剂盒还可包含可测定并与测试样品对比的对照样品或一系列对照样品。试剂盒的各组分可封装在单个容器中，不同容器连同说明一起全部在单独包装中，所述试剂盒用于分类和/或计数血液中的各类红细胞，如成熟红细胞和/或网织红细胞。
使用方法：
A：本申请还提供分类和/或计数血液中白细胞的方法。简单地说，在本申请的方法中，通过将血液样本与白细胞分类试剂混合，随后测定样品的至少一种散射光特性和至少一种荧光特性，并根据散射光特性和荧光特性将样 品分类和/或计数。
本申请方法中使用的血液样本可以是全血，也可以是成分血。可使血液样品先与红细胞溶解剂混合，使得红细胞被溶解，各亚群的白细胞产生不同程度的皱缩。该步骤同时在待测定白细胞的细胞膜上形成小孔，这种小孔足以允许荧光染料分子通过细胞膜。
随后加入荧光染料储存液，使得白细胞被荧光标记。在血液样本与试剂混合时，血液样本与试剂的总体积应保证足够的细胞浓度通过仪器的测量池。本申请的试剂组合物将血液样本稀释至1∶10、1∶50或1∶100或以上任何范围中的任何值，只要该稀释符合实际使用的要求。这种调整在本领域技术人员的范围内。此外，也可使血液样品与红细胞溶解剂和荧光染料同时混合。
样品混合物可在孵育池中进行温育，温育时间不超过60秒，优选30秒，24秒；温育温度可为任何合适的温度，例如40℃。可采用血液分析仪或流式细胞仪检测和分析白细胞，经过溶解、稀释并染色的血液样品导入测量仪器的流动室中，光源发出的光照射所述流动室中的粒子产生光学信息，检测器用于收集所述光学信息，所述光学信息包括至少一种散射光和至少一种荧光。
本申请中的散射光是指可由市售血液分析仪或类似的流式细胞仪检测的散射光。这种散射光包括但不限于侧向散射光、正向低角度散射光(接受光角度为约0-5度)和正向高角度散射光(接受光角度为约5-20度)。具有这种角度的散射光反映了白细胞大小或内部结构的信息，因此用作本发明的散射光。优选侧向散射光。
与细胞中核酸结合的荧光染料发射荧光。荧光特性是反映血液样本中细胞内荧光染料量的参数。由于不同亚群的细胞内代谢活动不同，而造成核酸含量的差异，因此不同亚群的白细胞的荧光特性在某些方面具有差异。取决于所使用的具体染料选择合适的激发光波长，并监测相应波长的发射光。本申请对白细胞检测时，可采用绿色或蓝色半导体激光器发射的绿色或蓝色波长区域激光为检测的光源。对所述绿色或蓝色波长的光源没有特殊的限制，只要能够发出所选择荧光染料的激发波长附近的绿色光(例如约波长501-560nm的光)或者或蓝色光(例如约波长400-500nm的光)。例如，光源可以配置为发射波长约为450nm或520nm的光。这类激光器相对于其它激光器成本较低，这样可降低设备的成本。
利用散射光特性和荧光特性识别各亚群白细胞，并对各聚类散点进行相 关的分类计数，计算各亚群白细胞的百分比。散射光反映细胞内部颗粒程度，细胞内部的颗粒程度大致为：嗜酸细胞内部有两分叶核并且内部有很多酸性染料染色的脆质微粒；中性粒细胞核(分叶或杆状)且内部颗粒较多；单核细胞为单大核内部颗粒较少；LYM细胞单大核基本没有颗粒。所以相同条件下各类白细胞的散射光强度特性顺序为EO(嗜酸细胞)＞NEUT(中性粒细胞)＞MONO(单核细胞)＞LYM(淋巴细胞)。
B：本申请还提供分类和/或计数血液中红细胞的方法。简单地说，在本申请的方法中，通过将血液样本与红细胞分析试剂混合，随后测定样品的至少一种散射光特性和至少一种荧光特性，并根据散射光特性和荧光特性将样品分类和/或计数。
本申请方法中使用的血液样本可以是全血，也可以是成分血。可使血液样品先与球形化试剂混合，使得红细胞和白细胞肿胀并轻微损伤细胞膜，在红细胞和白细胞的细胞膜上形成小孔，这种小孔足以允许荧光染料分子通过细胞膜。
随后加入荧光染料储存液，使得红细胞和白细胞被荧光标记。由于成熟红细胞、网织红细胞、和白细胞的核酸含量不同，所以荧光特性不同，可以将上述三种细胞区分开来。在血液样本与试剂混合时，血液样本与本申请试剂的总体积应保证足够的细胞浓度通过仪器的测量池。本申请的试剂组合物将血液样本稀释至1∶10、1∶50或1∶100或以上任何范围中的任何值，只要该稀释符合实际使用的要求。这种调整在本领域技术人员的范围内。此外，也可使血液样品与球形化试剂和荧光染料同时混合。
样品混合物可在孵育池中进行温育，温育时间不超过60秒，优选30秒、24秒；温育温度可为任何合适的温度，例如40℃。可采用血液分析仪或流式细胞仪检测和分析红细胞，经过球形化处理并染色的血液样品导入测量仪器的流动室中，光源发出的光照射所述流动室中的粒子产生光学信息，检测器用于收集所述光学信息，所述光学信息包括至少一种散射光和至少一种荧光。
本申请中的散射光是指可由市售血液分析仪或类似的流式细胞仪检测的散射光。这种散射光包括但不限于侧向散射光、正向低角度散射光(接受光角度为约0-5度)和正向高角度散射光(接受光角度为约5-20度)。具有这种角度的散射光反映了细胞大小或内部结构的信息，因此用作本发明的散射光。优选前向散射光。
本申请对红细胞检测时，同样可采用绿色或蓝色半导体激光器发射的绿色或蓝色波长区域激光为检测的光源。
利用散射光特性和荧光特性识别成熟红细胞、网织红细胞以及血小板，并对各聚类散点进行相关的分类计数，获得红细胞(包括成熟红细胞和网织红细胞)计数、网织红细胞计数以及血小板计数。前向散射光反应细胞体积大小的信息，血小板体积比红细胞小，其前向散射光强度相对较弱；成熟红细胞和网织红细胞核酸RNA含量不同，网织红细胞核酸RNA含量相对较高，其荧光强度相对较高。
实施例1
染料的合成
示例性的荧光染料通过以下方式合成，制备具有化学式I所示的结构的化合物A，
Figure PCTCN2020128839-appb-000014
在本实施方式中，X为S，R 1为H，R 2为H，R 3为苄基，R 4为苄基。
化合物A的具体制备方法如下：
第一步，根据下述反应式I制备2-甲硫基苯并噻唑(反应I右侧)，
Figure PCTCN2020128839-appb-000015
量取20mL的DMF放入容器中，将10mmol的2-巯基苯并噻唑(反应式I左侧)、11mmol的甲烷以及12mmol的碳酸钠加入容器。在氮气保护及温度为110℃的条件下搅拌反应8小时，之后停止反应。
将得到的混合物冷却至室温，之后将其倾入大量水中。使用适量的乙酸乙酯萃取3次，合并萃取的有机相。使用蒸馏水洗涤上述有机相两次，之后用无水硫酸镁对其进行干燥过夜。
然后将干燥过夜后的物质经过色谱柱纯化，得到约8.5mmol的橙黄色 固体粉末，该橙黄色固体粉末即为2-甲硫基苯并噻唑。反应式I的产率约为85％。
第二步，根据下述反应式II制备1-苄基-4-甲基吡啶季铵盐(反应式II右侧)，
Figure PCTCN2020128839-appb-000016
