WO2014015687A1 - 白细胞分类计数试剂和白细胞分类方法 - Google Patents
白细胞分类计数试剂和白细胞分类方法 Download PDFInfo
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- WO2014015687A1 WO2014015687A1 PCT/CN2013/074543 CN2013074543W WO2014015687A1 WO 2014015687 A1 WO2014015687 A1 WO 2014015687A1 CN 2013074543 W CN2013074543 W CN 2013074543W WO 2014015687 A1 WO2014015687 A1 WO 2014015687A1
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- white blood
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- 238000002738 Giemsa staining Methods 0.000 description 1
- IZWSFJTYBVKZNK-UHFFFAOYSA-O N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonic acid Chemical compound CCCCCCCCCCCC[N+](C)(C)CCCS(O)(=O)=O IZWSFJTYBVKZNK-UHFFFAOYSA-O 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- WTDRKSNJOHGRDG-UHFFFAOYSA-M [Br-].S[N+](C)(C)C Chemical compound [Br-].S[N+](C)(C)C WTDRKSNJOHGRDG-UHFFFAOYSA-M 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 125000003158 alcohol group Chemical group 0.000 description 1
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- 238000004458 analytical method Methods 0.000 description 1
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- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 238000010420 art technique Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
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- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- KRVSOGSZCMJSLX-UHFFFAOYSA-L chromic acid Substances O[Cr](O)(=O)=O KRVSOGSZCMJSLX-UHFFFAOYSA-L 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 238000010219 correlation analysis Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
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- XJWSAJYUBXQQDR-UHFFFAOYSA-M dodecyltrimethylammonium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)C XJWSAJYUBXQQDR-UHFFFAOYSA-M 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000002327 eosinophilic effect Effects 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- AWJWCTOOIBYHON-UHFFFAOYSA-N furo[3,4-b]pyrazine-5,7-dione Chemical compound C1=CN=C2C(=O)OC(=O)C2=N1 AWJWCTOOIBYHON-UHFFFAOYSA-N 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 125000005113 hydroxyalkoxy group Chemical group 0.000 description 1
- 239000000819 hypertonic solution Substances 0.000 description 1
- 229940021223 hypertonic solution Drugs 0.000 description 1
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- 210000002751 lymph Anatomy 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
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- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- KVBGVZZKJNLNJU-UHFFFAOYSA-N naphthalene-2-sulfonic acid Chemical compound C1=CC=CC2=CC(S(=O)(=O)O)=CC=C21 KVBGVZZKJNLNJU-UHFFFAOYSA-N 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
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- 230000001575 pathological effect Effects 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1456—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
- G01N15/1459—Electro-optical investigation, e.g. flow cytometers without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
-
- 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
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/49—Blood
-
- G01N2015/016—
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
Definitions
- the present invention relates to a cell sorting reagent and a method for classifying cells, and more particularly to a white cell sorting and counting reagent, a method for classifying white blood cells in the blood using the reagent, and a use of a zwitterionic surfactant.
- White blood cells in human blood can be divided into five subtypes of monocytes, lymphocytes, and granulocytes (including eosinophils, neutrophils, and basophils).
- the proportion of white blood cells in the blood of normal healthy people is relatively constant. When certain diseases occur in the human body, it will show an increase or decrease in the number of certain types or types of white blood cells.
- eosinophils are a kind of granulocytes which account for a small proportion in human whole blood.
- the proportion of white blood cells in healthy people is 0.5 ⁇ 5%.
- clinically allergic diseases or parasitic infections occur, eosinophilia
- the number of granulocytes will change significantly, so the accurate classification of eosinophils in human whole blood testing has direct guiding significance for the correct diagnosis of these diseases.
- eosinophils The size of eosinophils is close to that of neutrophils.
- whole blood samples from non-disease populations ie, normal mature eosinophils
- the particles in the cells are fully mature, allowing them to scatter light in the forward high angle. Underneath, it can be distinguished from neutrophils.
- a large number of immature eosinophils in the spinal cord enter the peripheral blood, making the proportion of eosinophils in the peripheral blood.
- the sharp increase, and the degree of differentiation between immature eosinophils and neutrophils is also significantly reduced.
- the prior art leukocyte classification reagent is not sufficient to distinguish immature eosinophils from neutrophils, making eosinophilic
- the detection accuracy of granulocytes is significantly reduced.
- the patient's blood sample When the patient's blood sample is analyzed and processed in the hospital, after the fresh anticoagulant sample is collected from the patient, it may be left for analysis for a period of time, or the blood sample of the patient who has been placed for a long time may be re-examined (48 hours at room temperature). Within). With the prolongation of the placement time, various cells undergo metabolism, and the fresh blood samples gradually age, and the cell morphology between the fresh blood and the aged blood of the same blood sample changes significantly, which tends to reduce the credibility of the white blood cell classification count of the aged blood. The classification accuracy of eosinophils, which account for a small proportion, is particularly severe.
- the reagent contains a polyoxyethylene structure-based nonionic surfactant with a pH range of 3-11.
- the Eos classification is performed by impedance method (DC).
- a method for classifying white blood cells after treatment of a blood sample containing polyoxyethylene-9-lauryl ether is also disclosed abroad. This method is suitable for distinguishing basophils in an acidic medium and for distinguishing eosinophils in an alkaline medium.
- a reagent for white blood cell classification which comprises at least one ionic surfactant, which may be one of a cationic or amphoteric surfactant, at least one organic compound having an anion, and a nonionic surface.
- the detection method is a laser scattering method.
- a reagent for five-class classification of white blood cells is also disclosed in foreign countries.
- the first reagent is a hypertonic solution of a diluted blood sample
- the second reagent is a solution containing an anionic surfactant and a nonionic surfactant.
- the detection method is direct current. Impedance and RF methods.
- a method and reagent system for five-class classification of white blood cells is disclosed in another foreign patent.
- the hemolytic agent is composed of two reagents, and the first reagent contains an alkali metal salt of an alkylsulfonic acid, an organic acid, an inorganic acid or a mixture thereof, and a nonionic surfactant.
- the second reagent contains a copolymer of a polyoxyethylene or polyoxypropylene surfactant, a highly alkaline solution of sodium dodecyl sulfate.
- the detection method is DC impedance and RF method.
- a method for accomplishing five classifications of white blood cells by two reagents is disclosed in the country.
- the two reagents contain, in addition to the nonionic surfactant and the buffer, the first reagent contains at least one ionic surfactant and one organic compound with an anion, and the blood cells are divided into lymphocytes, monocytes, and hobby. Acid granulocytes, as well as basophils and neutrophils, the second reagent contains at least one ionic surfactant, which divides blood cells into basophils and other cell groups.
- the detection method is a laser scattering method.
- a white blood cell differential counting reagent comprising a cation, an amphoteric surfactant, and a combination thereof, an organic compound containing a benzene ring or a heterocyclic ring, and a pH-adjusting buffer.
- the detection method is laser scattering.
- the hemolytic agent dissolves red blood cells in the whole blood sample, and performs non-specific effects on the white blood cells, and the combined detection method distinguishes the structural characteristics of various types of white blood cells, wherein eosinophils are used as One of the groups is classified and counted. Since the prior art does not contain components specific for eosinophils, the classification accuracy for whole blood samples containing high acidophilus or aged blood eosinophils is not ideal.
- the object of the present invention is to overcome the above-mentioned deficiencies of the prior art, and to provide a method for rapidly dissolving red blood cells, enhancing the classification of eosinophils, and making white blood cell classification results of fresh whole blood samples and aged whole blood samples stored at room temperature for 48 hours. Counting reagents for accurate white blood cell sorting.
- Another object of the present invention is to provide a method for classifying and counting white blood cells in aged whole blood samples which are convenient, fast, and accurate for fresh whole blood samples and stored at room temperature for 48 hours.
- a further object of the present invention is to provide a use of a zwitterionic surfactant in the preparation of a leukocyte differential counting reagent.
- a white fine classification counting reagent including
- a surfactant solution capable of dissolving red blood cells, partially damaging the leukocyte membrane, and stabilizing the cell membrane of eosinophils and aged cells, the surfactant solution containing at least one cationic surfactant and at least one a zwitterionic surfactant;
- the amphoteric surfactant has the structure shown in Formula I R ⁇ (CO-MH) -(CH 2 )- N ⁇ - (CH 2 ), wherein is an alkyl group of ( ⁇ 18 ; R 2 and R 3 are each independently selected from C 1-3 alkyl, C 1 -3 alkoxy or d. 3 alkylhydroxy; a is 0 or 1; n is an integer from 0 to 6; m is an integer from 1 to 5; X is selected from a carboxyl group or a sulfonic acid group;
- Or / and the zwitterionic surfactant is selected from the group consisting of bile acid derivative type amphoteric surfactants;
- a method for classifying white blood cells comprising the following steps:
- a method for counting white blood cells comprising the following steps:
- the white blood cells are divided into three groups corresponding to lymphocytes, monocytes, and eosinophils, and A group consisting of four groups corresponding to neutrophils and basophils counts the number of cells contained in each group.
- the present invention also provides a use of a zwitterionic surfactant having the structure of the formula I for the preparation of a white blood cell sorting and counting reagent,
- C 8-18 alkyl; R 2 and R 3 are each independently selected from C 1 -3 alkyl, C w alkoxy or CL 3 alkylhydroxy; a is 0 or 1; n is 0-6 An integer; m is an integer from 1 to 5; and X is selected from a carboxyl group or a sulfonic acid group.
- the cationic surfactant component of the above-mentioned white blood cell sorting and counting reagent functions as a hemolytic agent, can effectively dissolve red blood cells and platelets in the blood sample to be tested, and partially damage the white blood cell membrane; the zwitterionic surfactant can act as a hemolytic agent on the one hand; It helps to dissolve red blood cells and platelets. On the other hand, it can stabilize the leukocyte membrane due to its positive and negative ion groups, especially for eosinophils and white blood cells in aged samples. Excessive damage to the lysing agent component such as the agent.
- the above-mentioned white blood cell sorting and counting reagent can quickly dissolve red blood cells after contact with the blood sample to be tested, and specifically treat eosinophils, thereby enhancing the difference between eosinophils and neutrophils, thereby achieving enhanced hobby.
- the classification of acidic granulocytes makes the difference between the classification effect of various white blood cell subclasses in aging blood and the classification effect of fresh blood within an acceptable range, so that the reagent can simultaneously satisfy the aging of fresh whole blood and room temperature within 48 hours. The white blood cells in the whole blood sample are accurately classified.
- the above method for counting leukocytes can effectively enhance the relationship between eosinophils and neutrophils.
