WO2021087730A1 - 白细胞分类试剂及其使用方法 - Google Patents

白细胞分类试剂及其使用方法 Download PDF

Info

Publication number
WO2021087730A1
WO2021087730A1 PCT/CN2019/115631 CN2019115631W WO2021087730A1 WO 2021087730 A1 WO2021087730 A1 WO 2021087730A1 CN 2019115631 W CN2019115631 W CN 2019115631W WO 2021087730 A1 WO2021087730 A1 WO 2021087730A1
Authority
WO
WIPO (PCT)
Prior art keywords
blood cell
fluorescent dye
white blood
formula
solution
Prior art date
Application number
PCT/CN2019/115631
Other languages
English (en)
French (fr)
Inventor
陈庚文
张子千
李沿涛
Original Assignee
深圳迈瑞生物医疗电子股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳迈瑞生物医疗电子股份有限公司 filed Critical 深圳迈瑞生物医疗电子股份有限公司
Priority to PCT/CN2019/115631 priority Critical patent/WO2021087730A1/zh
Priority to CN201980101480.1A priority patent/CN114585921A/zh
Publication of WO2021087730A1 publication Critical patent/WO2021087730A1/zh

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • 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

Definitions

  • the invention relates to a white blood cell classification reagent and a method of use thereof, in particular to a reagent and method for automatically distinguishing each subgroup of white blood cells in the blood by an analyzer.
  • White blood cells in normal peripheral blood can generally be divided into five categories, namely lymphocytes, monocytes, neutrophils, eosinophils and basophils.
  • the analysis of white blood cell populations from blood samples can provide a lot of useful information for the clinical diagnosis of many diseases. For example, the proportion and number of various types of white blood cells in the blood may change when a disease occurs, and abnormal white blood cells such as abnormal lymphocytes and immature granulocytes may also appear. Therefore, classification and determination of different types of normal and abnormal white blood cells can obtain information about the incubation, initiation and development stages of the disease.
  • the instrument The analysis mainly uses a hemolytic agent to dissolve the red blood cells in the blood sample, causing the white blood cells to shrink differently to form a difference in size or scattered light intensity.
  • the fluorescence method has been widely used in blood analyzers in recent years.
  • US patent US6004816 discloses a method of labeling intracellular RNA with fluorescent dyes, thereby analyzing and counting leukocytes by side-scattered light and fluorescence intensity.
  • Chinese patent CN101349644 B discloses a method for staining leukocyte nucleic acid substances with cyanine dyes. Also analyze and count white blood cells by side-scattered fluorescence intensity, and count immature white blood cells at the same time.
  • the dyes used in the above methods are all cyanine dyes, which have a fatal shortcoming. They have poor light stability and are prone to fluorescence attenuation during laser irradiation, which affects the accuracy of detection.
  • This application provides a new reagent and method to distinguish and count leukocytes in the blood, and identify abnormal leukocytes at the same time.
  • the method should have good accuracy and precision, and low cost.
  • An embodiment provides a reagent for classification of leukocytes, the reagent comprising a fluorescent dye having the formula F:
  • A is selected from sulfur, selenium, tellurium, and oxygen;
  • R1, R2 and R3 are each independently selected from hydrogen and a substituted or unsubstituted C 1-20 alkyl group; the substituted alkyl group is any of the following groups Substitution: halogen, hydroxy, alkoxy, aldehyde, carbonyl, amino, carboxy, ester, amide, nitro or sulfonic acid group;
  • the X is selected from phosphate, sulfate, hydrogen sulfate, nitrate , Chloride anion, bromide anion, iodide anion or perchlorate.
  • R1, R2, and R3 in the formula F are each independently selected from hydrogen and C 1-14 substituted or unsubstituted alkyl.
  • R1, R2, and R3 in formula F are each independently selected from hydrogen and C 1-6 substituted or unsubstituted alkyl.
  • one of R1 and R2 in formula F is hydrogen.
  • R3 in formula F is hydrogen
  • a in formula F is oxygen, and formula F is:
  • the X is selected from chloride anion, bromide anion, iodide anion or perchlorate.
  • the substituted alkyl group is optionally substituted by the following groups: halogen, hydroxyl, amine, carboxy, or ester.
  • the compound is selected from F-1, F-2, F-3, F-4, F-5, and F-6:
  • the classification reagent further includes a red blood cell lysis agent.
  • the fluorescent dye is a separate fluorescent dye storage solution, or the fluorescent dye solution and the red blood cell lysis agent solution are combined for storage.
  • the fluorescent dye is a separate fluorescent dye storage solution, wherein the concentration of the fluorescent dye is 1-1000 mg/L, preferably 2-200 mg/L, more preferably 20-100 mg/L.
  • the fluorescent dye solution and the red blood cell lysis agent solution are combined and stored, wherein the concentration of the fluorescent dye is 0.02-20 mg/L, preferably 0.04-4 mg/L, more preferably 0.4-2 mg/L.
  • the red blood cell lysing agent comprises a cationic surfactant, a nonionic surfactant, an anionic surfactant, a buffer or any combination thereof;
  • the cationic surfactant is selected from the group consisting of dodecyltrimethyl Ammonium chloride, octyltrimethylammonium bromide, tetradecyltrimethylammonium chloride, tetradecyltrimethylammonium bromide, decyltrimethylammonium bromide and their mixtures, concentration range 300-800mg/L;
  • the nonionic surfactant is selected from the group consisting of long-chain fatty alcohol polyoxyethylene, alkylphenol polyoxyethylene ether, fatty acid polyoxyethylene ether, fatty amine polyoxyethylene ether and their mixtures, and the concentration range is 1-5 g/L
  • the buffer is selected from carboxylates, phosphates, citrates, Tris-HCl, 3-morpholinopropanesulfonic acid and their mixtures, which maintain the pH of the measurement system at 5-8;
  • the anionic surfactant is selected from the group consisting of dodecyl benzene sulfonic acid, sodium fatty alcohol acyl sulfate, sodium ethoxylated fatty acid methyl ester sulfonate, sodium secondary alkyl sulfonate, alcohol ether carboxylate and mixtures thereof, The concentration range is 0.1-2g/L.
  • it also contains alcohol in an appropriate ratio, preferably methanol.
  • Another embodiment provides a detection kit comprising the leukocyte classification reagent according to any one of claims 1-14.
  • Another embodiment also provides a method for classifying white blood cells, the method comprising:
  • the blood sample, the fluorescent dye of formula F, and the red blood cell lysis agent are mixed to form a mixture; wherein, the formula F is
  • A, R 1 , R 2 , R 3 and X are as defined in claim 1; measure at least one scattered light characteristic and at least one fluorescent characteristic of the mixture; classify and classify white blood cells according to the scattered light characteristic and fluorescence characteristic /Or count.
  • the fluorescent dye of formula F is: F-1, F-2, F-3, F-4, F-5, and F-6:
  • the mixing of the blood sample, the fluorescent dye of formula F, and the red blood cell lysis agent to form a mixture includes the following steps:
  • a fluorescent dye solution of formula F is added to the mixture.
  • the mixing of the blood sample, the fluorescent dye of formula F, and the red blood cell lysis agent to form a mixture includes the following steps: adding the red blood cell lysis agent solution and the fluorescent dye solution of formula F to the sample at the same time.
  • the white blood cell classification reagent of the present application contains an oxazine compound with the general formula F.
  • the reagent can not only distinguish and count white blood cells in the blood, but also identify abnormal white blood cells, with good accuracy and precision, and cost low.
  • Figure 1 is a graph showing the analysis results of a normal blood sample (No. 1 blood sample) using the composition of Example 2;
  • Figure 2 is a graph showing the analysis results of an abnormal blood sample (No. 2 blood sample) using the composition of Example 2;
  • Figure 3 is a graph showing the analysis results of a normal blood sample (No. 3 blood sample) using the composition of Example 3;
  • Figure 4 is a graph showing the analysis results of an abnormal blood sample (No. 4 blood sample) using the composition of Example 3;
  • Figure 5 is a graph showing the analysis results of a normal blood sample (No. 5 blood sample) using the composition of Example 4;
  • Figure 6 is a graph showing the analysis results of an abnormal blood sample (No. 6 blood sample) using the composition of Example 4;
  • Figure 7 is a graph showing the analysis result of a normal blood sample (No. 7 blood sample) using the composition of Example 5;
  • Figure 8 is a graph showing the analysis results of an abnormal blood sample (No. 8 blood sample) using the composition of Example 5;
  • Figure 9 is a graph showing the analysis results of a normal blood sample (No. 9 blood sample) using the composition of Example 6;
  • Figure 10 is a graph showing the analysis results of an abnormal blood sample (No. 10 blood sample) using the composition of Example 6;
  • Figure 11 is a graph showing the analysis results of a normal blood sample (blood sample No. 11) using the composition of Example 7;
  • Figure 12 is a graph showing the analysis results of an abnormal blood sample (No. 12 blood sample) using the composition of Example 7;
  • Fig. 13 is an accuracy test chart of a blood sample (blood sample No. 13) using the composition of Example 6.
  • blood or blood sample as used herein refers to a body fluid sample containing blood cells, such as peripheral blood, bone marrow fluid, and the like.
  • abnormal cells refers to cells that do not normally appear in the blood, including immature cells and abnormally mature cells, such as immature lymphocytes, immature myeloid cells (also referred to as Blast in this article), abnormal Mature lymphocytes, abnormal mature myeloid cells, nucleated red blood cells, etc.
  • alkyl as used herein includes straight chain alkyl groups and branched chain alkyl groups. References to a single alkyl group such as “propyl” refer only to straight chain alkyl groups, and reference to a single branched chain alkyl group such as “isopropyl” refers only to branched chain alkyl groups.
  • C 1-6 alkyl includes C 1-4 alkyl, C 1-3 alkyl, methyl, ethyl, n-propyl, isopropyl and tert-butyl. Similar rules apply to other groups used in this specification.
  • halogen as used herein includes fluorine, chlorine, bromine and iodine.
  • the fluorescent dye of the present invention can specifically bind to intracellular nucleic acid.
  • the dye is a red excitation fluorescent dye, which can be excited by a red laser emitted by a device that provides a red region laser, such as a red semiconductor laser, so that the reagent of the present invention can be used in a blood analyzer or a flow type using inexpensive equipment such as a semiconductor laser as a light source. Cytometer, etc.