在容器中量取50mL甲苯，将10mmol的4-甲基吡啶(反应式II左侧)和12mmol的苄溴加入到容器中。在氮气保护的条件下回流并搅拌8小时，之后停止反应。
对反应后的混合物进行抽滤，之后使用50mL甲苯洗涤滤饼3次，得到粗产物。
将上述粗产物经过色谱柱纯化后得到约7.3mmol的白色固体粉末，白色固体粉末即为1-苄基-4-甲基吡啶季铵盐。该反应式II的产率约为73％。
第三步，根据下述反应式III制备3-苄基-2-硫酮苯并噻唑(反应式III右侧)，
Figure PCTCN2020128839-appb-000017
量取30mL甲苯放入容器中，将3mmol的第一步反应得到的2-甲硫基苯并噻唑(反应式III左侧)、4mmol的苄溴以及5mmol的碳酸钾加入到容器中。在氮气保护及温度为110℃的条件下搅拌反应8小时。
对反应后的混合物进行抽滤，之后使用50mL甲苯洗涤滤饼3次，得粗产物。
将上述粗产物经过色谱柱纯化后得到约2.1mmol的白色固体粉末，该白色固体粉末即为3-苄基-2-硫酮苯并噻唑，反应式III的产率约为70％。
第四步，根据下述反应式IV制备3-苄基-2-乙硫基苯并噻唑(反应式IV右侧)，
Figure PCTCN2020128839-appb-000018
在容器中量取30mL二氯甲烷，将2mmol的第三步反应得到的3-苄基 -2-硫酮苯并噻唑以及2mmol的锳盐加入到容器中。在氮气保护的条件下回流搅拌24小时。
产物经过色谱柱纯化后得到约1.1mmol的白色固体粉末，该白色固体粉末即为3-苄基-2-乙硫基苯并噻唑。反应式IV的产率约为55％。
第五步，根据下述反应式V制备化合物A(反应式V右侧)，
Figure PCTCN2020128839-appb-000019
在容器中量取30mL的乙醇，称取第二步反应得到的的1-苄基-4-甲基吡啶季铵盐1mmol，称取第四步反应得到的3-苄基-2-乙硫基苯并噻唑1mmol，将二者加入到容器中。
在氮气保护地条件下回流搅拌24小时。产物经色谱柱纯化后得到约0.3mmol橙黄色固体粉末，该橙黄色固体粉末即为化合物A。反应式V的产率约为30％。
对化合物A进行核磁共振测试，结果如下。
1H-NMR(500MHz，DMSO，TMS)：δ4.00(s，3H)，5.59(s，2H)，6.39(s，1H)，7.26-7.31(m，4H)，7.35-7.38(t，2H)，7.40-7.42(d，2H)，7.45-7.48(t，1H)，7.52-7.54(d，1H)，7.94-7.96(d，1H)，8.33-8.35(d，2H)。
实施例2
制备具有化学式X所示的结构的化合物J，
Figure PCTCN2020128839-appb-000020
在本实施方式中，X为S，R 1为H，R 2为H，R 3为羧基戊基，R 4为甲基。
化合物J的具体制备方法如下：
第一步，在50mL容量的圆底烧瓶中装10mL乙酸乙酯，将5.37mmol的4-甲基吡啶和10.7mmol的6-溴己酸加入到上述圆底烧瓶中，并在80℃的条件下回流反应8h。使用乙酸乙酯对得到的混合物进行重结晶并抽滤，抽滤过程中使用乙酸乙酯充分洗涤。将得到的滤出物用甲醇溶解，之后进行蒸干。最终干燥后得到黄色油性液体，其为1-(5-羧基戊基)-4-甲基吡啶，其粗收率约为91.07％。
第二步，在50mL容量的圆底烧瓶中装10mL无水四氢呋喃，将11.84mmol的2-巯基苯并噻唑加入到该圆底烧瓶中，之后在搅拌的状态下缓慢向圆底烧瓶中滴加14.21mmol的碘甲烷，反应过夜。之后在反应得到的混合物中加入少量石油醚并静置沉淀。之后过滤，并用大量石油醚洗涤滤出物。最终将滤出物干燥后得到白色固体，其为2-甲硫基苯并噻唑，其粗收率约为98％。
第三步，在50mL容量的圆底烧瓶中盛装10mL甲苯，将10mmol的2-甲硫基苯并噻唑加入到上述圆底烧瓶中。之后在氮气保护下，向该圆底烧瓶中缓慢滴加12mmol的碘甲烷，并在110℃及回流的条件下持续反应24h。将反应得到的混合物冷却到室温，之后对混合物进行过滤，并用二氯甲烷洗涤滤出物。最终将滤出物干燥后得到微黄色固体粉末，其为3-甲基-2-甲硫基苯并噻唑，其粗收率约为30％。
第四步，在25mL容量的圆底烧瓶中加入5mL二氯甲烷。将0.500mmol的3-甲基-2-甲硫基苯并噻唑与0.509mmol的1-(5-羧基戊基)-4-甲基吡啶加入到上述圆底烧瓶中。在室温以及避光的条件下搅拌10min后，再向其中缓慢滴加756μL三乙胺，之后在室温及避光的条件下反应21h左右。之后将反应得到的混合物蒸干，剩余物通过中性硅胶柱进行分离，其中使用二氯甲烷和甲醇混合溶剂作为洗脱液。收集分离后的黄色组分并对其蒸干干燥，最终得到棕黄色粘稠物，即为化合物J，其粗收率50％。
对化合物J进行核磁共振测试，结果如下。
1H-NMR(400MHz，DMSO)：δ8.36(s，1H)，7.90(d，J＝7.6Hz，1H)，7.58(d，J＝8.0Hz，1H)，7.51(t，J＝7.4Hz，1H)，7.40(s，1H)，7.30(t，J＝7.1Hz，1H)，6.25(s，1H)，4.21(s，1H)，3.72(s，3H)，2.10(s，1H)，1.82(s，1H)，1.53(s，1H)，1.28(s，1H)。
实施例3：
试剂的配制
配制如下组成的试剂体系。
A：荧光染料储存液：
荧光染料(具有化学式I)  50mg
乙二醇   950ml
甲醇     50ml
B：红细胞溶解剂溶液
Figure PCTCN2020128839-appb-000021
C:球形化试剂溶液
Figure PCTCN2020128839-appb-000022
1)对血液样品中白细胞的测定和结果：
测试样本的过程在迈瑞自主研发的经改进的BC-6000血液分析仪器)中自动进行，设置吸样量为20微升，溶血剂溶液B进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml红细胞溶解剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的侧向散射光强度和荧光强度，得到白细胞散点图。对血液样本(1号血液样本)的测试结果如图1所示，表明本申请可以实现白细胞4个亚群 的分类识别，图1中标注的四团粒子分别为淋巴细胞，单核细胞、中性粒细胞和嗜酸性粒细胞，表明本申请白细胞分类试剂能够实现白细胞各亚群的有效分类。
2)对血液样品中红细胞的测定和结果：