- Figure 1 is a scatter diagram of forward low angle and high angle scattered light when a blood sample to be measured is subjected to white blood cell sorting by using the reagent of Example 1 of the present invention
- Figure 2 is a scatter diagram of forward low angle and high angle scattered light when the blood sample to be measured is subjected to leukocyte differential counting using the reagent of Example 2 of the present invention
- Figure 3 is a scatter diagram of forward low angle and high angle scattered light when the blood sample to be measured is subjected to leukocyte differential counting using the reagent of Example 3 of the present invention
- Figure 4 is a scatter diagram of forward low angle and high angle scattered light when the blood sample to be measured is subjected to leukocyte differential counting using the reagent of Example 4 of the present invention
- Figure 5 is a scatter diagram of forward low angle and high angle scattered light when the blood sample to be measured is subjected to leukocyte differential counting using the reagent of Example 5 of the present invention
- Figure 6 is a scatter diagram of forward low angle and high angle scattered light when the blood sample to be measured is counted by the reagent of Example 6 of the present invention
- Figure 7 is a scatter diagram of forward low angle and high angle scattered light when the blood sample to be measured is subjected to leukocyte differential counting using the reagent of Example 7 of the present invention
- Fig. 8 is a scatter diagram of forward low angle and high angle scattered light when the blood sample is subjected to white blood cell sorting by the reagent A of Comparative Example 1; wherein, Fig. 8-a is a blood sample test diagram, and Fig. 8-b is B Blood sample test chart, Figure 8-c is a C blood sample test chart;
- Fig. 9 is a scatter diagram of forward low angle and high angle scattered light when the blood sample to be measured is subjected to white blood cell sorting and counting by the reagent of the eighth embodiment of the present invention
- Fig. 9-a is a blood sample detection chart
- Fig. 9-b is a B blood sample.
- the test chart, Figure 9-c is a C blood test chart
- Figure 10 is a graph showing the correlation between the classification of four groups of white blood cells in the blood sample to be measured by the reagent of Example 9 of the present invention and the manual microscopic examination of the conventional Wright-Gemsa staining method; wherein, Figure 10-a is lymph The correlation between the cells and the results of manual microscopic examination of conventional Wright-Giemsa staining, Figure 10-b is the correlation between monocytes and the results of manual microscopy of conventional Wright-Giemsa staining.
- Figure 10-c shows the correlation between neutrophils and conventional Wright-Giemsa staining by manual microscopy.
- Figure 10-d shows eosinophils and conventional Wright-Giemsa staining. The correlation between the results of manual microscopy and counting;
- Figure 11 is a scatter diagram of forward low angle and high angle scattered light when the blood sample to be measured is subjected to leukocyte differential counting using the reagent of Example 11 of the present invention
- Fig. 12 is a scatter diagram of forward low angle and high angle scattered light when the blood sample to be measured is subjected to white blood cell sorting and counting using the reagent of the embodiment 12 of the present invention.
- the present invention provides the use of a zwitterionic surfactant having the structure of the formula I for the preparation of a white blood cell sorting and counting reagent.
- the formula I is as follows:
- R 2 and R 3 are each independently selected from an alkyl group of Ci -3 , a d. 3 alkoxy group or a d. 3 alkoxy group.
- a is 0 or 1
- n is an integer from 0 to 6
- m is an integer from 1 to 5
- X is selected from a carboxyl group or a sulfonic acid group.
- R 2 and R 3 in the above formula I are each independently selected from the group consisting of hydroxyethyl, ethoxy, hydroxypropyl; X is a sulfonic acid group, n is 3, and m is 3.
- R 2 , R 3 , n, m and X can be selectively combined in various ways.
- the amphoteric surfactant of the above formula I has at least the following molecular structures:
- n 3
- R 2 and R 3 are each independently selected from a C 1-3 alkyl group, a C 1-3 alkoxy group or a C 1-3 alkoxy group, and a is 0 or 1 and m is 1 An integer of 5, X is selected from a carboxyl group or a sulfonic acid group;
- R 2 and R 3 are each independently selected from hydroxyethyl or methyl, a is 0 or 1, n is an integer from 0 to 6, m is an integer from 1 to 5, and X is selected from a carboxyl group or a sulfonic acid group. ;
- m is 3
- X is a sulfonic acid group, and is an alkyl group of ( ⁇ 8-18 ;
- R 2 and R 3 are each independently selected from an alkyl group of C 1-3 , d. 3 alkoxy or ( ⁇ . 3 alkoxy, a is 0 or 1, n is an integer from 0 to 6;
- R 2 and R 3 are ethoxy groups, which are ( ⁇ 8-18 alkyl groups, a is 0 or 1, n is an integer of 0-6, m is an integer of 1-5, and X is selected from a carboxyl group. Or sulfonic acid group;
- R 2 and R 3 are hydroxypropyl, which is an alkyl group of ( 18 ), a is 0 or 1, n is an integer of 0-6, m is an integer of 1-5, and X is selected from a carboxyl group or a sulfonate.
- Acid base is an alkyl group of ( 18 )
- a is 0 or 1
- n is an integer of 0-6
- m is an integer of 1-5
- X is selected from a carboxyl group or a sulfonate.
- R 2 and R 3 are each independently selected from hydroxypropyl group, n is 3, X is a sulfonic acid group, is an alkyl group of ( 18 ), a is 0 or 1, and m is an integer of 1-5.
- amphoteric surfactant of the above formula I may be mercaptoamidopropyl betaine, lauryl amide propyl betaine, cocamidopropyl betaine, stearyl propyl betaine, lauryl di Hydroxyethyl betaine, tetradecyl dihydroxyethyl betaine, stearyl dihydroxyethyl betaine, lauryl sulfobetaine, tetradecyl sulfobetaine, cetyl sulfobeet Base, octadecylsulfobetaine, dodecyl ethoxy sulfobetaine, tetradecyl ethoxy sulfobetaine, cetyl ethoxy sultaine, octadecane Ethoxy sulphobetaine, dodecyl hydroxypropyl sulfobetaine, tetradecyl hydroxypropyl
- amphoteric surfactant When used in the white blood cell sorting and counting reagent, it can act as a hemolytic agent on the one hand, and assist the cationic surfactant to dissolve red blood cells and platelets, on the other hand, at the same time, it has two positive and negative ion groups, which can stabilize leukocyte membranes, especially leukocytes in eosinophils and aging blood samples, and relieve to some extent the excess of leukocytes by cationolytic components such as cationic surfactants.
- cationolytic components such as cationic surfactants.
- the embodiment of the invention further provides an effective enhancement of the difference between eosinophils and neutrophils, and can quickly and accurately classify white blood cells in fresh whole blood samples and aging whole blood samples stored within 48 hours at room temperature.
- Counting reagents includes:
- a surfactant solution capable of dissolving red blood cells, partially damaging the leukocyte membrane, and stabilizing the cell membrane of eosinophils and aged cells, the surfactant solution containing at least one cationic surfactant and at least one a zwitterionic surfactant;
- amphoteric surfactant is a structural amphoteric surfactant or / and a bile acid derivative type amphoteric surfactant represented by the above formula I; wherein, the formula I is as follows:
- R plant (CO-NH) (CH 2 )- ⁇ - (CH 2 ) m -X- I is an alkyl group of formula I which is C 8-18 (ie 8 to 18 C atoms); R 2 and R 3 is independently selected from alkyl d 3, d 3 d 3 alkoxy or hydroxy alkoxy;... a is 0 or 1; n is an integer of 0-6; m is an integer from 1-5; X is selected from From a carboxyl group or a sulfonic acid group;
- the amphoteric surfactant is a structural amphoteric surfactant of the above formula I
- R 2 and R 3 in the above formula I are each independently selected from hydroxyethyl , ethoxy, hydroxypropyl
- X is a sulfonic acid group
- n is 3, and m is 3.
- R 2 , R 3 , n, m and X can be selectively combined in various ways.
- the amphoteric surfactant of the above formula I has at least several molecular structures as described above, and for the sake of space saving, it will not be described herein.
- amphoteric surfactant may specifically be selected from the group consisting of mercaptoamidopropyl betaine, lauryl amide propyl betaine, cocamidopropyl betaine, stearyl propyl betaine, lauryl dihydroxyethyl beet Base, tetradecyl dihydroxyethyl betaine, stearyl dihydroxyethyl betaine, lauryl sulfobetaine, tetradecyl sulfobetaine, cetyl sulfobetaine, eighteen Alkyl sulfobetaine, dodecyl ethoxy sulfobetaine, tetradecyl ethoxy sultaine, cetyl ethoxy sultaine, octadecyl ethoxy Sulfobetaine, dodecyl hydroxypropyl sulfobetaine, tetradecyl hydroxypropyl sultaine
- bio-type amphoteric surfactant is preferably 3-[(3-cholamidopropyl)-dimethylammonium]-1-propanesulfonate ( CHAPS ), 3-[(3-cholanoamidopropyl) One or two intermixes of -dimethylammonium]-2-hydroxy-1-propanesulfonate ( CHAPSO ).
- CHAPS 3-[(3-cholamidopropyl)-dimethylammonium]-1-propanesulfonate
- CHAPSO 3-[(3-cholanoamidopropyl) One or two intermixes of -dimethylammonium]-2-hydroxy-1-propanesulfonate
- the above amphoteric surfactant may also be a complex of the structural amphoteric surfactant represented by the above formula I and a bile acid derivative type amphoteric surfactant.
- the structural amphoteric surfactant represented by the formula I and the bile acid derivative type amphoteric surfactant are preferably mixed at a mass ratio of 10:1 to 1:10. , ; ; , , , I , , I , , I , - - , ,
- the amphoteric surfactant in the above embodiments can act as a hemolytic agent on the one hand, assisting the cationic surfactant to dissolve red blood cells and platelets, and on the other hand, because it has both positive and negative ion groups, it can be especially suitable for leukocyte membranes.
- Eosinophils and leukocytes in aged blood samples stabilize the dysfunction of leukocytes by lytic components such as cationic surfactants to a certain extent, and significantly enhance the relationship between eosinophils and neutrophils. The difference is in order to accurately count the white blood cells.
- the above-mentioned preferred amphoteric surfactant content and the kind of lytic red blood cells and platelets are better assisted, and the excessive damage to the white blood cells by the cationic surfactant is better alleviated, so that the white cells damaged by the cationic surfactant are more stable and further enhance the tropism.
- the difference between acidic granulocytes and neutrophils makes the white blood cells completely separated in size and morphology, so that the white blood cells in the blood samples can be more conveniently and accurately identified and counted.
- the cationic surfactant in the above examples mainly functions as a hemolytic agent for dissolving red blood cells and platelets, and partially impairing the leukocyte membrane.