  • the fluorescent dye has a structure represented by formula F
  • A is selected from sulfur, selenium, tellurium, and oxygen;
  • R 1 , R 2 and R 3 are each independently selected from hydrogen and a substituted or unsubstituted C 1-20 alkyl group; the substituted alkyl group is as follows Groups are optionally substituted: halogen, hydroxy, alkoxy, aldehyde, carbonyl, amino, carboxy, ester, amide, nitro or sulfonic acid group; said X is an anion,
  • It can be selected from phosphate, sulfate, hydrogen sulfate, nitrate, chloride, bromide, iodide or perchlorate.
  • R 1 , R 2 and R 3 in the general formula F are each independently selected from hydrogen and C 1-14 substituted or unsubstituted alkyl.
  • said R 1 , R 2 and R 3 are each independently selected from hydrogen and a substituted or unsubstituted C 1-10 alkyl group; more preferably, said R 1 , R 2 and R 3 Each is independently selected from hydrogen and C 1-6 substituted or unsubstituted alkyl.
  • one of R 1 and R 2 in the general formula F is hydrogen.
  • R 3 in formula F is hydrogen
  • one of R 1 and R 2 in the general formula F is hydrogen, and the other is a substituted or unsubstituted C 1-6 alkyl group.
  • This feature can be combined with the technical feature that R 3 is hydrogen. It constitutes a further preferred technical solution.
  • a in formula F is oxygen, and formula F is:
  • the X is selected from chloride anion, bromide anion, iodide anion or perchlorate.
  • the substituted alkyl group is optionally substituted by the following groups: halogen, hydroxyl, amino, carboxy, or ester.
  • the fluorescent dye has a structure selected from the group consisting of F-1, F-2, F-3, F-4, F-5, and F-6:
  • the fluorescent dye of the present invention can be dissolved in methanol, ethanol, dimethyl sulfoxide, ethylene glycol and stored as a storage solution, and can also be dissolved in other non-aqueous solvents.
  • the fluorescent dye can be a separate fluorescent dye storage solution.
  • the concentration of the fluorescent dye is 1-1000 mg/L, preferably 2-200 mg/L, more preferably 20-100 mg/L.
  • the present invention preferably uses ethylene glycol as a solvent, and optionally contains 5-10% methanol as a component of the diluent.
  • the fluorescent dye of the present invention can be formulated with a red blood cell lysis agent.
  • the concentration of the fluorescent dye may be 0.02-20 mg/L, preferably 0.04-4 mg/L, more preferably 0.4-2 mg/L.
  • the red blood cell lysis agent used may include a cationic surfactant, a nonionic surfactant, an anionic surfactant, a buffer, or any combination thereof.
  • Cationic surfactants and non-ionic surfactants can be used as reagents to dissolve red blood cells. Their combination can dissolve red blood cells in the blood sample, and appropriately destroy the membrane structure of each subgroup of white blood cells, so that each subgroup of white blood cells in the blood shrinks into an appropriate The size of the white blood cells promotes the different aggregation of the internal structure of white blood cells, resulting in differences in the characteristics of scattered light.
  • the cationic surfactant can be octyl trimethyl ammonium bromide (OTAB), decyl trimethyl ammonium bromide (DTAB), dodecyl trimethyl ammonium chloride (LTAC), tetradecyl trimethyl ammonium bromide Benzyl ammonium bromide (CTAB), tetradecyl trimethyl ammonium chloride (CTAC), etc., preferably LTAC, with a concentration of 300-800 mg/L.
  • LTAC octyl trimethyl ammonium bromide
  • DTAB decyl trimethyl ammonium bromide
  • LTAC dodecyl trimethyl ammonium chloride
  • CTAB tetradecyl trimethyl ammonium bromide
  • CTAC tetradecyl trimethyl ammonium chloride
  • 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
  • the ionic surfactant is polyoxyethylene-2,3-dodecyl ether (Brij35) with a concentration of 1-5g/L.
  • the anionic surfactant is 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 use concentration is 0.1-2g /L.
  • the reagent of the present application may contain a buffer solution that maintains the pH and appropriately dilutes the blood sample.
  • the buffer keeps the pH in a constant range, thereby stabilizing the staining effect of each subgroup of leukocytes, and the use concentration of the buffer is in the range of 0.01-200mM.
  • 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 appropriate pH in the reagent of the present invention varies with the specific fluorescent dye selected, and is generally within the range of pH 5.0-9.0, and the preferred pH is 6.0-8.0. In the present invention, Tris-HCl or MOPS buffer system is preferred.
  • the red blood cell lysing agent of the present invention may optionally contain alcohol, such as methanol, in an appropriate ratio to promote the aggregation of the internal structure of white blood cells.
  • the present invention also provides a kit for distinguishing and counting normal white blood cells in the blood, and at the same time identifying abnormal white blood cells such as abnormal lymphocytes and immature granulocytes in the blood, the kit comprising a white blood cell classification reagent,
  • the white blood cell classification reagent includes a fluorescent dye having the following structure:
  • the fluorescent dye has the structure shown in formula F:
  • A is selected from sulfur, selenium, tellurium, and oxygen;
  • R1, R2 and R3 are each independently selected from hydrogen and C1-20 substituted or unsubstituted alkyl; the substituted alkyl is optionally substituted by the following groups :Halogen, hydroxyl, alkoxy, aldehyde, carbonyl, amino, carboxy, ester, amide, nitro or sulfonic acid group;
  • said X is an anion, which can be selected from phosphate, sulfate, hydrogen sulfate , Nitrate, chloride, bromide, iodide or perchlorate.
  • the fluorescent dye has a structure selected from the group consisting of F-1, F-2, F-3, F-4, F-5, and F-6:
  • the white blood cell sorting reagent further comprises the red blood cell lysis agent described herein.
  • the fluorescent dye exists as a storage solution and stored in a separate container.
  • the fluorescent dye is formulated as a mixed reagent solution together with the red blood cell lysis agent.
  • the kit preferably contains fluorescent dyes suitable for storage in compartments that are sealed and held in at least one container.
  • the kit may also include other white blood cell sorting reagents required for sorting white blood cells in the blood and instructions for a method for measuring white blood cells.
  • the kit may also include a control sample or a series of control samples that can be measured and compared with the test sample.
  • the components of the kit can be packaged in a single container, and the different containers are all in a separate package together with instructions.
  • the kit is used to classify and/or count various types of white blood cells in the blood.
  • this application also provides a method for classifying and/or counting leukocytes in blood.
  • the blood sample is mixed with the reagent of the present invention, and then at least one scattered light characteristic and at least one fluorescent characteristic of the sample are measured, and the sample is classified according to the scattered light characteristic and the fluorescent characteristic. And/or count.
  • the blood sample used in the method of this application may be whole blood or component blood.
  • the blood sample can first be mixed with the red blood cell lysis agent and buffer, so that the red blood cells are lysed, and the white blood cells of each subgroup shrink to different degrees. This step also forms small holes in the cell membrane of the leukocytes to be measured, which are sufficient to allow the fluorescent dye molecules to pass through the cell membrane.
  • a fluorescent dye storage solution is added so that the white blood cells are fluorescently labeled.
  • the total volume of the blood sample and the reagent of the application should ensure that a sufficient cell concentration passes 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, as long as the dilution meets the requirements of actual use. Such adjustments are within the scope of those skilled in the art.
  • the sample mixture can be incubated in an incubation tank, and the incubation time does not exceed 40 seconds, preferably 24 seconds; the incubation temperature can be any suitable temperature, such as 42°C. Subsequently, the diluted and stained blood sample is introduced into the flow chamber, and at least one scattered light characteristic and at least one fluorescence characteristic of the white blood cells are detected and analyzed through the detection channel of a blood analyzer or a similar flow cytometer.
  • the scattered light in this application refers to the scattered light that can be detected by a commercially available blood analyzer or similar flow cytometer.
  • Such scattered light includes, but is not limited to, side scattered light, forward low-angle scattered light (receiving light angle of about 0-5 degrees), and forward high-angle scattered light (receiving light angle of about 5-20 degrees).
  • the scattered light having such an angle reflects the information of the size or internal structure of white blood cells, and therefore is used as the scattered light of the present invention. Preference is given to side-scattered light.
  • Fluorescent dyes that bind to nucleic acids in cells emit fluorescence. Fluorescence characteristics are parameters that reflect the amount of fluorescent dyes in cells in a blood sample. Due to the different intracellular metabolic activities of different subgroups, which result in differences in nucleic acid content, the fluorescence characteristics of different subgroups of white blood cells are different in some aspects.
  • the red wavelength region laser emitted by a red semiconductor laser is preferably used as the light source for detection. There is no special restriction on the red wavelength light source, as long as it can emit red light near the excitation wavelength of the selected fluorescent dye, for example, light with a wavelength of about 600-680 nm.
  • a He-Ne laser, a semiconductor laser in the red region, etc. can be used. It is preferable to use a semiconductor laser because it has a lower cost compared to other lasers and a smaller volume, which can reduce the cost and volume of the device.
  • the scattered light characteristics and fluorescence characteristics are used to identify the white blood cells of each subgroup, as well as the abnormal information such as possible abnormal leaching, immature granulocytes, etc., and perform relevant classification and counting of the scattered points of each cluster to calculate the percentage of white blood cells in each subgroup.
  • Scattered light reflects the degree of granularity inside the cell.