测试样本的过程在迈瑞自主研发的血液分析仪器中自动进行，设置吸样量为4微升，球形化试剂溶液C进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml球形化试剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的前向散射光强度和荧光强度，得到包含红细胞的散点图。对血液样本(1号血液样本)的测试结果如图2所示，表明本申请可以实现红细胞和血小板的的分类识别，图2中标注的三团粒子分别为红细胞(即成熟红细胞)，网织红细胞(RET)和血小板，表明本申请红细胞检测试剂能够实现红细胞和血小板的分类和识别，尤其是RET的识别。
3)相同血液样品在迈瑞BC-6800仪器分别对白细胞和红细胞的测定和结果。
用同样一支血样(1号血液样本)在迈瑞BC-6800仪器上按照CDR模式进行测试，采用BC-6800仪器配套试剂，分别检测得到的白细胞和红细胞相关参数如下表所示：
Figure PCTCN2020128839-appb-000023
从表中可以看出，具有化学式I的荧光染料对白细胞分群和网织红细胞的测试结果与BC-6800的测试结果较为一致。
实施例4：
试剂的配制
配制如下组成的试剂体系。
A：荧光染料储存液：
荧光染料(具有化学式II)  50mg
乙二醇   950ml
甲醇     50ml
B：红细胞溶解剂溶液
Figure PCTCN2020128839-appb-000024
C:球形化试剂溶液
Figure PCTCN2020128839-appb-000025
1)对血液样品中白细胞的测定和结果：
测试样本的过程在迈瑞自主研发的经改进的BC-6000血液分析仪器中自动进行，设置吸样量为20微升，溶血剂溶液B进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml红细胞溶解剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的侧向散射光强度和荧光强度，得到白细胞散点图。对血液样本(2 号血液样本)的测试结果如图3所示，表明本申请可以实现白细胞4个亚群的分类识别，图3中标注的四团粒子分别为淋巴细胞，单核细胞、中性粒细胞和嗜酸性粒细胞，表明本申请白细胞分类试剂能够实现白细胞各亚群的有效分类。
2)对血液样品中红细胞的测定和结果：
测试样本的过程在迈瑞自主研发的血液分析仪器中自动进行，设置吸样量为4微升，球形化试剂溶液C进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml球形化试剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的前向散射光强度和荧光强度，得到包含红细胞的散点图。对血液样本(2号血液样本)的测试结果如图4所示，表明本申请可以实现红细胞和血小板的的分类识别，图4中标注的三团粒子分别为红细胞(即成熟红细胞)，网织红细胞(RET)和血小板，表明本申请红细胞检测试剂能够实现红细胞和血小板的分类和识别，尤其是RET的识别。
3)相同血液样品在迈瑞BC-6800仪器分别对白细胞和红细胞的测定和结果。
用同样一支血样(2号血液样本)在迈瑞BC-6800仪器上按照CDR模式进行测试，采用BC-6800仪器配套试剂，分别检测得到的白细胞和红细胞相关参数如下表所示：
Figure PCTCN2020128839-appb-000026
从表中可以看出，具有化学式II的荧光染料对白细胞分群和网织红细胞的测试结果与BC-6800的测试结果较为一致。
实施例5：
试剂的配制
配制如下组成的试剂体系。
A：荧光染料储存液：
荧光染料(具有化学式III)  50mg
乙二醇   950ml
甲醇     50ml
B：红细胞溶解剂溶液
Figure PCTCN2020128839-appb-000027
C:球形化试剂溶液
Figure PCTCN2020128839-appb-000028
1)对血液样品中白细胞的测定和结果：
测试样本的过程在迈瑞自主研发的经改进的BC-6000血液分析仪器中自动进行，设置吸样量为20微升，溶血剂溶液B进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml红细胞溶解剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定 测试样本的侧向散射光强度和荧光强度，得到白细胞散点图。对血液样本(3号血液样本)的测试结果如图5所示，表明本申请可以实现白细胞4个亚群的分类识别，图5中标注的四团粒子分别为淋巴细胞，单核细胞、中性粒细胞和嗜酸性粒细胞，表明本申请白细胞分类试剂能够实现白细胞各亚群的有效分类。
2)对血液样品中红细胞的测定和结果：
测试样本的过程在迈瑞自主研发的血液分析仪器中自动进行，设置吸样量为4微升，球形化试剂溶液C进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml球形化试剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的前向散射光强度和荧光强度，得到包含红细胞的散点图。对血液样本(3号血液样本)的测试结果如图6所示，表明本申请可以实现红细胞和血小板的的分类识别，图6中标注的三团粒子分别为红细胞(即成熟红细胞)，网织红细胞(RET)和血小板，表明本申请红细胞检测试剂能够实现红细胞和血小板的分类和识别，尤其是RET的识别。
3)相同血液样品在迈瑞BC-6800仪器分别对白细胞和红细胞的测定和结果。
用同样一支血样(3号血液样本)在迈瑞BC-6800仪器上按照CDR模式进行测试，采用BC-6800仪器配套试剂，分别检测得到的白细胞和红细胞相关参数如下表所示：
Figure PCTCN2020128839-appb-000029
从表中可以看出，具有化学式III的荧光染料对白细胞分群和网织红细 胞的测试结果与BC-6800的测试结果较为一致。
实施例6：
试剂的配制
配制如下组成的试剂体系。
A：荧光染料储存液：
荧光染料(具有化学式IV)  100mg
乙二醇   950ml
甲醇     50ml
B：红细胞溶解剂溶液
Figure PCTCN2020128839-appb-000030
C:球形化试剂溶液
Figure PCTCN2020128839-appb-000031
1)对血液样品中白细胞的测定和结果：