- it employs a quaternary ammonium salt cationic surfactant having the structure of Formula II:
- R 4 in formula II is an alkyl or alkenyl group of C 6 -14 , preferably octyl, decyl or lauryl; R 5 and R 6 are each independently selected from alkyl or alkenyl, preferably methyl, ethyl; of 17 ( ⁇ 4 alkyl, alkenyl or benzyl; Y is a halogen.
- the concentration of the cationic surfactant in the above-mentioned white blood cell sorting and counting reagent is 10 to 5000 mg, preferably 50 to 3000 mg / L.
- the above buffer having a pH adjusted in the range of 4 to 6 can effectively adjust and stabilize the pH of the white blood cell sorting and counting reagent of the above embodiment to exert the action of each component in the reagent and the synergistic effect between the components.
- the eosinophil classification effect can be enhanced, so that the reagent can accurately count the white blood cells.
- the pH of the reagent is adjusted to 4.5 to 5.5 using a pH adjusting buffer; meanwhile, the pH adjusting buffer is selected from formic acid, At least one of phthalic acid, acetic acid, phosphoric acid, citric acid, succinic acid, 2-morpholineethanesulfonic acid (MES), and glycine.
- the pH adjusting buffer may also employ other pH buffers commonly used in the art. It is also possible to combine the bases commonly used in the art, such as NaOH, to adjust the pH of the white blood cell sorting and counting reagent to an appropriate pH range.
- the above-mentioned white blood cell sorting and counting reagent is further included An organic compound having an anionic group selected from the group consisting of 8-anilino-1-naphthalenesulfonic acid, 8-anilino-1-naphthalenesulfonate, 6-(p-toluidine) At least one of 2-naphthalenesulfonic acid, 6-(p-tolylamino)-2-naphthalenesulfonate, chromic acid, and naphthalenesulfonic acid.
- the concentration of the organic compound having an anionic group in the white blood cell sorting and counting reagent is 100 to 6000 mg/L, preferably 500 to 4000 mg/L.
- the organic group with anionic groups is negatively charged in water, can bind to leukocytes (especially white cells damaged by cationic surfactants), or enters white blood cells, and combines with positively charged particles to help Acidic granulocytes are separated from other granulocytes in size and morphology, enhancing the classification accuracy of eosinophils.
- the above-described white blood cell sorting and counting reagent further comprises a cosolvent component.
- the co-solvent component enhances the solubility properties of the various components of the reagent and enhances the stability of the reagent, which is selected from, but not limited to, at least one of an alcohol, a cyclodextrin, and a cyclodextrin derivative.
- the concentration of the cosolvent in the white blood cell sorting and counting reagent is 5 to 100 ml/L; when the cosolvent is at least one of a cyclodextrin and a cyclodextrin derivative, the cosolvent The concentration is 5 ⁇ 50 g / L.
- the alcohol is preferably at least one selected from the group consisting of methanol, ethanol, ethylene glycol, glycerin, phenoxyethanol, and isopropanol
- the cyclodextrin is preferably selected from ⁇ -cyclodextrin, ⁇ .
- Red blood cells and platelets, and specific treatment of eosinophils enhance the difference between eosinophils and neutrophils, in order to enhance the classification of eosinophils, and to make various white blood cell subtypes in aging blood
- the difference between the classification effect and the classification effect of fresh blood is within an acceptable range, so that the reagent can simultaneously accurately classify white blood cells in aged whole blood samples stored in fresh whole blood and room temperature for 48 hours.
- the embodiment of the invention further provides a method for conveniently and quickly, and accurately classifying and counting white blood cells in fresh whole blood samples and aging whole blood samples stored within 48 hours at room temperature.
- the method for counting leukocyte counts includes the following steps:
- S02 detecting at least two kinds of light signals of the white blood cells to obtain shape and size information of the white blood cells;
- the white blood cells are divided into three groups corresponding to lymphocytes, monocytes, eosinophils, and a group corresponding to neutrophils and basophils. A total of 4 groups were counted and the number of cells contained in each group was counted.
- the white blood cell sorting and counting reagent and the blood sample to be tested are preferably mixed in a ratio of 10 to 200:1 by volume to carry out a reaction for 10 to 40 seconds, and then the next step is performed.
- the white blood cell sorting and counting reagent is as described above for the white blood cell sorting and counting reagent.
- the blood sample to be tested may be fresh whole blood and an aging whole blood sample stored within 48 hours at room temperature.
- the size information of the white blood cells is preferably measured by low-angle scattered light having a measurement angle of 2 to 6 degrees, and the shape information of the white blood cells is preferably a high angle with a measurement angle of 6 to 20 degrees.
- the detection of the scattered light can be done by a conventional photodiode sensor.
- the above method for counting and counting white blood cells can effectively enhance the difference between eosinophils and neutrophils by using the white blood cell sorting and counting reagent described above, and can classify various white blood cell subclasses in aged blood with new ones.
- the difference between the classification effects of blood is within an acceptable range, so that eosinophils in high eosinophil samples are better differentiated from neutrophils, which enhances the accuracy of white blood cell classification and improves the classification count. s efficiency. At the same time, this can effectively enhance the difference between eosinophils and neutrophils, so that the method for counting and counting white blood cells is stronger for different samples.
- the method for counting white blood cells in the embodiment of the present invention is also applicable to a laser detection method using two or more detection angles. For example, in the embodiment of the present invention, fluorescent signal detection is added, a three-dimensional scattergram is obtained, and white blood cell differential counting is performed. Or increase the scattered light signal detection at the third angle to obtain a three-dimensional scattergram for white blood cell sorting and counting.
- a white blood cell sorting and counting reagent and a method for classifying white blood cells :
- the white blood cell sorting and counting reagent contains the following components:
- Zwitterionic surfactant mercaptoamidopropyl betaine 7000mg/L
- Anionic compound 6-(p-toluidine)-2-naphthalenesulfonic acid 1500mg/L
- Cationic surfactant Octyltrimethylammonium chloride 50mg/L
- pH adjustment buffer MES 20mM
- the pH was adjusted to 5.0 with NaOH.
- a white blood cell sorting and counting reagent and a method for classifying white blood cells :
- Fig. 2 Method for classifying white blood cells: Refer to the detection classification method in Example 1. The results are shown in Fig. 2. In Fig. 2, Neu ⁇ shows neutrophils and basophils, Lym means lymphocytes, Mon means monocytes, and Eos means eosinophils.
- a white blood cell sorting and counting reagent and a method for classifying white blood cells contains components: the zwitterionic surfactant component in Example 1 is replaced by tetradecylsulfobetaine 1500 mg/L, and the other components and contents and the reagent pH are unchanged.
- a white blood cell sorting and counting reagent and a method for classifying white blood cells :
- Fig. 4 Method for classifying white blood cells: Refer to the detection classification method in Example 1. The results are shown in Fig. 4. In Fig. 4, Neu ⁇ shows neutrophils and basophils, Lym means lymphocytes, Mon means monocytes, and Eos means eosinophils.
- a white blood cell sorting and counting reagent and a method for classifying white blood cells :
- a white blood cell sorting and counting reagent and a method for classifying white blood cells :
- a white blood cell sorting and counting reagent and a method for classifying white blood cells :
- the white blood cell sorting and counting reagent contains components: the zwitterionic surfactant component of Example 1 is replaced by CHAPS 500 mg/L, and the other components and contents and the reagent pH are unchanged.
- Reagent A a reagent used in the white fine four classification, which consists of the following components:
- the pH was adjusted to 3.8 with NaOH.
- a normal human anticoagulated whole blood sample A blood sample (microscopic examination of 3 eosinophils per 100 white blood cells) and a patient anticoagulated whole blood sample B blood sample (microscopic examination of 7 eosinophils per 100 white blood cells) Cell), anti-coagulated whole blood sample C blood sample (65 eosinophils per 100 white blood cells), each mixed with lml of the above reagents, 20 to 30 seconds after laser detection
- the white blood cells were detected, and the size information of the cells was measured by forward low-angle scattered light with an angle of 2 to 6 degrees, and the morphology information of the cells was measured by forward high-angle scattered light with an angle of 8 to 20 degrees.
- Fig. 8-a is a blood sample test chart
- Fig. 8-b is a B blood sample test chart
- Fig. 8-c is a C blood sample test chart.
- the comparative reagent A can well distinguish between lymphocytes, monocytes, eosinophils, neutrophils and basophils, and four The boundary between the scatter plots of the cell-like cells is obvious.
- Fig. 8-b and Fig. 8-c for eosinophils in whole blood samples of patients with disease, the distance between neutrophils and neutrophils is obviously shortened (Fig.
- Comparative Reagent A does not perform a good classification of eosinophils in whole blood samples of disease patients.
- a white blood cell sorting and counting reagent and a method for classifying white blood cells :
- the white blood cell sorting and counting reagent contains the following components:
- the pH was adjusted to 4.0 with NaOH.
- Fig. 9-a is a blood sample test chart
- Fig. 9-b is a B blood sample test chart
- Fig. 9-c is a C blood sample test chart.
- the reagents in the present embodiment can effectively divide white blood cells of each sample into lymphocytes, monocytes, eosinophils, neutrophils, and basophils. And each of the four types has its own scatter plot area, and the position is fixed, and the clustering is obvious.
- eosinophils and neutrophil populations are distinct, especially C blood samples, eosinophils and neutrophils have obvious boundaries, and their Eos classification results are 60%, close to the results of microscopic examination.
- a white blood cell sorting and counting reagent and a method for classifying white blood cells :
- the white blood cell sorting and counting reagent contains the following components:
- the pH was adjusted to 5.7 with NaOH.
- lymphocytes 125 clinical anticoagulated whole blood samples were randomly selected, and their lymphocytes, monocytes, neutrophils and eosinophils were classified and counted according to the following methods.
- each blood sample was mixed with 1 ml of the reagent of the present Example 9, and the white blood cells were detected by laser detection after 20 to 30 seconds, and the cells were measured by forward low angle scattered light with an angle of 2 to 6 degrees.
- the classification results of the taxa were compared with the results of manual microscopic examination of the conventional Wright-Gemsa staining method to make a correlation analysis.
- Fig. 10-a is the correlation between the lymphocytes and the results of manual microscopic examination of the conventional Wright-Giemsa staining method
- Fig. 10-b shows the relationship between the monocytes and the conventional Wright.
- Figure 10-c is the correlation between neutrophils and conventional Wright-Gemsa staining results
- Figure 4- d is the correlation between eosinophils and the results of manual microscopic examination of conventional Wright-Giemsa staining.
- the classification results of the four types of lymphocytes, monocytes, eosinophils, neutrophils and basophils using the white blood cell sorting and counting reagent of the present invention are compared with the conventional Wright-Gemsa
- the manual microscopic count results of the staining method have a good correlation.