  • the degree of granularity inside the cell is roughly as follows: eosinophils have two lobed nuclei and many acid dye-stained fragile particles inside; neutrophil nuclei (lobed or rod-shaped) and inside There are more granules; monocytes have single large nuclei with fewer internal particles; LYM cells have almost no large nuclei. Therefore, the order of the scattered light intensity characteristics of various types of white blood cells under the same conditions is EO (eosinophils)> NEUT (neutrophils)> MONO (monocytes)> LYM (lymphocytes).
  • the oxazine compounds described in this application are synthesized by the following method: an azo compound formed by an aromatic amine or its derivative is condensed with 8-hydroxyjulonidine in acid-containing DMF to prepare the target oxazine dye .
  • the synthesis method has a simple process and a high conversion rate. More specifically, the synthetic route of compound F of the general formula of the present application is:
  • the preparation method of the compound of general formula F represented by the above route includes the following steps:
  • An exemplary fluorescent dye is synthesized in the following manner, wherein the dye has the following structure:
  • the preparation method includes the following steps:
  • the chlorinated p-nitrodiazobenzene and the compound of 1-I are reacted at 25 ⁇ 35°C for 0.5 ⁇ 2 hours according to the molar ratio of 1:1. After the reaction is completed, a brick red color is obtained after suction filtration and washing operation.
  • the crude solid powder product yields the compound of formula 1-II with a yield of 95%.
  • the intermediate 1-II and 8-hydroxyjulolin prepared by the above reaction (1) were added to a round bottom flask containing DMF, and 1 mL of perchloric acid solution was dropped. After the dropwise addition was completed, the system was stirred for 2.5 hours and then the reaction was stopped, and the target probe compound F-1 with metallic luster dark blue needle-like crystals was obtained after separation and purification by column chromatography, with a yield of 78.2%.
  • the process of testing samples is automatically carried out in Mindray's BC-6800 instrument. Set the aspiration volume to 20 microliters, the hemolytic solution injection volume to 1ml, the fluorescent dye storage solution injection volume to 20 microliters, and the blood sample to be anticoagulated. Processed blood.
  • test results of a normal blood sample (blood sample No. 1) is shown in Figure 1, which shows that the application can realize the classification and identification of 4 subgroups of white blood cells.
  • the four clusters marked on the lower left corner are lymphocytes and monocytes. , Neutrophils and Eosinophils.
  • the test results of the abnormal blood sample (No.
  • FIG 2 blood sample are shown in Figure 2, which shows that this application can not only realize the effective classification of the subgroups of white blood cells (the upper four clusters marked in the lower left corner of Figure 2 are lymphocytes). Nuclear cells, neutrophils and eosinophils), and can effectively distinguish abnormal white blood cells, including immature granulocytes, abnormal lymphocytes (the upper left box in Figure 2 is abnormal lymphocytes, the upper right side of the figure The box is naive granulocytes).
  • the process of testing samples is automatically carried out in Mindray's BC-6800 instrument. Set the aspiration volume to 20 microliters, the hemolytic solution injection volume to 1ml, the fluorescent dye storage solution injection volume to 20 microliters, and the blood sample to be anticoagulated. Processed blood.
  • red blood cell lysing agent ie, hemolytic agent
  • 20 microliters of blood treated with anticoagulant 20 microliters of fluorescent dye stock solution is added immediately, and mixed and incubated in an incubator at 42°C for 24 seconds ,
  • the intensity of side scattered light and the intensity of side fluorescence of the measurement pattern are measured.
  • the test result of a normal blood sample (No. 3 blood sample) is shown in Figure 3, which shows that the application can realize the classification and identification of 4 subgroups of white blood cells.
  • the four clusters of particles on the upper side marked in the lower left corner are lymphocytes and monocytes. , Neutrophils and Eosinophils.
  • the test result of the abnormal blood sample No.
  • FIG. 4 blood sample is shown in Figure 4, indicating that the application can not only effectively classify the subgroups of white blood cells (the four clusters marked on the upper left corner of Figure 4 are lymphocytes and mononuclear cells). Cells, neutrophils and eosinophils), and can effectively distinguish abnormal white blood cells, including immature granulocytes, abnormal lymphocytes (the upper left box in Figure 4 is abnormal lymphocytes, the upper right box in the figure For immature granulocytes).
  • the process of testing samples is automatically carried out in Mindray's BC-6800 instrument. Set the aspiration volume to 20 microliters, the hemolytic solution injection volume to 1ml, the fluorescent dye storage solution injection volume to 20 microliters, and the blood sample to be anticoagulated. Processed blood.
  • Example 2 For the specific steps of forming the sample for measurement.
  • the intensity of side scattered light and the intensity of side fluorescence of the measurement pattern are measured.
  • the test result of a normal blood sample (No. 5 blood sample) is shown in Figure 5, which shows that the application can realize the classification and identification of 4 subgroups of white blood cells.
  • the four clusters of particles on the upper side marked in the lower left corner are lymphocytes and monocytes. , Neutrophils and Eosinophils.
  • the test result of the abnormal blood sample (No. 6 blood sample) is shown in Figure 6, which shows that this application can not only realize the effective classification of the subgroups of white blood cells (the four clusters marked on the upper left corner of Figure 6 are lymphocytes and mononuclear cells).
  • the process of testing samples is automatically carried out in Mindray's BC-6800 instrument. Set the aspiration volume to 20 microliters, the hemolytic solution injection volume to 1ml, the fluorescent dye storage solution injection volume to 20 microliters, and the blood sample to be anticoagulated. Processed blood.
  • Example 2 For the specific steps of forming the sample for measurement.
  • the intensity of side scattered light and the intensity of side fluorescence of the measurement pattern are measured.
  • the test result of the normal blood sample (No. 7 blood sample) is shown in Figure 7, which shows that the application can realize the classification and identification of the 4 subgroups of white blood cells.
  • the four clusters of particles on the upper side marked in the lower left corner are lymphocytes and monocytes. , Neutrophils and Eosinophils.
  • the test results of the abnormal blood sample (No. 8 blood sample) are shown in Figure 8, which shows that this application can not only realize the effective classification of the subgroups of white blood cells (the four clusters marked on the upper left corner of Figure 8 are lymphocytes and mononuclear cells).
  • Fluorescent dye and red blood cell lysing agent are prepared together as a mixed reagent.
  • the dye is pre-dissolved in methanol, and then mixed with ethylene glycol to prepare a dye solution. Then the rest of the materials are added according to the formula. After adjusting the pH value, add the previously prepared dye solution. Just mix evenly, the formula is as follows:
  • the process of testing samples is automatically carried out in Mindray's BC-6800 instrument. Set the aspiration volume to 20 microliters, the hemolytic solution injection volume to 1ml, the fluorescent dye storage solution injection volume to 20 microliters, and the blood sample to be anticoagulated. Processed blood.
  • test results of a normal blood sample (blood sample No. 9) is shown in Figure 9, which shows that the application can realize the classification and identification of the 4 subgroups of white blood cells.
  • the four clusters marked on the upper left corner are lymphocytes, monocytes, and monocytes. Neutrophils and eosinophils.
  • the test results of the abnormal blood sample (blood sample No.
  • FIG 10 10) are shown in Figure 10, indicating that the application can not only realize the effective classification of the subgroups of white blood cells, but also effectively distinguish abnormal white blood cells, including immature granulocytes and abnormal lymphocytes ( In Figure 10, the upper left box is abnormal lymphocytes, and the upper right box in the figure is immature granulocytes).
  • the process of testing samples is automatically carried out in Mindray's BC-6800 instrument. Set the aspiration volume to 20 microliters, the hemolytic solution injection volume to 1ml, the fluorescent dye storage solution injection volume to 20 microliters, and the blood sample to be anticoagulated. Processed blood.
  • Example 2 for the specific steps of forming the sample for measurement.
  • the intensity of side scattered light and the intensity of side fluorescence of the measurement pattern were measured.
  • the test result of a normal blood sample (blood sample No. 11) is shown in Figure 11, which shows that the application can realize the classification and identification of 4 subgroups of white blood cells.
  • the four clusters marked on the upper left corner are lymphocytes, monocytes, and monocytes. Neutrophils and eosinophils.
  • the test result of the abnormal blood sample No.
  • Figure 12 blood sample
  • Figure 12 shows that the application can not only realize the effective classification of the subgroups of white blood cells, but also can effectively distinguish abnormal white blood cells, including immature granulocytes and abnormal lymphocytes
  • the upper left box is abnormal lymphocytes
  • the upper right box in the figure is immature granulocytes
  • Example 6 Configure the mixed reagents according to Example 6. After emptying the matching staining solution and hemolytic agent in the Mindray BC-6800DIFF channel, replace with the newly prepared reagents. Set the measurement mode to CD, and the test sample is a random blood sample (blood sample No. 13). After the test is completed, use the BC-6800 blood cell analyzer (normal matching reagent) to retest the above sample (blood sample No. 13), and calculate the correlation of the test results of the random blood sample between the two reagents. As shown in Figure 13, it can be seen from the test results that, in terms of the performance of white blood cell classification, the test result of the mixed reagent has a high correlation with the test result of BC-6800, indicating that the mixed reagent can accurately identify white blood cells. Classification and counting.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