测试样本的过程在迈瑞自主研发的经改进的BC-6000血液分析仪器中自动进行，设置吸样量为20微升，溶血剂溶液B进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml红细胞溶解剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成 测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的侧向散射光强度和荧光强度，得到白细胞散点图。对血液样本(4号血液样本)的测试结果如图7所示，表明本申请可以实现白细胞4个亚群的分类识别，图7中标注的四团粒子分别为淋巴细胞，单核细胞、中性粒细胞和嗜酸性粒细胞，表明本申请白细胞分类试剂能够实现白细胞各亚群的有效分类。
2)对血液样品中红细胞的测定和结果：
测试样本的过程在迈瑞自主研发的血液分析仪器中自动进行，设置吸样量为4微升，球形化试剂溶液C进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml球形化试剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的前向散射光强度和荧光强度，得到包含红细胞的散点图。对血液样本(4号血液样本)的测试结果如图8所示，表明本申请可以实现红细胞和血小板的的分类识别，图8中标注的三团粒子分别为红细胞(即成熟红细胞)，网织红细胞(RET)和血小板，表明本申请红细胞检测试剂能够实现红细胞和血小板的分类和识别，尤其是RET的识别。
3)相同血液样品在迈瑞BC-6800仪器分别对白细胞和红细胞的测定和结果。
用同样一支血样(4号血液样本)在迈瑞BC-6800仪器上按照CDR模式进行测试，采用BC-6800仪器配套试剂，分别检测得到的白细胞和红细胞相关参数如下表所示：
Figure PCTCN2020128839-appb-000032
从表中可以看出，具有化学式IV的荧光染料对白细胞分群和网织红细胞的测试结果与BC-6800的测试结果较为一致。
实施例7：
试剂的配制
配制如下组成的试剂体系。
A：荧光染料储存液：
荧光染料(具有化学式V)  500mg
乙二醇   950ml
甲醇     50ml
B：红细胞溶解剂溶液
Figure PCTCN2020128839-appb-000033
C:球形化试剂溶液
Figure PCTCN2020128839-appb-000034
1)对血液样品中白细胞的测定和结果：
测试样本的过程在迈瑞自主研发的经改进的BC-6000血液分析仪器中自动进行，设置吸样量为20微升，溶血剂溶液B进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml红细胞溶解剂与经过抗凝剂处理的20微升血液混匀，并立 即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的侧向散射光强度和荧光强度，得到白细胞散点图。对血液样本(5号血液样本)的测试结果如图9所示，表明本申请可以实现白细胞4个亚群的分类识别，图9中标注的四团粒子分别为淋巴细胞，单核细胞、中性粒细胞和嗜酸性粒细胞，表明本申请白细胞分类试剂能够实现白细胞各亚群的有效分类。
2)对血液样品中红细胞的测定和结果：
测试样本的过程在迈瑞自主研发的血液分析仪器中自动进行，设置吸样量为4微升，球形化试剂溶液C进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml球形化试剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的前向散射光强度和荧光强度，得到包含红细胞的散点图。对血液样本(5号血液样本)的测试结果如图10所示，表明本申请可以实现红细胞和血小板的的分类识别，图10中标注的三团粒子分别为红细胞(即成熟红细胞)，网织红细胞(RET)和血小板，表明本申请红细胞检测试剂能够实现红细胞和血小板的分类和识别，尤其是RET的识别。
3)相同血液样品在迈瑞BC-6800仪器分别对白细胞和红细胞的测定和结果。
用同样一支血样(5号血液样本)在迈瑞BC-6800仪器上按照CDR模式进行测试，采用BC-6800仪器配套试剂，分别检测得到的白细胞和红细胞相关参数如下表所示：
Figure PCTCN2020128839-appb-000035
Figure PCTCN2020128839-appb-000036
从表中可以看出，具有化学式V的荧光染料对白细胞分群和网织红细胞的测试结果与BC-6800的测试结果较为一致。
实施例8：
试剂的配制
配制如下组成的试剂体系。
A：荧光染料储存液：
荧光染料(具有化学式VI)  50mg
乙二醇  950ml
甲醇    50ml
B：红细胞溶解剂溶液
Figure PCTCN2020128839-appb-000037
C:球形化试剂溶液
Figure PCTCN2020128839-appb-000038
1)对血液样品中白细胞的测定和结果：
测试样本的过程在迈瑞自主研发的经改进的BC-6000血液分析仪器中自动进行，设置吸样量为20微升，溶血剂溶液B进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml红细胞溶解剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的侧向散射光强度和荧光强度，得到白细胞散点图。对血液样本(6号血液样本)的测试结果如图11所示，表明本申请可以实现白细胞4个亚群的分类识别，图11中标注的四团粒子分别为淋巴细胞，单核细胞、中性粒细胞和嗜酸性粒细胞，表明本申请白细胞分类试剂能够实现白细胞各亚群的有效分类。
2)对血液样品中红细胞的测定和结果：
测试样本的过程在迈瑞自主研发的血液分析仪器中自动进行，设置吸样量为4微升，球形化试剂溶液C进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml球形化试剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的前向散射光强度和荧光强度，得到包含红细胞的散点图。对血液样本(6号血液样本)的测试结果如图12所示，表明本申请可以实现红细胞和血小板的的分类识别，图12中标注的三团粒子分别为红细胞(即成熟红细胞)，网织红细胞(RET)和血小板，表明本申请红细胞检测试剂能够实现红细胞和血小板的分类和识别，尤其是RET的识别。
3)相同血液样品在迈瑞BC-6800仪器分别对白细胞和红细胞的测定和结果。
用同样一支血样(6号血液样本)在迈瑞BC-6800仪器上按照CDR模式进行测试，采用BC-6800仪器配套试剂，分别检测得到的白细胞和红细胞相关参数如下表所示：
Figure PCTCN2020128839-appb-000039
Figure PCTCN2020128839-appb-000040