- a white blood cell sorting and counting reagent and a method for classifying white blood cells :
- the white blood cell sorting and counting reagent contains the following components:
- the pH was adjusted to 6.0 with NaOH.
- Methods for classifying white blood cells Ten anticoagulated whole blood samples were taken, and the reagent A in the above Comparative Example 1 and the reagent in the present Example 10 were simultaneously used in the first day (24 hours) in vitro and in the second day (48 hours) in vitro. Test the blood sample. The results of the assay are shown in Table 1, where the first day is the test result of the whole blood sample within 1 day, and the second day is the test result within 2 days of the in vitro (within 48 hours). Among them, Neu% indicates the proportion of neutrophils and basophils, Lym% indicates the proportion of lymphocytes, Mon% indicates the proportion of monocytes, and £08% indicates the proportion of eosinophils.
- the first day is synonymous with the aforementioned fresh whole blood sample.
- a white blood cell sorting and counting reagent and a method for classifying white blood cells :
- the white blood cell sorting and counting reagent contains the following components:
- Zwitterionic surfactant mercaptoamidopropyl betaine 5000mg/L
- Cationic surfactant Octyltrimethylammonium chloride 50mg/L
- pH adjustment buffer MES 20mM
- the pH was adjusted to 5.0 with NaOH.
- Example 12 Method for classifying white blood cells: Refer to the detection classification method in Example 1. The test results are shown in Figure 11. In Fig. 11, Neu represents neutrophils and basophils, Lym represents lymphocytes, Mon represents monocytes, and Eos represents eosinophils. Example 12
- a white blood cell sorting and counting reagent and a method for classifying white blood cells :
- the white blood cell sorting and counting reagent contains the following components:
- Zwitterionic surfactant tetradecyl sulfobetaine 1000mg/L
- Cationic surfactant Octyltrimethylammonium chloride 50mg/L
- pH adjustment buffer MES 20mM
- the pH was adjusted to 5.0 with NaOH.
Abstract
一种白细胞分类计数试剂、对白细胞分类计数的方法和两性离子表面活性剂的应用。该白细胞分类计数试剂包括至少一种阳离子表面活性剂和至少一种两性离子表面活性剂以及pH缓冲剂，该两性离子表面活性为本文通式I所示的两性离子表面活性剂或为胆汁酸衍生物型两性表面活性剂。该白细胞分类计数试剂能对嗜酸性粒细胞进行特异性处理，增强了嗜酸性粒细胞与中性粒细胞之间的差异，以达到增强嗜酸性粒细胞分类作用，使老化血中各个白细胞亚类分类效果与新鲜血的分类效果之间差异在可接受的范围内，从而对白细胞分类计数的方法快速、准确。
Description
白细胞分类计数试剂和白细胞分类方法 技术领域
本发明涉及一种细胞分类试剂和对细胞分类的方法， 具体的涉及一种白细 胞分类计数试剂和利用该试剂对血液中白细胞分类的方法以及一种两性离子表 面活性剂的用途。
背景技术
人体血液中的白细胞可以分为单核细胞、 淋巴细胞和粒细胞（包括嗜酸性 粒细胞、 中性粒细胞和嗜碱性粒细胞）五个亚类。 正常健康人血液中白细胞各 个亚类的比例是相对恒定的， 当人体出现某些疾病时， 则会表现出某一类或几 类白细胞数量的增多或减少。 其中嗜酸性粒细胞是在人全血中占比例很少的一 种粒细胞， 健康人血液中占白细胞比例为 0.5 ~ 5%, 临床上出现过敏性疾病或 寄生虫感染等疾病时， 嗜酸性粒细胞的数量会出现明显的变化， 因此在人全血 检测中嗜酸性粒细胞的准确分类计数对这些疾病的正确诊断有直接的指导意 义。
嗜酸性粒细胞大小与中性粒细胞接近， 在非疾病人群的全血样本中， 即正 常成熟的嗜酸性粒细胞， 细胞中的颗粒已经完全成熟， 使其在前向高角度光散 射的作用下， 能够与中性粒细胞得以区分。 而在疾病条件下， 尤其是寄生虫感 染的病人全血样本中， 由于病理性作用， 脊髓中大量不成熟嗜酸性粒细胞进入 外周血中， 使嗜酸性粒细胞在外周血中的所占比例急剧增加， 并且不成熟嗜酸 性粒细胞与中性粒细胞的区分程度也明显降低， 应用现有技术的白细胞分类试 剂不足以将不成熟嗜酸性粒细胞与中性粒细胞区分开， 使得嗜酸性粒细胞的检 测准确性明显减低。
在医院对病人血液样本进行分析处理时，从病人体内采集新鲜抗凝血样后， 有时会放置一段时间后进行分析，或会对放置较长时间的病人血样进行复检（室 温条件下存储 48小时以内） 。 随着放置时间的延长， 各种细胞经过新陈代谢， 新鲜血样逐渐老化， 而同一血样的新鲜血和老化血之间细胞形态会出现明显变 化， 往往会造成老化血的白细胞分类计数可信度降低， 其中所占比例较少的嗜 酸性粒细胞的分类准确性降低的尤为严重。
目前， 国内外对白细胞分类相关内容进行了研究，也出现了一下研究成果。 在国外公开了一种用于测量血红蛋白 （HGB )和嗜酸性粒细胞（Eos ) 的试剂 和方法。 试剂含有聚氧乙烯结构为基础的非离子表面活性剂， pH范围 3-11 , 通 过阻抗法 （DC ) 实现 Eos分类计数。
国外还公开了一种用含有聚氧乙烯 -9-月桂醚对血样进行处理后对白细胞 进行分类的方法。 这种方法在酸性介质下适于区分嗜碱性粒细胞， 在碱性介质 下适用于区分嗜酸性粒细胞。
上述两种现有技术均是从白细胞中单独分离出嗜酸性粒细胞， 但不能同时 对其他类型的白细胞进行分类。
另外， 国外公开了一种白细胞分类的试剂， 该试剂包括至少一种离子表面 活性剂， 可以是阳离子或两性表面活性剂中一种， 至少含一种带阴离子的有机 化合物， 一种非离子表面活性剂和用来调整 pH值的緩冲剂。 检测方法为激光 散射法。
国外还公开了一种白细胞五分类的试剂， 通过两种试剂， 第一试剂为稀释 血样的高渗溶液，第二试剂为含阴离子表面活性剂和非离子表面活性剂的溶液， 检测方法为直流阻抗和射频法。
在国外另一专利中公开了一种白细胞五分类的方法和试剂系统。 溶血剂由 两种试剂组成， 第一试剂含烷基磺酸的碱金属盐类、 有机酸、 无机酸或其混合 物及非离子表面活性剂。 第二试剂含聚氧乙烯或聚氧丙烯的共聚物类表面活性 剂、 十二烷基磺酸钠的高深碱性溶液。 检测方法为直流阻抗和射频法。
在国内公开了一种通过两种试剂完成白细胞五分类的方法。 该两种试剂除 都含有非离子表面活性剂和緩冲液外， 第一种试剂至少含有一种离子表面活性 剂和一种带阴离子的有机化合物， 将血细胞分为淋巴细胞、 单核细胞、 嗜酸性 粒细胞以及嗜碱性粒细胞和中性粒细胞四类， 第二种试剂至少含有一种离子表 面活性剂， 将血细胞分成嗜碱性粒细胞和其他细胞类群。 检测方法为激光散射 法。 国内还公开了一种白细胞分类计数试剂， 该试剂包含阳离子、 两性表面活 性剂以及其组合， 包含苯环或者杂环的有机化合物， 调节 pH值的緩冲液。 检 测方法为激光散射法。
以上现有技术的分类试剂以及方法中， 溶血剂溶解全血样本中的红细胞， 并对白细胞进行非特异性作用， 结合检测方法通过各种类型白细胞的结构特点 进行区分， 其中嗜酸性粒细胞是作为其中一个类群进行分类和计数。 由于现有 技术并不含有针对嗜酸性粒细胞特异性处理的成分， 因此对于含有高嗜酸的全 血样本或老化血样 嗜酸性粒细胞的分类准确性不够理想。