一种白细胞分类试剂，包含具有通式F的噁嗪类化合物，该试剂对白细胞进行分析时不仅能区分和计数血液中的白细胞，同时识别异常白细胞，具有良好的准确度和精密度，并且成本低廉。

Description

白细胞分类试剂及其使用方法 技术领域
本发明涉及白细胞分类试剂和其使用方法，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和方法。
背景技术
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。
传统血液样本分析主要是将血液样本涂片，用特异染料进行细胞内部结构的着色，并于显微镜下观察，计数白细胞各亚群的种类和数目。这种方法费时，而且操作人员的个体识别差异产生不同的结果，因此发展了利用流式技术进行白细胞计数来自动分析白细胞亚群的方法。
目前对血液样本中的白细胞进行分类的方法很多，包括采用射频、高低角度散射，阻抗，侧向散射、侧向荧光等分别组合的方法，或采用几步的方法实现对白细胞的分类，目前仪器分析主要采用溶血剂将血液样本中的红细胞溶解，使得白细胞产生不同皱缩而形成大小或散射光强度的差异。荧光方法作为一种高灵敏度的方法，近年来被广泛的使用于血液分析仪中。
美国专利US6004816公开了一种采用荧光染料标记细胞内RNA的方法，从而通过侧向散射光喝荧光强度来分析和计数白细胞，中国专利CN101349644 B公开了一种采用菁染料染色白细胞核酸物质的方法，同样通过侧向散射荧光强度来分析和计数白细胞，并同时计数未成熟白细胞。以上方法中所使用的染料都为菁类染料，存在一个致命的缺点，其光稳定性较差，容易在激光照射过程中产生荧光衰减，从而影响检测的准确度。
发明内容
本申请提供一种新的试剂和方法来区分和计数血液中的白细胞，同时识别异常白细胞，该方法应具有良好的准确度和精密度，并且成本低廉。
一个实施例中提供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 PCTCN2019115631-appb-000001
其中，A选自硫，硒，碲、氧元素；R1、R2和R3各自独立地选自氢和C 1-20的取代或未取代烷基；所述的取代烷基由下述基团任意取代：卤素、羟基、烷氧基、醛基、羰基、氨基、羧基、酯基、酰胺基、硝基或磺酸基；所述的X选自磷酸根，硫酸根，硫酸氢根，硝酸根，氯负离子，溴负离子，碘负离子或高氯酸根。
一个实施例中，所述式F中所述的R1、R2和R3各自独立地选自氢和C 1-14的取代或未取代烷基。
一个实施例中，式F中所述的R1、R2和R3各自独立地选自氢和C 1-6的取代或未取代烷基。
一个实施例中，式F中所述的R1和R2其中之一是氢。
一个实施例中，式F中所述的R3是氢。
一个实施例中，式F中所述的A是氧，式F为：
Figure PCTCN2019115631-appb-000002
一个实施例中，所述的X选自氯负离子，溴负离子，碘负离子或高氯酸根。
一个实施例中，所述的取代烷基由下述基团任意取代：卤素、羟基、胺基、羧基、或酯基。
一个实施例中，所述化合物选自F-1、F-2、F-3、F-4、F-5、和F-6：
Figure PCTCN2019115631-appb-000003
Figure PCTCN2019115631-appb-000004
一个实施例中，所述分类试剂还包括红细胞溶解剂。
一个实施例中，所述荧光染料为单独的荧光染料储存液，或所述荧光染料溶液与红细胞溶解剂溶液合并储存。
一个实施例中，所述荧光染料为单独的荧光染料储存液，其中所述荧光染料的浓度为1-1000mg/L，优选2-200mg/L，更优选20-100mg/L。
一个实施例中，所述荧光染料溶液与红细胞溶解剂溶液合并储存，其中所述荧光染料的浓度为0.02-20mg/L，优选0.04-4mg/L，更优选0.4-2mg/L。
一个实施例中，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、缓冲剂或它们的任何组合；所述阳离子表面活性剂选自十二烷基三甲基氯化铵、辛基三甲基溴化铵、十四烷基三甲基氯化铵、十四烷基三甲基溴化铵、癸基三甲基溴化铵和它们的混合物，浓度范围为300-800mg/L；
所述非离子表面活性剂选自长链脂肪醇聚氧乙烯、烷基酚聚氧乙烯醚、脂肪酸聚氧乙烯醚、脂肪胺聚氧乙烯醚和它们的混合物，浓度范围为1-5g/L；所述缓冲剂选自羧酸盐类、磷酸盐类、柠檬酸盐类、Tris-HCl、3-吗啉代丙磺酸和它们的混合物，它们将测定体系的pH保持在5-8；所述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、醇醚羧酸盐和它们的混合物，浓度范围为0.1-2g/L。
一个实施例中，其中还包含适当比例的醇，优选甲醇。
另一个实施例中提供一种检测试剂盒，所述试剂盒包含权利要求1-14中任一项所述的白细胞分类试剂。
另一个实施例中还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 PCTCN2019115631-appb-000005
其中，A、R 1、R 2、R 3和X如权利要求1中定义；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；根据散射光特性和荧光特性将白细胞分类和/或计数。
另一个实施例中，所述式F的荧光染料为：F-1、F-2、F-3、F-4、F-5、和F-6：
Figure PCTCN2019115631-appb-000006
另一个实施例中，其中所述将血液样品、具有式F的荧光染料、和红细胞溶解剂混合形成混合物，包括以下步骤：
将血液样品与红细胞溶解剂溶液混合形成混合物；
将具有式F的荧光染料溶液加入所述混合物中。
另一个实施例中，其中所述将血液样品、具有式F的荧光染料、和红细胞溶解剂混合形成混合物，包括以下步骤：将红细胞溶解剂溶液和具有式F的荧光染料溶液同时加入样品中。
本申请的白细胞分类试剂包含具有通式F的噁嗪类化合物，该试剂对白细胞进行分析时不仅能区分和计数血液中的白细胞，同时识别异常白细胞，具有良好的准确度和精密度，并且成本低廉。
附图说明
图1为使用实施例2的组合物对正常血液样本(1号血液样本)的分析结果图；
图2为使用实施例2的组合物对异常血液样本(2号血液样本)的分析结果图；
图3为使用实施例3的组合物对正常血液样本(3号血液样本)的分析结果图；
图4为使用实施例3的组合物对异常血液样本(4号血液样本)的分析结果图；
图5为使用实施例4的组合物对正常血液样本(5号血液样本)的分析结果图；
图6为使用实施例4的组合物对异常血液样本(6号血液样本)的分析结果图；
图7为使用实施例5的组合物对正常血液样本(7号血液样本)的分析结果图；
图8为使用实施例5的组合物对异常血液样本(8号血液样本)的分析结果图；
图9为使用实施例6的组合物对正常血液样本(9号血液样本)的分析结果图；
图10为使用实施例6的组合物对异常血液样本(10号血液样本)的分析结果图；
图11为使用实施例7的组合物对正常血液样本(11号血液样本)的分析结果图；
图12为使用实施例7的组合物对异常血液样本(12号血液样本)的分析结果图；
图13为使用实施例6的组合物对血液样本(13号血液样本)的准确性测试图。
具体实施方式
除另有说明外，本文中使用的术语具有以下含义。
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。
本文中使用的术语“异常细胞”是指通常不出现在血液中的细胞，包括不成熟细胞和异常成熟细胞，例如不成熟淋巴细胞、不成熟髓细胞(在本文中也称为Blast)、异常成熟淋巴细胞、异常成熟髓细胞、有核红细胞等。
本文中使用的术语“烷基”包括直链烷基和支链烷基。如提及单个烷基如“丙基”，则只特指直链烷基，如提及单个支链烷基如“异丙基”，则只特指支链烷基。例如，“C 1-6烷基”包括C 1-4烷基、C 1-3烷基、甲基、乙基、正丙基、异丙基和叔丁基。类似的规则也适用于本说明书中使用的其它基团。
本文中使用的术语“卤素”包括氟、氯、溴和碘。
荧光染料
本发明的荧光染料能够特异性结合细胞内核酸。优选所述染料为红色激发荧光染料，能够被红色半导体激光器等提供红色区域激光的器件发出的红色激光所激发，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。
在一个实施方案中，荧光染料具有式F所示的结构
Figure PCTCN2019115631-appb-000007