从表中可以看出，具有化学式VI的荧光染料对白细胞分群和网织红细胞的测试结果与BC-6800的测试结果较为一致。
实施例9：
试剂的配制
配制如下组成的试剂体系。
A：荧光染料储存液：
荧光染料(具有化学式VII)  250mg
乙二醇  950ml
甲醇    50ml
B：红细胞溶解剂溶液
Figure PCTCN2020128839-appb-000041
C:球形化试剂溶液
Figure PCTCN2020128839-appb-000042
1)对血液样品中白细胞的测定和结果：
测试样本的过程在迈瑞自主研发的经改进的BC-6000血液分析仪器中自动进行，设置吸样量为20微升，溶血剂溶液B进样量为1ml，荧光染料存储 液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml红细胞溶解剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的侧向散射光强度和荧光强度，得到白细胞散点图。对血液样本(7号血液样本)的测试结果如图13所示，表明本申请可以实现白细胞4个亚群的分类识别，图13中标注的四团粒子分别为淋巴细胞，单核细胞、中性粒细胞和嗜酸性粒细胞，表明本申请白细胞分类试剂能够实现白细胞各亚群的有效分类。
2)对血液样品中红细胞的测定和结果：
测试样本的过程在迈瑞自主研发的血液分析仪器中自动进行，设置吸样量为4微升，球形化试剂溶液C进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml球形化试剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的前向散射光强度和荧光强度，得到包含红细胞的散点图。对血液样本(7号血液样本)的测试结果如图14所示，表明本申请可以实现红细胞和血小板的的分类识别，图14中标注的三团粒子分别为红细胞(即成熟红细胞)，网织红细胞(RET)和血小板，表明本申请红细胞检测试剂能够实现红细胞和血小板的分类和识别，尤其是RET的识别。
3)相同血液样品在迈瑞BC-6800仪器分别对白细胞和红细胞的测定和结果。
用同样一支血样(7号血液样本)在迈瑞BC-6800仪器上按照CDR模式进行测试，采用BC-6800仪器配套试剂，分别检测得到的白细胞和红细胞相关参数如下表所示：
Figure PCTCN2020128839-appb-000043
Figure PCTCN2020128839-appb-000044
从表中可以看出，具有化学式VII的荧光染料对白细胞分群和网织红细胞的测试结果与BC-6800的测试结果较为一致。
实施例10：
试剂的配制
配制如下组成的试剂体系。
A：荧光染料储存液：
荧光染料(具有化学式VIII)  50mg
乙二醇  950ml
甲醇    50ml
B：红细胞溶解剂溶液
Figure PCTCN2020128839-appb-000045
C:球形化试剂溶液
Figure PCTCN2020128839-appb-000046
1)对血液样品中白细胞的测定和结果：
测试样本的过程在迈瑞自主研发的经改进的BC-6000血液分析仪器中自 动进行，设置吸样量为20微升，溶血剂溶液B进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml红细胞溶解剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的侧向散射光强度和荧光强度，得到白细胞散点图。对血液样本(8号血液样本)的测试结果如图15所示，表明本申请可以实现白细胞5个亚群的分类识别，图15中标注的五团粒子分别为淋巴细胞，单核细胞、中性粒细胞、嗜酸性粒细胞和嗜碱性细胞，表明本申请白细胞分类试剂能够实现白细胞各亚群的有效分类。
2)对血液样品中红细胞的测定和结果：
测试样本的过程在迈瑞自主研发的血液分析仪器中自动进行，设置吸样量为4微升，球形化试剂溶液C进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml球形化试剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的前向散射光强度和荧光强度，得到包含红细胞的散点图。对血液样本(8号血液样本)的测试结果如图16所示，表明本申请可以实现红细胞和血小板的的分类识别，图16中标注的三团粒子分别为红细胞(即成熟红细胞)，网织红细胞(RET)和血小板，表明本申请红细胞检测试剂能够实现红细胞和血小板的分类和识别，尤其是RET的识别。
3)相同血液样品在迈瑞BC-6800仪器分别对白细胞和红细胞的测定和结果。
用同样一支血样(8号血液样本)在迈瑞BC-6800仪器上按照CDR模式进行测试，采用BC-6800仪器配套试剂，分别检测得到的白细胞和红细胞相关参数如下表所示：
Figure PCTCN2020128839-appb-000047
Figure PCTCN2020128839-appb-000048
从表中可以看出，具有化学式VIII的荧光染料对白细胞分群和网织红细胞的测试结果与BC-6800的测试结果较为一致。
实施例11：
试剂的配制
配制如下组成的试剂体系。
A：荧光染料储存液：
荧光染料(具有化学式IX)  50mg
乙二醇  950ml
甲醇    50ml
B：红细胞溶解剂溶液
Figure PCTCN2020128839-appb-000049
C:球形化试剂溶液
Figure PCTCN2020128839-appb-000050
1)对血液样品中白细胞的测定和结果：
测试样本的过程在迈瑞自主研发的经改进的BC-6000血液分析仪器中自动进行，设置吸样量为20微升，溶血剂溶液B进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml红细胞溶解剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的侧向散射光强度和荧光强度，得到白细胞散点图。对血液样本(9号血液样本)的测试结果如图17所示，表明本申请可以实现白细胞4个亚群的分类识别，图17中标注的四团粒子分别为淋巴细胞，单核细胞、中性粒细胞和嗜酸性粒细胞，表明本申请白细胞分类试剂能够实现白细胞各亚群的有效分类。
2)对血液样品中红细胞的测定和结果：