发明内容
本发明的目的在于克服现有技术的上述不足， 提供一种能够快速溶解红细 胞， 能增强嗜酸性粒细胞分类作用， 使新鲜全血样本和室温存储 48小时的老化 全血样本的白细胞分类结果更为准确的白细胞分类计数试剂。
本发明另一目的是提供一种方便、 快捷， 且能准确对新鲜全血样本和室温 存储 48小时以内的老化全血样本中的白细胞进行分类计数的方法。
本发明又一目的是提供一种两性离子表面活性剂在制备白细胞分类计数试 剂中的用途。
为了实现上述发明目的， 本发明的技术方案如下：
一种白细 分类计数试剂， 包括
( 1 )表面活性剂溶液， 该表面活性剂溶液能够溶解红细胞， 使白细胞膜部 分损伤， 稳定嗜酸性粒细胞和老化细胞的细胞膜， 该表面活性剂溶液含有至少 一种阳离子表面活性剂和至少一种两性离子表面活性剂；
该两性表面活性剂具有通式 I所示的结构
R^(CO-MH) -(CH2)— N÷- (CH2), 式中 为（^18的烷基； R2和 R3各自独立选自 C 1-3的烷基、 C 1-3烷氧基或 d.3烷羟基； a为 0或 1 ; n为 0-6的整数； m为 1-5的整数； X选自羧基或磺酸 基；
或 /和该两性离子表面活性剂选自胆汁酸衍生物型两性表面活性剂；
( 2 ) 能够将 pH值调整在 4 ~ 6范围内的緩冲剂。
以及， 一种对白细胞分类的方法， 包括如下步骤：
一种对白细胞分类计数的方法， 包括如下步骤：
将上述的白细胞分类计数试剂与待测血样混合反应，溶解红细胞和血小板， 损伤白细胞；
检测该白细胞的至少两种光信号， 得到白细胞的形态和大小信息； 根据该白细胞的形态和大小信息， 将白细胞分为淋巴细胞、 单核细胞、 嗜 酸性粒细胞相对应的 3个集团以及与中性粒细胞和嗜碱性粒细胞相对应的 1个 集团共 4个集团， 并对该各集团中所含细胞数目进行计数。
本发明还提供一种具有通式 I所示结构的两性离子表面活性剂在制备白细 胞分类计数试剂中的用途，
R2
R厂 (〔:0 H) ( ) ― (C¾)m-X- I
R,
式中 为 C8-18的烷基； R2和 R3各自独立选自 C 1-3的烷基、 C w烷氧基或 C L3烷羟基； a为 0或 1 ; n为 0-6的整数； m为 1-5的整数； X选自羧基或磺 酸基。
上述白细胞分类计数试剂中的阳离子表面活性剂组分起到溶血剂作用， 能 有效的溶解待测血样中红细胞和血小板， 并对白细胞膜进行部分损伤； 两性离 子表面活性剂一方面能够起到溶血剂作用， 协助溶解红细胞和血小板， 另一方 面， 由于其同时带有正负两个离子集团， 能够对白细胞膜起到稳定作用， 尤其 对嗜酸性粒细胞以及老化样本中的白细胞， 能緩解阳离子表面活性剂等溶血剂 组分对其的过度损伤。 因此， 上述白细胞分类计数试剂与待测血样接触后， 能 快速的溶解红细胞， 对嗜酸性粒细胞进行特异性处理， 增强了嗜酸性粒细胞与 中性粒细胞之间的差异， 以达到增强嗜酸性粒细胞分类作用， 使老化血中各个 白细胞亚类分类效果与新鲜血的分类效果之间差异在可接受的范围内， 使得该 试剂同时能够满足对新鲜全血和室温存储 48 小时以内的老化全血样本中白细 胞准确分类。
上述对白细胞分类计数的方法能有效增强嗜酸性粒细胞与中性粒细胞之间
3
的差异， 可以使老化血中各个白细胞亚类分类效果与新鲜血的分类效果之间差 异在可接受的范围内， 使高嗜酸性粒细胞样本中的嗜酸性粒细胞更好地与中性 粒细胞区分， 增强了白细胞分类的方法准确度。 同时， 该方法能有效增强嗜酸 性粒细胞与中性粒细胞之间的差异， 对不同血液样本的适应性强。
附图说明
下面将结合附图及实施例对本发明作进一步说明， 附图中：
图 1是利用本发明实施例 1的试剂对待测血样进行白细胞分类计数时的前 向低角度和高角度散射光的散射图；
图 2是利用本发明实施例 2的试剂对待测血样进行白细胞分类计数时的前 向低角度和高角度散射光的散射图；
图 3是利用本发明实施例 3的试剂对待测血样进行白细胞分类计数时的前 向低角度和高角度散射光的散射图；
图 4是利用本发明实施例 4的试剂对待测血样进行白细胞分类计数时的前 向低角度和高角度散射光的散射图；
图 5是利用本发明实施例 5的试剂对待测血样进行白细胞分类计数时的前 向低角度和高角度散射光的散射图；
图 6是利用本发明实施例 6的试剂对待测血样进行白细胞分类计数时的前 向低角度和高角度散射光的散射图；
图 7是利用本发明实施例 7的试剂对待测血样进行白细胞分类计数时的前 向低角度和高角度散射光的散射图；
图 8是利用对比实例 1的试剂 A对待测血样进行白细胞分类计数时的前向 低角度和高角度散射光的散射图； 其中， 图 8-a是 A血样检测图、 图 8-b是 B 血样检测图、 图 8-c是 C血样检测图；
图 9是利用本发明实施例 8的试剂对待测血样进行白细胞分类计数时的前 向低角度和高角度散射光的散射图； 图 9-a是 A血样检测图、 图 9-b是 B血样 检测图、 图 9-c是 C血样检测图；
图 10是利用本发明实施例 9的试剂对待测血样中四类群白细胞的分类与常 规瑞氏 -姬姆萨染色法的手工镜检计数结果的相关性关系图； 其中， 图 10-a是 淋巴细胞与常规瑞氏-姬姆萨染色法的手工镜检计数结果的相关性关系，图 10-b 是单核细胞与常规瑞氏 -姬姆萨染色法的手工镜检计数结果的相关性关系， 图 10-c 是中性粒细胞与常规瑞氏 -姬姆萨染色法的手工镜检计数结果的相关性关 系， 图 10-d是嗜酸性粒细胞与常规瑞氏-姬姆萨染色法的手工镜检计数结果的 相关性关系；
图 11是利用本发明实施例 11的试剂对待测血样进行白细胞分类计数时的 前向低角度和高角度散射光的散射图；
图 12是利用本发明实施例 12的试剂对待测血样进行白细胞分类计数时的 前向低角度和高角度散射光的散射图。
具体实施方式
为了使本发明的目的、 技术方案及优点更加清楚明白， 以下结合附图及实
施例， 对本发明进行进一步详细说明。 应当理解， 此处所描述的具体实施例仅 仅用以解释本发明， 并不用于限定本发明。
本发明实施例提供了一种具有通式 I所示结构的两性离子表面活性剂在制 备白细胞分类计数试剂中的用途， 该通式 I如下：
R3
R厂 (CCH H) (CH2)— ÷— (CH2)n— X- I
3
式中， 为 C8-18 (即 C原子数为 8 ~ 18 ) 的烷基； R2和 R3各自独立选自 Ci-3的烷基、 d.3烷氧基或 d.3烷羟基； a为 0或 1 ; n为 0-6的整数； m为 1-5 的整数； X选自羧基或磺酸基。
在优选实施例中， 上述通式 I中的 R2和 R3各自独立选自羟乙基、 乙氧基、 羟 丙基； X为磺酸基， n为 3 , m为 3。 因此， 在该优选实施例中， 、 R2、 R3、 n、 m和 X可以多种选择性组合。 这样， 上述通式 I的两性表面活性剂至少有如下几 种分子结构：
第一种： n为 3 , R2和 R3各自独立选自 C1-3的烷基、 C1-3烷氧基或 C1-3烷羟基， a为 0或 1 , m为 1-5的整数， X选自羧基或磺酸基；
第二种： R2和 R3各自独立选自羟乙基或甲基， a为 0或 1 , n为 0-6的整数， m 为 1-5的整数， X选自羧基或磺酸基；
第三种： m为 3 , X为磺酸基， 为(^8-18的烷基； R2和 R3各自独立选自 C1-3 的烷基、 d.3烷氧基或(^.3烷羟基， a为 0或 1 , n为 0-6的整数；
第四种： R2和 R3为乙氧基， 为(^8-18的烷基， a为 0或 1 , n为 0-6的整数， m 为 1-5的整数， X选自羧基或磺酸基；
第五种： R2和 R3为羟丙基， 为(^18的烷基， a为 0或 1 , n为 0-6的整数， m 为 1-5的整数， X选自羧基或磺酸基；
第六种： R2和 R3各自独立选自羟丙基， n为 3 , X为磺酸基， 为(^18的烷基， a为 0或 1 , m为 1-5的整数。
具体地， 上述通式 I的两性表面活性剂可以是癸基酰胺丙基甜菜碱、 月桂 基酰胺丙基甜菜碱、 椰油酰胺丙基甜菜碱、 硬脂基酰胺丙基甜菜碱、 月桂基二 羟乙基甜菜碱、 十四烷基二羟乙基甜菜碱、 硬脂基二羟乙基甜菜碱、 月桂基磺 基甜菜碱、 十四烷基磺基甜菜碱、 十六烷基磺基甜菜碱、 十八烷基磺基甜菜碱、 十二烷基乙氧基磺基甜菜碱、 十四烷基乙氧基磺基甜菜碱、 十六烷基乙氧基磺 基甜菜碱、 十八烷基乙氧基磺基甜菜碱、 十二烷基羟丙基磺基甜菜碱、 十四烷 基羟丙基磺基甜菜碱、 十六烷基羟丙基磺基甜菜碱、 十八烷基羟丙基磺基甜菜 碱、 月桂酰胺丙基羟丙基磺基甜菜碱、 椰油酰胺丙基羟丙基磺基甜菜碱、 十四 烷基酰胺丙基羟丙基磺基甜菜碱、 十六烷基酰胺丙基羟丙基磺基甜菜碱或十八 烷基酰胺丙基羟丙基磺基甜菜碱。
上述两性表面活性剂在白细胞分类计数试剂中应用时， 其一方面能够起到 溶血剂作用， 协助阳离子表面活性剂溶解红细胞和血小板， 另一方面， 由于其
同时带有正负两个离子集团， 能够对白细胞膜尤其是对嗜酸性粒细胞以及老化 血样本中的白细胞起到稳定作用， 在一定程度上緩解阳离子表面活性剂等溶血 剂组分对白细胞的过度损伤， 达到显著增强嗜酸性粒细胞与中性粒细胞之间的 差异， 以便对白细胞的准确分类计数。
本发明实施例还提供了一种有效增强嗜酸性粒细胞与中性粒细胞之间的差 异，能对新鲜全血样本和室温存储 48小时以内的老化全血样本中的白细胞进行 快速， 准确分类计数的试剂。 该白细胞分类计数试剂包括：
( 1 )表面活性剂溶液， 该表面活性剂溶液能够溶解红细胞， 使白细胞膜部 分损伤， 稳定嗜酸性粒细胞和老化细胞的细胞膜， 该表面活性剂溶液含有至少 一种阳离子表面活性剂和至少一种两性离子表面活性剂；