其中，A选自硫，硒，碲、氧元素；R 1、R 2和R 3各自独立地选自氢和C 1-20的取代或未取代烷基；所述的取代烷基由下述基团任意取代：卤素、羟基、烷氧基、醛基、羰基、氨基、羧基、酯基、酰胺基、硝基或磺酸基；所述的X是阴离子，
可选自磷酸根，硫酸根，硫酸氢根，硝酸根，氯负离子，溴负离子，碘负离子或高氯酸根。
一个实施例中，所述的通式F中的R 1、R 2和R 3各自独立地选自氢和C 1-14的取代或未取代烷基。其中作为优选地，所述的R 1、R 2和R 3各自独立地选自氢和C 1-10的取代或未取代烷基；更优选地，所述的R 1、R 2和R 3各自独立地选自氢和C 1-6的取代或未取代烷基。
一个实施例中，所述通式F中的R 1和R 2其中之一是氢。
一个实施例中，式F中所述的R 3是氢。
一个实施例中，所述通式F中的R 1和R 2其中之一是氢，另一个是C 1-6的取代或未取代烷基，该特征可以结合R 3是氢的技术特征，构成进一步优选的技术方案。
一个实施例中，式F中所述的A是氧，式F为：
Figure PCTCN2019115631-appb-000008
一个实施例中，所述的X选自氯负离子，溴负离子，碘负离子或高氯酸根。
一个实施例中，所述的取代烷基由下述基团任意取代：卤素、羟基、胺基、羧基、或 酯基。
一个实施例中，优选荧光染料具有选自F-1、F-2、F-3、F-4、F-5、和F-6式的结构：
Figure PCTCN2019115631-appb-000009
本发明的荧光染料可以溶解在甲醇、乙醇、二甲亚砜、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。所述荧光染料可为单独的荧光染料储存液。其中，所述荧光染料的浓度为1-1000mg/L，优选2-200mg/L，更优选20-100mg/L。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。
或者，本发明的荧光染料可与红细胞溶解剂配制在一起。其中，荧光染料浓度可为0.02-20mg/L，优选0.04-4mg/L,更优选0.4-2mg/L。
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、缓冲剂或它们的任何组合。
可采用阳离子表面活性剂和非离子表面活性剂作为溶解红细胞的试剂，它们的组合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溴化辛基三甲基铵(OTAB)、溴化癸基三甲基铵(DTAB)、氯化十二 烷基三甲基铵(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 5.0-9.0范围内，优选的pH是6.0-8.0。本发明优选Tris-HCl或MOPS缓冲体系。
本发明的红细胞溶解剂中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。
检测试剂盒
在另一个方面，本发明还提供用于能够区分和计数血液中的正常白细胞，同时识别血液中的异常白细胞如异常淋巴细胞和未成熟粒细胞的试剂盒，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以下结构的荧光染料：
荧光染料具有式F所示的结构：
Figure PCTCN2019115631-appb-000010
其中，A选自硫，硒，碲、氧元素；R1、R2和R3各自独立地选自氢和C1-20的取代或未取代烷基；所述的取代烷基由下述基团任意取代：卤素、羟基、烷氧基、醛基、羰基、氨基、羧基、酯基、酰胺基、硝基或磺酸基；所述的X是阴离子，可选自磷酸根，硫酸根，硫酸氢根，硝酸根，氯负离子，溴负离子，碘负离子或高氯酸根。
优选荧光染料具有选自F-1、F-2、F-3、F-4、F-5、和F-6式的结构：
Figure PCTCN2019115631-appb-000011
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。
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。
使用方法
在另一个方面，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，通过将血液样本与本发明的试剂混合，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。可使血液样品首先与红细胞溶解剂和缓冲剂混合，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。
样品混合物可在孵育池中进行温育，温育时间不超过40秒，优选24秒；温育温度可为任何合适的温度，例如42℃。随后稀释并染色的血液样品导入流动室中，通过血液分析仪或类似的流式细胞仪的检测通道，检测和分析白细胞的至少一种散射光特性和至少一种荧光特性。
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。本发明优选采用红色半导体激光器发射的红色波长区域激光为检测的光源。对所述红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红色光，例如约波长600-680nm的光。例如可以使用He-Ne激光器、红色区域的半导体激光器等。优选使用半导体激光器，因为它相对于其它激光器成本较低，并且体积较小，这样可降低设备的成本和体积。
利用散射光特性和荧光特性识别各亚群白细胞，以及可能的异淋、幼稚粒细胞等异常信息，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)。
染料的合成
本申请中所述及的噁嗪类化合物通过下述方法合成：使用芳胺或其衍生物形成的偶氮化合物与8-羟基久洛尼定在含酸DMF中缩合，制备得到目标噁嗪染料。该合成方法工艺 简洁，转化率高。更为具体的，本申请通式化合物F合成路线为：
Figure PCTCN2019115631-appb-000012
上述路线所表示的通式F的化合物的制备方法包括如下步骤：
(1)盐酸酸化体系中，氯化对硝基重氮苯与式I的化合物按照摩尔比1:1在25～35℃条件下反应0.5～2小时，制备式II化合物；
(2)式II化合物与8-羟基久洛里啶按照摩尔比1:1在酸性DMF中于135～145℃条件下反应2～4小时制备通式F的化合物。
实施例1
示例性的荧光染料通过以下方式合成，其中所述染料具有以下结构：
Figure PCTCN2019115631-appb-000013
更为具体的，本申请化合物F-1合成路线为：
Figure PCTCN2019115631-appb-000014
制备方法包括以下步骤：
(1)中间体1-II的合成
盐酸酸化体系中，氯化对硝基重氮苯与1-I的化合物按照摩尔比1:1在25～35℃条件下反应0.5～2小时，反应完毕，经过抽滤洗涤操作后得到砖红色固体粉末粗产品得式1-II的化合物，收率95％。
(2)化合物F-1的合成
将上述反应(1)制备得到的中间体1-II与8-羟基久洛里定加入到含有DMF的圆底烧瓶中，滴入1mL高氯酸溶液。滴加完毕，体系搅拌2.5h后停止反应，经柱色谱分离提纯得具金属光泽的深蓝色针状晶体目标探针化合物F-1，收率78.2％。
1H NMR(400MHz,DMSO-d6)δ9.40(d,J＝5.3Hz,1H),8.64(d,J＝8.0Hz,1H),8.42(d,J＝8.3Hz,1H),7.88(t,J＝7.6Hz,1H),7.79(t,J＝7.6Hz,1H),7.38(s,1H),6.93(s,1H),5.08(t,J＝4.9Hz,1H),3.82 3.67(m,4H),3.58(d,J＝4.7Hz,4H),2.83(dt,J＝12.2,5.9Hz,4H),1.98(d,J＝4.8Hz,4H)。
实施例2
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F-1 50mg
乙二醇      950ml
甲醇    50ml
B：红细胞溶解剂溶液
Figure PCTCN2019115631-appb-000015
最后将溶液的pH调整为7.0。
各种血液样品的测定和结果：