测试样本的过程在迈瑞自主研发的血液分析仪器中自动进行，设置吸样量为4微升，球形化试剂溶液C进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml球形化试剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的前向散射光强度和荧光强度，得到包含红细胞的散点图。对血液样本(9号血液样本)的测试结果如图18所示，表明本申请可以实现红细胞和血小板的的分类识别，图18中标注的三团粒子分别为红细胞(即成熟红细胞)，网织红细胞(RET)和血小板，表明本申请红细胞检测试剂能够实现红细胞和血小板的分类和识别，尤其是RET的识别。
3)相同血液样品在迈瑞BC-6800仪器分别对白细胞和红细胞的测定和结果。
用同样一支血样(9号血液样本)在迈瑞BC-6800仪器上按照CDR模式进行测试，采用BC-6800仪器配套试剂，分别检测得到的白细胞和红细胞相关参数如下表所示：
Figure PCTCN2020128839-appb-000051
Figure PCTCN2020128839-appb-000052
从表中可以看出，具有化学式IX的荧光染料对白细胞分群和网织红细胞的测试结果与BC-6800的测试结果较为一致。
实施例12：
试剂的配制
配制如下组成的试剂体系。
A：荧光染料储存液：
荧光染料(具有化学式X)  50mg
乙二醇  950ml
甲醇    50ml
B：红细胞溶解剂溶液
Figure PCTCN2020128839-appb-000053
C:球形化试剂溶液
Figure PCTCN2020128839-appb-000054
Figure PCTCN2020128839-appb-000055
1)对血液样品中白细胞的测定和结果：
测试样本的过程在迈瑞自主研发的经改进的BC-6000血液分析仪器中自动进行，设置吸样量为20微升，溶血剂溶液B进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml红细胞溶解剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的侧向散射光强度和荧光强度，得到白细胞散点图。对血液样本(10号血液样本)的测试结果如图19所示，表明本申请可以实现白细胞4个亚群的分类识别，图19中标注的四团粒子分别为淋巴细胞，单核细胞、中性粒细胞和嗜酸性粒细胞，表明本申请白细胞分类试剂能够实现白细胞各亚群的有效分类。
2)对血液样品中红细胞的测定和结果：
测试样本的过程在迈瑞自主研发的血液分析仪器中自动进行，设置吸样量为4微升，球形化试剂溶液C进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml球形化试剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的前向散射光强度和荧光强度，得到包含红细胞的散点图。对血液样本(10号血液样本)的测试结果如图20所示，表明本申请可以实现红细胞和血小板的的分类识别，图20中标注的三团粒子分别为红细胞(即成熟红细胞)，网织红细胞(RET)和血小板，表明本申请红细胞检测试剂能够实现红细胞和血小板的分类和识别，尤其是RET的识别。
3)相同血液样品在迈瑞BC-6800仪器分别对白细胞和红细胞的测定和结果。
用同样一支血样(10号血液样本)在迈瑞BC-6800仪器上按照CDR模式进行测试，采用BC-6800仪器配套试剂，分别检测得到的白细胞和红细胞相关参数如下表所示：
Figure PCTCN2020128839-appb-000056
从表中可以看出，具有化学式X的荧光染料对白细胞分群和网织红细胞的测试结果与BC-6800的测试结果较为一致。
实施例13：
试剂的配制
配制如下组成的试剂体系。
A：荧光染料储存液：
荧光染料(具有化学式XI)  50mg
乙二醇  950ml
甲醇    50ml
B：红细胞溶解剂溶液
Figure PCTCN2020128839-appb-000057
C:球形化试剂溶液
Figure PCTCN2020128839-appb-000058
Figure PCTCN2020128839-appb-000059
1)对血液样品中白细胞的测定和结果：
测试样本的过程在迈瑞自主研发的经改进的BC-6000血液分析仪器中自动进行，设置吸样量为20微升，溶血剂溶液B进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml红细胞溶解剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的侧向散射光强度和荧光强度，得到白细胞散点图。对血液样本(11号血液样本)的测试结果如图21所示，表明本申请可以实现白细胞4个亚群的分类识别，图21中标注的四团粒子分别为淋巴细胞，单核细胞、中性粒细胞和嗜酸性粒细胞，表明本申请白细胞分类试剂能够实现白细胞各亚群的有效分类。
2)对血液样品中红细胞的测定和结果：
测试样本的过程在迈瑞自主研发的血液分析仪器中自动进行，设置吸样量为4微升，球形化试剂溶液C进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml球形化试剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的前向散射光强度和荧光强度，得到包含红细胞的散点图。对血液样本(11号血液样本)的测试结果如图22所示，表明本申请可以实现红细胞和血小板的的分类识别，图22中标注的三团粒子分别为红细胞(即成熟红细胞)，网织红细胞(RET)和血小板，表明本申请红细胞检测试剂能够实现红细胞和血小板的分类和识别，尤其是RET的识别。
3)相同血液样品在迈瑞BC-6800仪器分别对白细胞和红细胞的测定和结果。
用同样一支血样(11号血液样本)在迈瑞BC-6800仪器上按照CDR模式进行测试，采用BC-6800仪器配套试剂，分别检测得到的白细胞和红细胞 相关参数如下表所示：
Figure PCTCN2020128839-appb-000060
从表中可以看出，具有化学式XI的荧光染料对白细胞分群和网织红细胞的测试结果与BC-6800的测试结果较为一致。
实施例14：
试剂的配制
配制如下组成的试剂体系。
A：荧光染料储存液：
荧光染料(具有化学式XII)  1000mg
乙二醇  950ml
甲醇    50ml
B：红细胞溶解剂溶液
Figure PCTCN2020128839-appb-000061
C:球形化试剂溶液
Figure PCTCN2020128839-appb-000062
Figure PCTCN2020128839-appb-000063
1)对血液样品中白细胞的测定和结果：