其中， 该两性表面活性剂为上文所述的通式 I所示结构两性表面活性剂或 / 和胆汁酸衍生物型两性表面活性剂； 其中， 通式 I如下：
R厂 (CO- NH) (CH2)-^- (CH2)m-X- I 通式 I中的 为 C8-18 (即 C原子数为 8 ~ 18 ) 的烷基； R2和 R3各自独立 选自 d.3的烷基、 d.3烷氧基或 d.3烷羟基； a为 0或 1 ; n为 0-6的整数； m 为 1-5的整数； X选自羧基或磺酸基；
( 2 ) 能够将 pH值调整在 4 ~ 6范围内的緩冲剂。
具体地， 当两性表面活性剂为上文所述的通式 I所示结构两性表面活性剂 时， 在优选实施例中， 上述通式 I中的 R2和 R3各自独立选自羟乙基、 乙氧基、 羟 丙基； X为磺酸基， n为 3 , m为 3。 因此， 在该优选实施例中， 、 R2、 R3、 n、 m和 X可以多种选择性组合。 这样， 上述通式 I的两性表面活性剂至少有如上文 所述的几种分子结构， 为了节约篇幅， 在此不再赘述。
上述两性表面活性剂具体地可以选自癸基酰胺丙基甜菜碱、 月桂基酰胺丙 基甜菜碱、 椰油酰胺丙基甜菜碱、 硬脂基酰胺丙基甜菜碱、 月桂基二羟乙基甜 菜碱、 十四烷基二羟乙基甜菜碱、硬脂基二羟乙基甜菜碱、 月桂基磺基甜菜碱、 十四烷基磺基甜菜碱、 十六烷基磺基甜菜碱、 十八烷基磺基甜菜碱、 十二烷基 乙氧基磺基甜菜碱、十四烷基乙氧基磺基甜菜碱、十六烷基乙氧基磺基甜菜碱、 十八烷基乙氧基磺基甜菜碱、 十二烷基羟丙基磺基甜菜碱、 十四烷基羟丙基磺 基甜菜碱、 十六烷基羟丙基磺基甜菜碱、 十八烷基羟丙基磺基甜菜碱、 月桂酰 胺丙基羟丙基磺基甜菜碱、 椰油酰胺丙基羟丙基磺基甜菜碱、 十四烷基酰胺丙 基羟丙基磺基甜菜碱、 十六烷基酰胺丙基羟丙基磺基甜菜碱、 十八烷基酰胺丙 基羟 ^基磺基甜菜碱中的一种或两种以上互配。 、 、 生物型两性表面活性剂优选为 3-[(3-胆烷酰胺丙基) -二甲基铵] -1-丙磺酸盐 ( CHAPS ) 、 3-[(3-胆烷酰胺基丙基) -二甲基铵] -2-羟 -1-丙磺酸盐 （ CHAPSO ) 中的一种或两种互配。
当然，上述两性表面活性剂还可以是上述的通式 I所示的结构两性表面活性 剂与胆汁酸衍生物型两性表面活性剂的复合物。 此时， 通式 I所示的结构两性表 面活性剂与胆汁酸衍生物型两性表面活性剂优选按质量比为 10: 1 ~ 1 :10进行混 合。 、； ；、、、 I 、 、 I 、、 I 、 - - 、、
50 ~ 8000mg/L, 优选为 100 ~ 7000mg/L。
上述实施例中的两性表面活性剂一方面能够起到溶血剂作用， 协助阳离子 表面活性剂溶解红细胞和血小板， 另一方面， 由于其同时带有正负两个离子集 团， 能够对白细胞膜尤其是对嗜酸性粒细胞以及老化血样本中的白细胞起到稳 定作用， 在一定程度上緩解阳离子表面活性剂等溶血剂组分对白细胞的过度损 伤， 达到显著增强嗜酸性粒细胞与中性粒细胞之间的差异， 以便对白细胞的准 确分类计数。 上述优选的两性表面活性剂含量和种类溶解红细胞和血小板的协 助作用更好， 更好的緩解对阳离子表面活性剂对白细胞的过度损伤， 使得经阳 离子表面活性剂损伤的白细胞更加稳定， 进一步增强嗜酸性粒细胞与中性粒细 胞之间的差异， 使得各类白细胞从大小和形态上完全分离， 以更加方便准确的 对血样中的白细胞进行识别， 并分类计数。
上述实施例中的阳离子表面活性剂主要起到溶血剂作用， 用于溶解红细胞 和血小板， 并对白细胞膜进行部分损伤。 为了使得该阳离子表面活性剂组分溶 血效果更好， 并对白细胞膜进行适度损伤， 在优选实施例中， 其选用具有通式 II结构的季铵盐阳离子表面活性 ：
式 II中的 R4为 C6_14的烷基或链烯基， 优选辛基、 癸基或月桂基； R5和 R6 各自独立选自 的烷基或链烯基， 优选甲基、 乙基； 1 7为（^4的烷基、 链烯 基或苄基； Y为卤素。
该阳离子表面活性剂在上述白细胞分类计数试剂中的浓度为 10 ~ 5000mg, 优选为 50 ~ 3000mg /L。
上述将 pH值调整在 4 ~ 6范围内的緩冲剂能有效的调节和稳定上述实施例 白细胞分类计数试剂的 pH, 以发挥该试剂中各组分作用以及组分之间协同作 用。 同时通过调整并稳定 pH值可以满足增强嗜酸性粒细胞分类效果， 使得该 试剂准确对白细胞分类计数。 为了更加准确的对白细胞， 特别是对嗜酸性粒细 胞分类并计数，在优选实施例中，采用 pH调节緩冲剂将该试剂的 pH调整 4.5 ~ 5.5; 同时， 该 pH调节緩冲剂选自甲酸、 邻苯二甲酸、 乙酸、 磷酸、 柠檬酸、 琥珀酸、 2-吗啉乙磺酸（MES )、 甘氨酸中的至少一种。 当然， 该 pH调节緩冲 剂还可以采用本领域常用的其他 pH緩冲剂。 也可以结合本领域常用的碱如 NaOH共同作用， 已达到将白细胞分类计数试剂的 pH调整至适当的 pH范围。
进一步优选地， 在上述各实施例的基础上， 上述白细胞分类计数试剂还包
括带阴离子基团的有机化合物， 该带阴离子基团的有机化合物选自 8-苯胺基 -1- 萘磺酸、 8-苯胺基 -1-萘磺酸盐、 6- (对甲苯胺基 )-2-萘磺酸、 6- (对甲苯胺基 )-2- 萘磺酸盐、 铬变酸、 萘磺酸中的至少一种。 另外带阴离子基团的有机化合物在 白细胞分类计数试剂中的浓度为 100 ~ 6000mg/L, 优选为 500 ~ 4000 mg/L。 该 带阴离子基团的有机化由于在水中带负电荷， 能与白细胞结合（特别是被阳离 子表面活性剂损伤后的白细胞）， 或进入白细胞内， 与带正电的颗粒结合， 有助 于嗜酸性粒细胞从大小和形态上与其他粒细胞分离， 增强嗜酸性粒细胞的分类 准确性。
进一步优选地， 在上述各实施例的基础上， 上述白细胞分类计数试剂还包 含助溶剂组分。 该助溶剂组分能提高该试剂中各种组分的溶解性能， 增强试剂 稳定性， 其选自但不限于醇、 环糊精、 环糊精衍生物中的至少一种。 当助溶剂 为醇溶剂时， 该助溶剂在白细胞分类计数试剂中的浓度为 5 ~ 100 ml/L; 当助溶 剂为环糊精、 环糊精衍生物中的至少一种时， 该助溶剂的浓度为 5 ~ 50 g/L。
具体地， 上述优选实施例中， 醇优选选自甲醇、 乙醇、 乙二醇、 丙三醇、 苯氧乙醇、 异丙醇中的至少一种， 环糊精优选自 α-环糊精、 β-环糊精中的至少 一种， 环糊精衍生物优选自二甲基 -β-环糊精、 羟乙基 -β-环糊精、 羟丙基 -β-环糊 精、 羧甲基 -β-环糊精中的至少一种。
由上述可知， 本发明实施例白细胞分类计数试剂中各组分发挥独自作用的 基础上， 各组分之间还发挥协同作用， 使得上述白细胞分类计数试剂与待测血 样接触后， 能快速的溶解红细胞和血小板， 并对嗜酸性粒细胞进行特异性处理， 增强了嗜酸性粒细胞与中性粒细胞之间的差异， 以达到增强嗜酸性粒细胞分类 作用， 并使老化血中各个白细胞亚类分类效果与新鲜血的分类效果之间差异在 可接受的范围内，使得该试剂同时能够满足对新鲜全血和室温存储 48小时以内 的老化全血样本中白细胞准确分类。
本发明实施例进一步提供了一种方便、 快捷， 且能准确对新鲜全血样本和 室温存储 48小时以内的老化全血样本中的白细胞进行分类计数的方法。该对白 细胞分类计数的方法包括如下步骤：
S01 : 将上述实施例中的白细胞分类计数试剂与待测血样混合反应， 溶解 红细 和血小板， 损伤白细月包；
S02: 检测该白细胞的至少两种光信号， 得到白细胞的形态和大小信息；
S03 : 根据该白细胞的形态和大小信息， 将白细胞分为淋巴细胞、 单核细 胞、 嗜酸性粒细胞相对应的 3个集团以及与中性粒细胞和嗜碱性粒细胞相对应 的 1个集团共 4个集团， 并对该各集团中所含细胞数目进行计数。
具体地， 上述步骤 S01中， 白细胞分类计数试剂与待测血样优选按体积比 10 ~ 200:1的比例混合进行反应 10 ~ 40秒后， 再进行下步的操作。 其中， 白细 胞分类计数试剂如上文所述的白细胞分类计数试剂， 为了节约篇幅， 在此不再 赘述； 该待测血样可以是新鲜全血和室温存储 48小时以内的老化全血样本。
上述步骤 S02中， 白细胞的大小信息优选采用测定角度为 2 ~ 6度的低角 度散射光来测定获取， 白细胞的形态信息优选采用测定角度为 6 ~ 20度的高角
度散射光测定获取。 该散射光的检测可以通过常用的光电二极管传感器完成。 上述对白细胞分类计数的方法由于采用上文所述的白细胞分类计数试剂， 因此能有效增强嗜酸性粒细胞与中性粒细胞之间的差异， 可以使老化血中各个 白细胞亚类分类效果与新鲜血的分类效果之间差异在可接受的范围内， 使高嗜 酸性粒细胞样本中的嗜酸性粒细胞更好地与中性粒细胞区分， 增强了白细胞分 类的方法准确度， 提高了分类计数的效率。 同时， 该能有效增强嗜酸性粒细胞 与中性粒细胞之间的差异， 使得该对白细胞分类计数的方法对不同样本的适应 行强。 本发明实施例对白细胞分类计数的方法同样适用于采用两个以上检测角 度的激光检测法。 例如， 在本发明实施例上增加荧光信号检测， 获得三维散点 图， 进行白细胞分类计数。 或者增加第三个角度的散射光信号检测， 获得三维 散点图， 进行白细胞分类计数。
以下通过多个实施例来举例说明上述白细胞分类计数试剂和白细胞分类的 方法， 以及其性能等方面。
实施例 1
一种白细胞分类计数试剂和对白细胞分类的方法：
白细胞分类计数试剂含有如下组分：
两性离子表面活性剂： 癸基酰胺丙基甜菜碱 7000mg/L
阴离子化合物： 6- (对甲苯胺基 )-2-萘磺酸 1500mg/L
阳离子表面活性剂： 辛基三甲基氯化铵 50mg/L
助溶剂： 乙二醇 10ml/L
pH调节緩冲剂： MES 20mM;
用 NaOH将 pH调节到 5.0。
对白细胞分类的方法：