测试样本的过程在迈瑞BC-6800仪器中自动进行，设置吸样量为20微升，溶血剂溶液进样量为1ml，荧光染料存储液的进样量为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42℃下的孵育池中混匀孵育24秒，形成测定用试样。测定测定用式样的侧向散射光强度和侧向荧光强度。对正常血液样本(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，左下角标注的上方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。对异常血液样本(2号血液样本)的测试结果如图2所示，表明本申请不仅能够实现白细胞各亚群的有效分类(图2左下角标注的上方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)，而且可有效的区分异常白细胞，包括幼稚粒细胞，异常淋巴细胞(图2中上方左侧方框为异常淋巴细胞，图中上方右侧方框为幼稚粒细胞)。
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F-2 100mg
乙二醇      950ml
甲醇        50ml
B：红细胞溶解剂溶液
Figure PCTCN2019115631-appb-000016
最后将溶液的pH调整为7.5。
各种血液样品的测定和结果：
测试样本的过程在迈瑞BC-6800仪器中自动进行，设置吸样量为20微升，溶血剂溶液进样量为1ml，荧光染料存储液的进样量为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(即溶血剂)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42℃下的孵育池中混匀孵育24秒，形成测定用试样。测定测定用式样的侧向散射光强度和侧向荧光强度。对正常血液样本(3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，左下角标注的上方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。对异常血液样本(4号血液样本)的测试结果如图4所示，表明本申请不仅能够实现白细胞各亚群的有效分类(图4左下角标注上方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)，而且可有效的区分异常白细胞，包括幼稚粒细胞，异常淋巴细胞(图4中上方左侧方框为异常淋巴细胞，图中上方右侧方框为幼稚粒细胞)。
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F-3 20mg
乙二醇      950ml
甲醇        50ml
B：红细胞溶解剂溶液
Figure PCTCN2019115631-appb-000017
Figure PCTCN2019115631-appb-000018
最后将溶液的pH调整为7.0。
各种血液样品的测定和结果：
测试样本的过程在迈瑞BC-6800仪器中自动进行，设置吸样量为20微升，溶血剂溶液进样量为1ml，荧光染料存储液的进样量为20微升，血样为经过抗凝处理的血液。
形成测定用试样的具体步骤可参考实施例2。测定测定用式样的侧向散射光强度和侧向荧光强度。对正常血液样本(5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，左下角标注的上方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。对异常血液样本(6号血液样本)的测试结果如图6所示，表明本申请不仅能够实现白细胞各亚群的有效分类(图6左下角标注上方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)，而且可有效的区分异常白细胞，包括幼稚粒细胞，异常淋巴细胞(图6中上方左侧方框为异常淋巴细胞，图中上方右侧方框为幼稚粒细胞)。
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F-4 50mg
乙二醇      950ml
甲醇        50ml
B：红细胞溶解剂溶液
Figure PCTCN2019115631-appb-000019
Figure PCTCN2019115631-appb-000020
最后将溶液的pH调整为7.5。
各种血液样品的测定和结果：
测试样本的过程在迈瑞BC-6800仪器中自动进行，设置吸样量为20微升，溶血剂溶液进样量为1ml，荧光染料存储液的进样量为20微升，血样为经过抗凝处理的血液。
形成测定用试样的具体步骤可参考实施例2。测定测定用式样的侧向散射光强度和侧向荧光强度。对正常血液样本(7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，左下角标注的上方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。对异常血液样本(8号血液样本)的测试结果如图8所示，表明本申请不仅能够实现白细胞各亚群的有效分类(图8左下角标注上方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)，而且可有效的区分异常白细胞，包括幼稚粒细胞，异常淋巴细胞(图8中上方左侧方框为异常淋巴细胞，图中上方右侧方框为幼稚粒细胞)。
实施例6
荧光染料与红细胞溶解剂一起配制为混合试剂，染料先用甲醇预先溶解，然后加入乙二醇混合，制备成染液，然后其余物料按照配方加入，调整好pH值后加入此前制备的染液，混合均匀即可，配方如下：
Figure PCTCN2019115631-appb-000021
最后将溶液的pH调整为7.0。
各种血液样品的测定和结果：
测试样本的过程在迈瑞BC-6800仪器中自动进行，设置吸样量为20微升，溶血剂溶液进样量为1ml，荧光染料存储液的进样量为20微升，血样为经过抗凝处理的血液。
将1ml上述染料与红细胞溶解剂形成的混合试剂溶液与经过抗凝剂处理的20微升血液混匀，在42℃下的孵育池中混匀孵育24秒，形成测定用试样。测定测定用式样的侧向散射光强度和侧向荧光强度。对正常血液样本(9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，左下角标注上方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。对异常血液样本(10号血液样本)的测试结果如图10所示，表明本申请不仅能够实现白细胞各亚群的有效分类，而且可有效的区分异常白细胞，包括幼稚粒细胞，异常淋巴细胞(图10中上方左侧方框为异常淋巴细胞，图中上方右侧方框为幼稚粒细胞)。
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F-6 200mg
乙二醇      950ml
甲醇        50ml
B：红细胞溶解剂溶液
Figure PCTCN2019115631-appb-000022
最后将溶液的pH调整为7.0。
各种血液样品的测定和结果：
测试样本的过程在迈瑞BC-6800仪器中自动进行，设置吸样量为20微升，溶血剂溶液进样量为1ml，荧光染料存储液的进样量为20微升，血样为经过抗凝处理的血液。
形成测定用试样的具体步骤可参考实施例2。测定测定用式样的侧向散射光强度和侧 向荧光强度。对正常血液样本(11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，左下角标注上方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。对异常血液样本(12号血液样本)的测试结果如图12所示，表明本申请不仅能够实现白细胞各亚群的有效分类，而且可有效的区分异常白细胞，包括幼稚粒细胞，异常淋巴细胞(图12中上方左侧方框为异常淋巴细胞，图中上方右侧方框为幼稚粒细胞)。
实施例8
白细胞四分群准确性测试
按照实施例6配置混合试剂，将迈瑞BC-6800DIFF通道中配套的染色液和溶血剂排空后，换入新配制好的试剂。设置测量模式为CD，测试样本为随机血样(13号血液样本)。测试完成后，用BC-6800血细胞分析仪(正常配套试剂)重新测试一次上述样本(13号血液样本)，统计随机血样的在两种试剂间的测试结果相关性。如图13所示，从测试结果可以看出，在对白细胞分类的性能上，混合试剂的测试结果与BC-6800测试结果具有很高的相关性，说明混合试剂能够对白细胞进行准确的识别、分类和计数。
本文中的所有数据、图像、仪器、试剂和步骤应理解为说明性而非限制性的。虽然已结合上述具体实施方案描述了本发明，许多修改和其它变化对于本领域技术人员是显而易见的。所有这种修改和其它变化也落入本发明的精神和范围内。