测试样本的过程在迈瑞自主研发的经改进的BC-6000血液分析仪器中自动进行，设置吸样量为20微升，溶血剂溶液B进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml红细胞溶解剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的侧向散射光强度和荧光强度，得到白细胞散点图。对血液样本(12号血液样本)的测试结果如图23所示，表明本申请可以实现白细胞4个亚群的分类识别，图23中标注的四团粒子分别为淋巴细胞，单核细胞、中性粒细胞和嗜酸性粒细胞，表明本申请白细胞分类试剂能够实现白细胞各亚群的有效分类。
2)对血液样品中红细胞的测定和结果：
测试样本的过程在迈瑞自主研发的血液分析仪器中自动进行，设置吸样量为4微升，球形化试剂溶液C进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml球形化试剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的前向散射光强度和荧光强度，得到包含红细胞的散点图。对血液样本(12号血液样本)的测试结果如图24所示，表明本申请可以实现红细胞和血小板的的分类识别，图24中标注的三团粒子分别为红细胞(即成熟红细胞)，网织红细胞(RET)和血小板，表明本申请红细胞检测试剂能够实现红细胞和血小板的分类和识别，尤其是RET的识别。
3)相同血液样品在迈瑞BC-6800仪器分别对白细胞和红细胞的测定和结果。
用同样一支血样(12号血液样本)在迈瑞BC-6800仪器上按照CDR模 式进行测试，采用BC-6800仪器配套试剂，分别检测得到的白细胞和红细胞相关参数如下表所示：
Figure PCTCN2020128839-appb-000064
从表中可以看出，具有化学式XII的荧光染料对白细胞分群和网织红细胞的测试结果与BC-6800的测试结果较为一致。
实施例15：
试剂的配制
配制如下组成的试剂体系。
A：荧光染料储存液：
荧光染料(具有化学式XIII)  50mg
乙二醇  950ml
甲醇    50ml
B：红细胞溶解剂溶液
Figure PCTCN2020128839-appb-000065
C:球形化试剂溶液
Figure PCTCN2020128839-appb-000066
Figure PCTCN2020128839-appb-000067
1)对血液样品中白细胞的测定和结果：
测试样本的过程在迈瑞自主研发的经改进的BC-6000血液分析仪器中自动进行，设置吸样量为20微升，溶血剂溶液B进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml红细胞溶解剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的侧向散射光强度和荧光强度，得到白细胞散点图。对血液样本(13号血液样本)的测试结果如图25所示，表明本申请可以实现白细胞4个亚群的分类识别，图25中标注的四团粒子分别为淋巴细胞，单核细胞、中性粒细胞和嗜酸性粒细胞，表明本申请白细胞分类试剂能够实现白细胞各亚群的有效分类。
2)对血液样品中红细胞的测定和结果：
测试样本的过程在迈瑞自主研发的血液分析仪器中自动进行，设置吸样量为4微升，球形化试剂溶液C进样量为1ml，荧光染料存储液A的进样量为20微升，血样为经过抗凝处理的血液。
具体为将1ml球形化试剂与经过抗凝剂处理的20微升血液混匀，并立即加入20微升荧光染料储存液，在40℃下的孵育池中混匀孵育30秒，形成测定用试样。所述血液分析仪器带有蓝色激光器，激发波长为450nm，测定测试样本的前向散射光强度和荧光强度，得到包含红细胞的散点图。对血液样本(13号血液样本)的测试结果如图26所示，表明本申请可以实现红细胞和血小板的的分类识别，图26中标注的三团粒子分别为红细胞(即成熟红细胞)，网织红细胞(RET)和血小板，表明本申请红细胞检测试剂能够实现红细胞和血小板的分类和识别，尤其是RET的识别。
3)相同血液样品在迈瑞BC-6800仪器分别对白细胞和红细胞的测定和结果。
用同样一支血样(13号血液样本)在迈瑞BC-6800仪器上按照CDR模式进行测试，采用BC-6800仪器配套试剂，分别检测得到的白细胞和红细胞相关参数如下表所示：
Figure PCTCN2020128839-appb-000068
从表中可以看出，具有化学式XIII的荧光染料对白细胞分群和网织红细胞的测试结果与BC-6800的测试结果较为一致。
本发明已经通过上述实施例进行了说明，但应当理解的是，上述实施例只是用于举例和说明的目的，而非意在将本发明限制于所描述的实施例范围内。此外本领域技术人员可以理解的是，本发明并不局限于上述实施例，根据本发明的教导还可以做出更多种的变型和修改，这些变型和修改均落在本发明所要求保护的范围以内。本发明的保护范围由附属的权利要求书及其等效范围所界定。

Claims (25)

  1. 一种白细胞分类试剂，所述试剂包含具有式F的荧光染料：
    Figure PCTCN2020128839-appb-100001
    其中，X选自由C(CH 3) 2、O、S和Se组成的组；
    R 1和R 2各自独立地选自由H、C 1-C 18烷基、苯基、OR 6和卤素组成的组；
    R 3和R 4各自独立地选自由C 1-C 18烷基、C 1-C 18羧基、C 1-C 18羟基、C 1-C 18NR 5R 6、苄基和取代苄基组成的组，其中所述取代苄基的取代基选自由C 1-C 18烷基、CN、COOH、NH 2、NO 2、OH、SH、C 1-C 6烷氧基、C 1-C 6烷基氨基、C 1-C 6酰氨基、卤素和C 1-C 6卤代烷基组成的组；
    R 5和R 6各自独立地选自由H和C 1-C 18烷基组成的组；
    Y -为负离子。
  2. 根据权利要求1所述的白细胞分类试剂，其特征在于，所述X选自由C(CH 3) 2和S组成的组。
  3. 根据权利要求1所述的白细胞分类试剂，其特征在于，所述R 1和所述R 2各自独立地选自由H、C 1-C 12烷基、苯基、OR 6和卤素组成的组；
    优选地，所述R 1和所述R 2各自独立地选自由H、C 1-C 6烷基、苯基、OR 6和卤素组成的组；
    更优选地，所述R 1选自由H、C 1-C 6烷基、苯基、和卤素组成的组，所述R 2为H。
  4. 根据权利要求1所述的白细胞分类试剂，其特征在于，所述R 3和所述R 4各自独立地选自由C 1-C 12烷基、C 1-C 12羧基、C 1-C 12羟基、C 1-C 12NR 5R 6、苄基和取代苄基组成的组，其中所述取代苄基的取代基选自由C 1-C 12烷基、CN、COOH、NH 2、NO 2、OH、SH、C 1-C 6烷氧基、C 1-C 6烷基氨基、C 1-C 6酰氨基、卤素和C 1-C 6卤代烷基组成的组；