将取 lml本实施例白细胞分类计数试剂与 20ul待测血液混合， 20 ~ 30秒后用 激光检测法检测白细胞。 采用测定角度为 2 ~ 6度的前向低角度散射光测定细胞 的大小信息，并采用测定角度为 8 ~ 20度的前向高角度散射光测定细胞的形态信 息。 检测结果如图 1所示， 图 1中， Neu表示中性粒细胞及嗜碱性粒细胞， Lym 表示淋巴细胞， Mon表示单核细胞， Eos表示嗜酸性粒细胞。
实施例 2
一种白细胞分类计数试剂和对白细胞分类的方法：
白细胞分类计数试剂含有的组分：将实施例 1中的两性离子表面活性剂组分 换成月桂基二羟乙基甜菜碱 5000mg/L, 其他组分和含量以及试剂 pH不变。
对白细胞分类的方法： 参照实施例 1中的检测分类方法。 检测结果如图 2所 示， 图 2中， Neu^示中性粒细胞及嗜碱性粒细胞， Lym表示淋巴细胞， Mon表 示单核细胞， Eos表示嗜酸性粒细胞。
实施例 3
一种白细胞分类计数试剂和对白细胞分类的方法：
白细胞分类计数试剂含有的组分：将实施例 1中的两性离子表面活性剂组分 换成十四烷基磺基甜菜碱 1500mg/L, 其他组分和含量以及试剂 pH不变。
对白细胞分类的方法： 参照实施例 1中的检测分类方法。 检测结果如图 3所 示， 图 3中， Neu^示中性粒细胞及嗜碱性粒细胞， Lym表示淋巴细胞， Mon表 示单核细胞， Eos表示嗜酸性粒细胞。
实施例 4
一种白细胞分类计数试剂和对白细胞分类的方法：
白细胞分类计数试剂含有的组分：将实施例 1中的两性离子表面活性剂组分 换成十二烷基乙氧基磺基甜菜碱 2500mg/L,其他组分和含量以及试剂 pH不变。
对白细胞分类的方法： 参照实施例 1中的检测分类方法。 检测结果如图 4所 示， 图 4中， Neu^示中性粒细胞及嗜碱性粒细胞， Lym表示淋巴细胞， Mon表 示单核细胞， Eos表示嗜酸性粒细胞。
实施例 5
一种白细胞分类计数试剂和对白细胞分类的方法：
白细胞分类计数试剂含有的组分：将实施例 1中的两性离子表面活性剂组分 换成十四烷基羟丙基磺基甜菜碱 800mg/L, 其他组分和含量以及试剂 pH不变。
对白细胞分类的方法： 参照实施例 1中的检测分类方法。 检测结果如图 5所 示， 图 5中， Neu^示中性粒细胞及嗜碱性粒细胞， Lym表示淋巴细胞， Mon表 示单核细胞， Eos表示嗜酸性粒细胞。
实施例 6
一种白细胞分类计数试剂和对白细胞分类的方法：
白细胞分类计数试剂含有的组分：将实施例 1中的两性离子表面活性剂组分 换成月桂酰胺丙基羟磺基甜菜碱 4000mg/L,其他组分和含量以及试剂 pH不变。
对白细胞分类的方法： 参照实施例 1中的检测分类方法。 检测结果如图 6所 示， 图 6中， Neu^示中性粒细胞及嗜碱性粒细胞， Lym表示淋巴细胞， Mon表 示单核细胞， Eos表示嗜酸性粒细胞。
实施例 7
一种白细胞分类计数试剂和对白细胞分类的方法：
白细胞分类计数试剂含有的组分：将实施例 1中的两性离子表面活性剂组分 换成 CHAPS 500mg/L , 其他组分和含量以及试剂 pH不变。
对白细胞分类的方法： 参照实施例 1中的检测分类方法。 检测结果如图 7所 示， 图 7中， Neu^示中性粒细胞及嗜碱性粒细胞， Lym表示淋巴细胞， Mon表 示单核细胞， Eos表示嗜酸性粒细胞。
由上述实施例 1 ~ 7以及图 1 ~ 7可知， 换成不同的两性离子表面活性剂都可 以得到大致相同的散点图结果， 均可以满足白细胞四分类。
对比实例 1
试剂 A, —种用于白细 四分类的试剂， 该试剂由以下成分组成：
NaCl 300mg/L
乙醇 2000mg/L
丙三醇 750mg/L
癸基三甲基溴化铵 6100mg/L
1-萘乙酸 400mg/L
乙酸 1950mg/L
酸性蓝 150mg/L;
用NaOH将pH调节到3.8。
利用对比试剂 A对白细胞进行检测分类：
分别选取正常人抗凝全血样本 A血样（镜检每 100个白细胞中有 3个嗜酸性 粒细胞）、 病人抗凝全血样本 B血样（镜检每 100个白细胞中有 7个嗜酸性粒细 胞）、 患寄生虫感染病人抗凝全血样本 C血样（镜检每 100个白细胞中有 65个嗜 酸性粒细胞）各 20ul, 分别与 lml上述试剂混合， 20 ~ 30秒后用激光检测法检测 白细胞， 采用测定角度为 2 ~ 6度的前向低角度散射光测定细胞的大小信息， 并 采用测定角度为 8 ~ 20度的前向高角度散射光测定细胞的形态信息。
检测结果如图 8所示， 其中， 图 8-a是 A血样检测图、 图 8-b是 B血样检测图、 图 8-c是 C血样检测图。 由图 8-a可知， 对于正常人样本， 对比例试剂 A能够很好 的实现淋巴细胞、 单核细胞、 嗜酸性粒细胞、 中性粒细胞及嗜碱性粒细胞四类 的区分， 并且四类细胞的散点图之间界很明显。 由图 8-b、 图 8-c可知， 对于疾 病患者的全血样本中的嗜酸性粒细胞， 明显与中性粒细胞之间距离缩短（如图 8-b ), 甚至混合在一起（如图 8-c ), 未能实现四类细胞的正确分类。 由此可知， 对比试剂 A不能很好对疾病患者的全血样本中的嗜酸性粒细胞进行分类计数。
实施例 8
一种白细胞分类计数试剂和对白细胞分类的方法：
白细胞分类计数试剂含有如下组分：
月桂基酰胺丙基甜菜碱 3000mg/L
8-苯胺基 -1-萘磺酸 N¾盐 2500mg/L
癸基三甲基氯化铵 500mg/L
甲醇 20ml
甘氨酸 30mM;
用 NaOH将 pH调节到 4.0。
对白细胞分类的方法：
选取与前述对比实例 1中所用的八、 B、 C三份血样各 20ul, 分别与 lml的本 实施例 8中试剂混合， 20 ~ 30秒后用激光检测法检测白细胞，采用测定角度为 2 ~ 6度的前向低角度散射光测定细胞的大小信息， 并采用测定角度为 8 ~ 20度的前 向高角度散射光测定细胞的形态信息。
检测结果如图 9所示， 其中， 图 9-a是 A血样检测图、 图 9-b是 B血样检测图、 图 9-c是 C血样检测图。 由图 9可知， 本实施例中的试剂能有效的将各个样本的白 细胞均被分为淋巴细胞、 单核细胞、 嗜酸性粒细胞、 中性粒细胞及嗜碱性粒细 胞四类。 并且四种类型均有各自独立的散点图区域， 且位置固定， 聚类性明显，
其中嗜酸性粒细胞与中性粒细胞群区分明显， 尤其是 C血样， 嗜酸性粒细胞与 中性粒细胞有明显的界限， 其 Eos分类结果为 60%, 与镜检结果艮接近。
实施例 9
一种白细胞分类计数试剂和对白细胞分类的方法：
白细胞分类计数试剂含有如下组分：
月桂酰胺丙基羟磺基甜菜碱 4000mg/L
铬变酸 3000mg/L
月桂基三甲基溴化铵 100mg/L
乙醇 15ml/L
MES 15mM;
用 NaOH将 pH调节到 5.7。
对白细胞分类的方法：
随机选取 125例临床抗凝全血样本，按照下述方法对其淋巴细胞、单核细胞、 中性粒细胞和嗜酸性粒细胞进行分类计数。
每支血样中取 20 μΐ, 分别与 lml的本实施例 9中试剂混合， 20 ~ 30秒后用激 光检测法检测白细胞， 采用测定角度有为 2 ~ 6度的前向低角度散射光测定细胞 的大小信息，并采用测定角度为 8 ~ 20度的前向高角度散射光测定细胞的形态信 息， 获得淋巴细胞、 单核细胞、 嗜酸性粒细胞、 中性粒细胞及嗜碱性粒细胞四 类群的分类结果， 并和常规瑞氏 -姬姆萨染色法的手工镜检计数结果进行比较， 做出相关性分析。
检测结果如图 10所示， 其中， 图 10-a是淋巴细胞与常规瑞氏-姬姆萨染色法 的手工镜检计数结果的相关性关系， 图 10-b是单核细胞与常规瑞氏 -姬姆萨染色 法的手工镜检计数结果的相关性关系， 图 10-c是中性粒细胞与常规瑞氏-姬姆萨 染色法的手工镜检计数结果的相关性关系， 图 4-d是嗜酸性粒细胞与常规瑞氏- 姬姆萨染色法的手工镜检计数结果的相关性关系。 由图 10可知， 采用本发明实 施例白细胞分类计数试剂对淋巴细胞、 单核细胞、 嗜酸性粒细胞、 中性粒细胞 及嗜碱性粒细胞四类群的分类结果与常规瑞氏 -姬姆萨染色法的手工镜检计数 结果有良好的相关性。
实施例 10
一种白细胞分类计数试剂和对白细胞分类的方法：
白细胞分类计数试剂含有如下组分：
椰油酰胺丙基甜菜碱 2000mg/L
8-苯胺基 -1-萘磺酸 N¾盐 1000mg/L
癸基三甲基氯化铵 500mg/L
羧甲基 -β-环糊精 40g/L
磷酸 50mM;
用 NaOH将 pH调节到 6.0。
对白细胞分类的方法：
取 10例抗凝全血样本， 分别在离体第 1天（24小时） 内、 离体第 2天（48小 时内） 内， 使用前述对比实例 1中的试剂 A和本实施例 10试剂同时对血样进行测 试。 测定结果见表 1 , 其中第 1天为全血样本离体 1天内测试结果， 第二天为离体 2天内测试结果（48小时内）。 其中， Neu%表示中性粒细胞及嗜碱性粒细胞所占 比例、 Lym%表示淋巴细胞所占比例、 Mon%表示单核细胞所占比例、 £08%表 示嗜酸性粒细胞所占比例。 由表 1中测定结果的对比可以明显看出，采用对比例 试剂 A进行测定时， 随全血样本离体时间的延长， 各个白细胞亚类， 尤其是嗜 酸性粒细胞的分类结果明显变化，偏差比较大。 而当使用试剂 10的进行测定时， 随全血样本离体时间延长， 各个白细胞亚类的分类结果均无太大的变化， 可以 满足临床上对全血白细胞分类准确性的要求。
表 1
样 对比例试剂 A 试剂 10
本 第一天※ 第二天 第一天 第二天 编
Neu Lym Mon Eos Neu Lym Mon Eos Neu Lym Mon Eos Neu Lym Mon Eos 号
% % % % % % % % % % % % % % % %
1 56.1 29.1 6.1 8.2 47.8 31.5 5.9 14.3 55.7 29.8 6.0 8.5 54.3 31.3 5.5 8.9