Claims (20)

  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 PCTCN2019115631-appb-100001
    其中，A选自硫，硒，碲、氧元素；
    R 1、R 2和R 3各自独立地选自氢和C 1-20的取代或未取代烷基；
    所述的取代烷基由下述基团任意取代：卤素、羟基、烷氧基、醛基、羰基、氨基、羧基、酯基、酰胺基、硝基或磺酸基；所述的X选自磷酸根，硫酸根，硫酸氢根，硝酸根，氯负离子，溴负离子，碘负离子或高氯酸根。
  2. 根据权利要求1所述的白细胞分类试剂，其特征在于，式F中所述的R 1、R 2和R 3各自独立地选自氢和C 1-14的取代或未取代烷基。
  3. 根据权利要求2所述的白细胞分类试剂，其特征在于，式F中所述的R 1、R 2和R 3各自独立地选自氢和C 1-6的取代或未取代烷基。
  4. 根据权利要求3所述的白细胞分类试剂，其特征在于，式F中所述的R 1和R 2其中之一是氢。
  5. 根据权利要求3所述的白细胞分类试剂，其特征在于，式F中所述的R 3是氢。
  6. 根据权利要求1-5任一项所述的白细胞分类试剂，其特征在于，式F中所述的A是氧，式F为：
    Figure PCTCN2019115631-appb-100002
  7. 根据权利要求6所述的白细胞分类试剂，其特征在于，所述的X选自氯负离子，溴负离子，碘负离子或高氯酸根。
  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，所述的取代烷基由下述基团任意取代：卤素、羟基、胺基、羧基、或酯基。
  9. 根据权利要求1所述的白细胞分类试剂，其特征在于，所述化合物选自F-1、F-2、F-3、F-4、F-5、和F-6：
    Figure PCTCN2019115631-appb-100003
  10. 根据权利要求1-9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。
  11. 根据权利要求1-10任一项所述的白细胞分类试剂，其特征在于，所述荧光染料为单独的荧光染料储存液，或所述荧光染料溶液与红细胞溶解剂溶液合并储存。
  12. 根据权利要求1-11任一项所述的白细胞分类试剂，其特征在于，所述荧光染料为单独的荧光染料储存液，其中所述荧光染料的浓度为1-1000mg/L，优选2-200mg/L，更优选20-100mg/L。
  13. 根据权利要求1-12任一项所述的白细胞分类试剂，其特征在于，所述荧光染料溶液与红细胞溶解剂溶液合并储存，其中所述荧光染料的浓度为0.02-20mg/L，优选0.04-4mg/L，更优选0.4-2mg/L。
  14. 根据权利要求10、11或13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、缓冲剂或它们的任何组合；
    所述阳离子表面活性剂选自十二烷基三甲基氯化铵、辛基三甲基溴化铵、十四烷基三甲基氯化铵、十四烷基三甲基溴化铵、癸基三甲基溴化铵和它们的混合物，浓度范围为300-800mg/L；
    所述非离子表面活性剂选自长链脂肪醇聚氧乙烯、烷基酚聚氧乙烯醚、脂肪酸聚氧乙烯醚、脂肪胺聚氧乙烯醚和它们的混合物，浓度范围为1-5g/L；
    所述缓冲剂选自羧酸盐类、磷酸盐类、柠檬酸盐类、Tris-HCl、3-吗啉代丙磺酸和它们的混合物，它们将测定体系的pH保持在5-8；
    所述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、醇醚羧酸盐和它们的混合物，浓度范围为0.1-2g/L。
  15. 根据权利要求1-14任一项所述的白细胞分类试剂，其特征在于：其中还 包含适当比例的醇，优选甲醇。
  16. 一种检测试剂盒，所述试剂盒包含权利要求1-15中任一项所述的白细胞分类试剂。
  17. 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 PCTCN2019115631-appb-100004
    其中，A、R 1、R 2、R 3和X如权利要求1中定义；
    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；
    根据散射光特性和荧光特性将白细胞分类和/或计数。
  18. 根据权利要求17的所述方法，其中所述式F的荧光染料为：F-1、F-2、F-3、F-4、F-5、和F-6：
    Figure PCTCN2019115631-appb-100005
    Figure PCTCN2019115631-appb-100006
  19. 根据权利要求18的所述方法，其中所述将血液样品、具有式F的荧光染料、和红细胞溶解剂混合形成混合物，包括以下步骤：
    将血液样品与红细胞溶解剂溶液混合形成混合物；
    将具有式F的荧光染料溶液加入所述混合物中。
  20. 根据权利要求19的所述方法，其中所述将血液样品、具有式F的荧光染料、和红细胞溶解剂混合形成混合物，包括以下步骤：
    将红细胞溶解剂溶液和具有式F的荧光染料溶液同时加入样品中。
PCT/CN2019/115631 2019-11-05 2019-11-05 白细胞分类试剂及其使用方法 WO2021087730A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2019/115631 WO2021087730A1 (zh) 2019-11-05 2019-11-05 白细胞分类试剂及其使用方法
CN201980101480.1A CN114585921A (zh) 2019-11-05 2019-11-05 白细胞分类试剂及其使用方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2019/115631 WO2021087730A1 (zh) 2019-11-05 2019-11-05 白细胞分类试剂及其使用方法