    优选地，所述R 3和所述R 4各自独立地选自由C 1-C 6烷基、C 1-C 6羧基、C 1-C 6羟基、C 1-C 6NR 5R 6、苄基和取代苄基组成的组，其中所述取代苄基的取代基选自由C 1-C 6烷基、CN、COOH、NH 2、NO 2、OH、SH、C 1-C 6烷氧基、C 1-C 6烷基氨基、C 1-C 6酰氨基、卤素和C 1-C 6卤代烷基组成的组；
    更优选地，所述R 3选自由C 1-C 6烷基、C 1-C 6羟基、C 1-C 6羧基、C 1-C 6NR 5R 6和苄基组成的组；所述R 4选自由C 1-C 6烷基、C 1-C 6羟基、C 1-C 6羧基、和苄基组成的组。
  5. 根据权利要求1所述的白细胞分类试剂，其特征在于，所述R 5和所述R 6各自独立地选自由H和C 1-C 12烷基组成的组；
    优选地，所述R 5和所述R 6各自独立地选自由H和C 1-C 6烷基组成的组；
    更优选地，所述R 5和所述R 6各自独立地为C 1-C 6烷基。
  6. 根据权利要求1所述的白细胞分类试剂，其特征在于，所述Y -选自由卤素负离子、ClO 4 -、PF 6 -、BF 4 -、CH 3COO -或OTs -组成的组。
  7. 根据权利要求1所述的白细胞分类试剂，其特征在于，所述荧光染料包含化学式I、化学式II、化学式III、化学式IV、化学式V、化学式VI、化学式VII、化学式VIII、化学式IX、化学式X、化学式XI、化学式XII和化学式XIII中的一种所示的结构，
    Figure PCTCN2020128839-appb-100002
    Figure PCTCN2020128839-appb-100003
  8. 根据权利要求1-7任一项所述的白细胞分类试剂，其特征在于，所述分类试剂还包括红细胞溶解剂。
  9. 根据权利要求1-8任一项所述的白细胞分类试剂，其特征在于，所述荧光染料的浓度为：0.1-1000mg/L,优选20-500mg/L，更优选50-200mg/L。
  10. 根据权利要求8所述的白细胞分类试剂，其特征在于，所述红细胞溶解剂包含阳离子表面活性剂、非离子表面活性剂、阴离子表面活性剂或它们的任何组合，以及缓冲剂，将测定体系的pH保持在5-11。
  11. 一种白细胞分析试剂盒，所述试剂盒包含权利要求1-10中任一项所述的白细胞分类试剂。
  12. 一种白细胞分类方法，所述方法包括：将血液样品、具有式F的 荧光染料、和红细胞溶解剂混合形成待测试样；其中，式F为
    Figure PCTCN2020128839-appb-100004
    其中，X、R 1、R 2、R 3、R 4和Y -如权利要求1-6中任一项所定义；
    用光照射所述待测试样中的粒子，测定所述待测试样的至少一种散射光特性和至少一种荧光特性；
    根据所述散射光特性和荧光特性对白细胞进行分类和/或计数。
  13. 根据权利要求12的所述方法，其中所述式F的荧光染料为：化学式I、化学式II、化学式III、化学式IV、化学式V、化学式VI、化学式VII、化学式VIII、化学式IX、化学式X、化学式XI、化学式XII和化学式XIII中的一种所示的结构，
    Figure PCTCN2020128839-appb-100005
    Figure PCTCN2020128839-appb-100006
  14. 根据权利要求12-13任一项的所述方法，其特征在于：照射所述待测试样中的粒子的光为：蓝光、绿光或发射波长范围为400-560nm的光。
  15. 一种红细胞分析试剂，其特征在于，所述试剂包含具有式F的荧光染料；其中，式F为
    Figure PCTCN2020128839-appb-100007
    其中，X、R 1、R 2、R 3、R 4和Y -如权利要求1-6中任一项所定义。
  16. 根据权利要求15所述的红细胞分析试剂，其特征在于，所述荧光 染料包含化学式I、化学式II、化学式III、化学式IV、化学式V、化学式VI、化学式VII、化学式VIII、化学式IX、化学式X、化学式XI、化学式XII和化学式XIII中的一种所示的结构，
    Figure PCTCN2020128839-appb-100008
  17. 根据权利要求15所述的红细胞分析试剂，其特征在于，所述红细胞分析试剂还包括球形化试剂。
  18. 根据权利要求17所述的红细胞分析试剂，其特征在于，所述球形化试剂包括两性表面活性剂、渗透压调节剂及缓冲剂。
  19. 根据权利要求15-18任一项所述的红细胞分析试剂，其特征在于，所述荧光染料的浓度为：0.1-1000mg/L，优选20-500mg/L，更优选50-200 mg/L。
  20. 一种红细胞分析试剂盒，所述试剂盒包含权利要求15-19任一项所述的红细胞分析试剂。
  21. 一种红细胞分析方法，包括：
    将血液样品、具有式F的荧光染料、和球形化试剂混合形成待测试样；
    其中，式F为
    Figure PCTCN2020128839-appb-100009
    其中，X、R 1、R 2、R 3、R 4和Y -如权利要求1-6中任一项所定义；
    用光照射所述待测试样中的粒子，测定所述待测试样的至少一种散射光特性和至少一种荧光特性；
    根据所述散射光特性和荧光特性获得红细胞分类和/或计数。
  22. 根据权利要求21所述的分析方法，其特征在于，所述红细胞分类包括：将红细胞区分为成熟红细胞和/或网织红细胞；所述红细胞计数包括：对成熟红细胞和/或网织红细胞的计数。
  23. 根据权利要求22所述的分析方法，其特征在于，根据所述散射光特性和荧光特性还能获得血小板的计数。
  24. 根据权利要求21-23任一项所述的分析方法，其特征在于，所述式F的荧光染料为：化学式I、化学式II、化学式III、化学式IV、化学式V、化学式VI、化学式VII、化学式VIII、化学式IX、化学式X、化学式XI、化学式XII和化学式XIII中的一种所示的结构，
    Figure PCTCN2020128839-appb-100010
    Figure PCTCN2020128839-appb-100011
  25. 根据权利要求21-24任一项的所述方法，其特征在于：照射所述待测试样中的粒子的光为：蓝光、绿光或发射波长范围为400-560nm的光。
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