2 44.7 44.9 4.9 5.5 38.9 47.2 3.0 10.9 43.1 44.0 5.5 6.5 43.1 46.3 4.3 5.7
3 62.3 30.4 5 1.6 53.9 34 4.2 7.5 62.5 31.2 4.5 1.8 61.0 33.0 4.0 2.0
4 56.7 36.7 5.4 0.7 48.2 39.8 4.8 6.6 55.6 38.4 5.0 1.0 55.0 38.4 4.8 1.8
5 55.8 35.2 6.2 2.8 49.2 44.6 5.7 0.5 54.6 35.7 6.1 3.2 54.1 37.3 5.6 2.6
6 63.5 26.7 4.9 4.4 64.5 27.7 4.9 2.4 63.1 27.0 5.1 4.8 61.8 28.2 5.0 5.0
7 56.7 35.7 5.6 2.0 50.6 35.8 5.0 8.6 57.2 36.0 4.6 1.9 55.9 36.4 4.5 2.9
8 63.1 22.7 10.4 3.8 57.6 28.1 8.9 5.4 63.6 23.0 9.6 3.3 61.9 27.8 7.1 2.7
9 46.4 41.9 8.8 1.9 43.6 44 5.9 6.1 46.0 43.5 8.5 2.0 45.1 45.6 7.0 2.3
10 64.8 28.0 5.9 1.3 59.4 28.7 5.0 6.9 64.4 27.6 6.1 1.5 62.3 30.7 5.7 1 平
57.0 33.1 51.3 36.1 56.5 33.6 55.4
均 6.32 3.22 5.33 6.92 6.1 3.45 35.5 5.35 3.49 1 3 7 4 8 2 5
值
※注： 第一天与前述新鲜全血样本同义
实施例 11
一种白细胞分类计数试剂和对白细胞分类的方法：
白细胞分类计数试剂含有如下组分：
两性离子表面活性剂： 癸基酰胺丙基甜菜碱 5000mg/L
阳离子表面活性剂： 辛基三甲基氯化铵 50mg/L
助溶剂： 乙二醇 10ml/L
pH调节緩冲剂： MES 20mM;
用 NaOH将 pH调节到 5.0。
对白细胞分类的方法：
对白细胞分类的方法： 参照实施例 1中的检测分类方法。 检测结果如图 11 所示。 图 11中， Neu表示中性粒细胞及嗜碱性粒细胞， Lym表示淋巴细胞， Mon 表示单核细胞， Eos表示嗜酸性粒细胞。
实施例 12
一种白细胞分类计数试剂和对白细胞分类的方法：
白细胞分类计数试剂含有如下组分：
两性离子表面活性剂： 十四烷基磺基甜菜碱 1000mg/L
阳离子表面活性剂： 辛基三甲基氯化铵 50mg/L
pH调节緩冲剂： MES 20mM;
用 NaOH将 pH调节到 5.0。
对白细胞分类的方法：
对白细胞分类的方法： 参照实施例 1中的检测分类方法。 检测结果如如图 12所示。 图 12中， Neu表示中性粒细胞及嗜碱性粒细胞， Lym表示淋巴细胞， Mon表示单核细胞， Eos表示嗜酸性粒细胞。 以上所述仅为本发明的较佳实施例而已， 并不用以限制本发明， 凡在本发 明的精神和原则之内所作的任何修改、 等同替换和改进等， 均应包括在本发明 的保护范围之内。
Claims
1. 一种白细胞分类计数试剂， 包括
( 1 )表面活性剂溶液， 所述表面活性剂溶液能够溶解红细胞， 使白细胞膜 部分损伤， 稳定嗜酸性粒细胞和老化细胞的细胞膜， 所述表面活性剂溶液含有 至少一种阳离子表面活性剂和至少一种两性离子表面活性剂；
所述两性 性表面活性剂， ι
式中 为（^18的烷基； R2和 R3各自独立选自 C1-3的烷基、 C1-3烷氧基或 d.3烷羟基； a为 0或 1 ; n为 0-6的整数； m为 1-5的整数； X选自羧基或磺酸 基；
或 /和所述两性离子表面活性剂选自胆汁酸衍生物型两性表面活性剂； ( 2 ) 能够将 pH值调整在 4 ~ 6范围内的緩冲剂。
2. 如权利要求 1 的白细胞分类计数试齐1 其特征在于， 所述 n为 3 , 和 / 或 R2和 R3各自独立选自羟乙基或甲基。
3. 如权利要求 1 的白细胞分类计数试齐 其特征在于， 所述 m为 3 , 和 / 或 X为磺酸基。
4. 如权利要求 1的白细胞分类计数试剂 其特征在于， 所述 R2和 R3为乙 氧基， 或所述 R2和 R3为羟丙基。
5. 如权利要求 1的白细胞分类计数试剂 其特征在于， 所述 n为 3 , 所述 X为磺酸基， 所述 R2和 R3各自独立选自羟丙基。
6. 如权利要求 1的白细胞分类计数试剂， 其特征在于， 所述两性离子表面 活性剂选自癸基酰胺丙基甜菜碱、 月桂基酰胺丙基甜菜碱、 椰油酰胺丙基甜菜 碱、 硬脂基酰胺丙基甜菜碱、 月桂基二羟乙基甜菜碱、 十四烷基二羟乙基甜菜 碱、 硬脂基二羟乙基甜菜碱、 月桂基磺基甜菜碱、 十四烷基磺基甜菜碱、 十六 烷基磺基甜菜碱、 十八烷基磺基甜菜碱、 十二烷基乙氧基磺基甜菜碱、 十四烷 基乙氧基磺基甜菜碱、 十六烷基乙氧基磺基甜菜碱、 十八烷基乙氧基磺基甜菜 碱、 十二烷基羟丙基磺基甜菜碱、 十四烷基羟丙基磺基甜菜碱、 十六烷基羟丙 基磺基甜菜碱、十八烷基羟丙基磺基甜菜碱、 月桂酰胺丙基羟丙基磺基甜菜碱、 椰油酰胺丙基羟丙基磺基甜菜碱、 十四烷基酰胺丙基羟丙基磺基甜菜碱、 十六 烷基酰胺丙基羟丙基磺基甜菜碱、 十八烷基酰胺丙基羟丙基磺基甜菜碱、 3-[(3- 胆烷酰胺丙基) -二甲基铵] -1-丙磺酸盐、 3-[(3-胆烷酰胺基丙基) -二甲基铵] -2-羟 -1-丙磺酸盐中的至少一种。
7. 如权利要求 1的白细胞分类计数试剂， 其特征在于， 所述阳离子表面活 性剂为具有通式 II结构的季铵盐阳离子表面活性剂：
8. 如权利要求 1的白细胞分类计数试剂， 其特征在于， 所述试剂还包括带 阴离子基团的有机化合物， 所述带阴离子基团的有机化合物优选 8-苯胺基 -1- 萘磺酸、 8-苯胺基 -1-萘磺酸盐、 6- (对甲苯胺基 )-2-萘磺酸、 6- (对甲苯胺基 )-2- 萘磺酸盐、 铬变酸、 萘磺酸中的至少一种。
9. 如权利要求 1的白细胞分类计数试剂， 其特征在于， 还包含助溶剂， 所 述助溶剂优选醇、 环糊精、 环糊精衍生物中的至少一种； 所述醇优选甲醇、 乙 醇、 乙二醇、 丙三醇、 苯氧乙醇、 异丙醇中的至少一种； 所述环糊精优选 α-环 糊精、 β-环糊精中的至少一种； 所述环糊精衍生物优选二甲基 -β-环糊精、 羟乙 基—β—环糊精、 羟丙基 -β-环糊精、 羧甲基 -β-环糊精中的至少一种。
10. 一种对白细胞分类计数的方法， 包括如下步骤：
将权利要求 1 ~ 9任一所述的白细胞分类计数试剂与待测血样混合反应，溶 解红细胞和血小板， 损伤白细胞；
检测所述白细胞的至少两种光信号， 得到所述白细胞的形态和大小信息； 根据所述白细胞的形态和大小信息， 将所述白细胞分为淋巴细胞、 单核细 胞、 嗜酸性粒细胞相对应的 3个集团以及与中性粒细胞和嗜碱性粒细胞相对应 的 1个集团共 4个集团， 并对所述各集团中所含细胞数目进行计数。
11. 如权利要求 10所述的用于白细胞分类的方法， 其特征在于， 采用测定 角度为 2 ~ 6度的低角度散射光来测定白细胞的大小信息， 采用测定角度为 6 ~ 20度的高角度散射光测定白细胞的形态信息。
12. 一种具有通式 I所示结构的两性离子表面活性剂在制备白细胞分类计 数试剂中的用途，
R2
R厂 (CO^[H) (C ) ^ (CH2)m-X- I
ί
式中 为（^18的烷基； R2和 R3各自独立选自 C 1-3的烷基、 C 1-3烷氧基或 C L3烷羟基； a为 0或 1; n为 0-6的整数； m为 1-5的整数； X选自羧基或磺 酸基。
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| CN104360050B (zh) * | 2014-09-22 | 2016-07-20 | 重庆医科大学附属儿童医院 | 一种淋巴细胞免疫分型的方法和试剂盒 |
| CN110231469A (zh) * | 2018-03-06 | 2019-09-13 | 深圳市帝迈生物技术有限公司 | 一种白细胞三分类溶血剂 |
| CN112424582A (zh) * | 2018-08-29 | 2021-02-26 | 深圳迈瑞生物医疗电子股份有限公司 | 血液样本检测的方法、血液样本检测仪和存储介质 |
| CN109580550A (zh) * | 2018-12-03 | 2019-04-05 | 迪瑞医疗科技股份有限公司 | 一种白细胞的分类处理方法及其装置 |
| CN109991052A (zh) * | 2019-03-12 | 2019-07-09 | 迪瑞医疗科技股份有限公司 | 一种用于白细胞、嗜碱性粒细胞计数和血红蛋白测定的溶血剂 |
| CN110031383A (zh) * | 2019-04-22 | 2019-07-19 | 深圳开立生物医疗科技股份有限公司 | 一种白细胞分类试剂及方法 |
| WO2021087730A1 (zh) * | 2019-11-05 | 2021-05-14 | 深圳迈瑞生物医疗电子股份有限公司 | 白细胞分类试剂及其使用方法 |
| CN111157431A (zh) * | 2020-01-14 | 2020-05-15 | 迪瑞医疗科技股份有限公司 | 用于有核红细胞、嗜碱性粒细胞分类的试剂 |
| CN114281283B (zh) * | 2020-09-27 | 2023-03-24 | 深圳市帝迈生物技术有限公司 | 散点图像的显示方法及样本分析设备、相关装置 |
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