Publications (1)

Publication Number Publication Date
WO2021087730A1 true WO2021087730A1 (zh) 2021-05-14

Family

ID=75849067

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/115631 WO2021087730A1 (zh) 2019-11-05 2019-11-05 白细胞分类试剂及其使用方法

Country Status (2)

Country Link
CN (1) CN114585921A (zh)
WO (1) WO2021087730A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114152556A (zh) * 2021-10-22 2022-03-08 深圳市国赛生物技术有限公司 一种白细胞溶血剂、制备方法及血细胞分析仪
CN114235769A (zh) * 2021-12-22 2022-03-25 深圳锐视生物科技有限公司 一种血细胞染色方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01199161A (ja) * 1987-04-13 1989-08-10 Ortho Pharmaceut Corp 白血球を定量且つ識別する方法
JP2000329762A (ja) * 1999-05-19 2000-11-30 Sysmex Corp 白血球測定用試薬及び白血球測定用試料調製方法
JP2002148261A (ja) * 2000-11-09 2002-05-22 Sysmex Corp 異常細胞分類計数方法
US20020098589A1 (en) * 2001-01-19 2002-07-25 Crews Harold Richardson Multi-purpose reagent system and method for enumeration of red blood cells, white blood cells and thrombocytes and differential determination of white blood cells
CN101349644A (zh) * 2007-07-20 2009-01-21 深圳迈瑞生物医疗电子股份有限公司 一种白细胞分类试剂和其使用方法
CN103424540A (zh) * 2012-05-18 2013-12-04 嘉善加斯戴克医疗器械有限公司 一种白细胞分类试剂盒及其分类方法
CN103575702A (zh) * 2012-07-27 2014-02-12 深圳迈瑞生物医疗电子股份有限公司 白细胞分类计数试剂和白细胞分类方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01199161A (ja) * 1987-04-13 1989-08-10 Ortho Pharmaceut Corp 白血球を定量且つ識別する方法
JP2000329762A (ja) * 1999-05-19 2000-11-30 Sysmex Corp 白血球測定用試薬及び白血球測定用試料調製方法
JP2002148261A (ja) * 2000-11-09 2002-05-22 Sysmex Corp 異常細胞分類計数方法
US20020098589A1 (en) * 2001-01-19 2002-07-25 Crews Harold Richardson Multi-purpose reagent system and method for enumeration of red blood cells, white blood cells and thrombocytes and differential determination of white blood cells
CN101349644A (zh) * 2007-07-20 2009-01-21 深圳迈瑞生物医疗电子股份有限公司 一种白细胞分类试剂和其使用方法
CN103424540A (zh) * 2012-05-18 2013-12-04 嘉善加斯戴克医疗器械有限公司 一种白细胞分类试剂盒及其分类方法
CN103575702A (zh) * 2012-07-27 2014-02-12 深圳迈瑞生物医疗电子股份有限公司 白细胞分类计数试剂和白细胞分类方法

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114152556A (zh) * 2021-10-22 2022-03-08 深圳市国赛生物技术有限公司 一种白细胞溶血剂、制备方法及血细胞分析仪
CN114235769A (zh) * 2021-12-22 2022-03-25 深圳锐视生物科技有限公司 一种血细胞染色方法
CN114235769B (zh) * 2021-12-22 2024-04-12 深圳市路美康尔医疗科技有限公司 一种血细胞染色方法

Also Published As

Publication number Publication date
CN114585921A (zh) 2022-06-03

Similar Documents

Publication Publication Date Title
US7960099B2 (en) White blood cell differentiation reagent containing an asymmetric cyanine fluorescent dye and method of use thereof
EP0882983B1 (en) Reagent and method for classification and counting of leukocytes
US10774359B2 (en) Cellular analysis of body fluids
AU750108B2 (en) Fully automated method and reagent composition therefor for rapid identification and characterization of reticulocytes, erythrocytes and platelets in whole blood
JP3886271B2 (ja) 赤芽球の分類計数用試薬及び分類計数方法
JP4831914B2 (ja) 未成熟赤血球細胞における核酸の検出用色素及び方法
Evenson et al. Rapid determination on sperm cell concentration in bovine semen by flow cytometry
EP0842410B1 (en) Compositions and methods for the rapid analysis of reticulocytes
US20050202400A1 (en) Method of classifying counting leucocytes
US10371640B2 (en) Compositions and methods for leukocyte differential counting
US20100143955A1 (en) Reagent for blood analysis and method of using the same
JP2014219420A (ja) 完全自動化したモノクローナル抗体による広範な識別法
JP2002501188A (ja) 有核の赤血球の識別方法
JPH0822967B2 (ja) 全血中の網状赤血球の定量測定における化合物、試薬組成物及びその用途
WO2010013678A1 (ja) 子宮頸部異常細胞検出用試薬及びそれを用いる子宮頸部異常細胞検出方法
WO2021087730A1 (zh) 白细胞分类试剂及其使用方法
CN102947266A (zh) 分析细胞或含有核酸的其它生物材料的方法
JP2002148261A (ja) 異常細胞分類計数方法
Dolník et al. Flow cytometry in assessment of sperm integrity and functionality–a review
KR102024497B1 (ko) 소변 시료 분석 방법, 소변 시료 분석용 시약 및 소변 시료 분석용 시약 키트
KR20180023061A (ko) 소변 시료 분석 방법, 소변 시료 분석용 시약 및 소변 시료 분석용 시약 키트
WO2022099659A1 (zh) 白细胞分类试剂、红细胞分析试剂、试剂盒以及分析方法
CN117233067B (zh) 一种白细胞检测试剂盒及其应用
JP3421111B2 (ja) マラリア感染細胞の検出方法及び試薬
US11709116B2 (en) Liquid flourescent dye concentrate for flow cytometry evaluation of virus-size particles and related products and methods

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19951348

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19951348

Country of ref document: EP

Kind code of ref document: A1