CN115655829A - Improved hemolysin, application thereof and method for special hemolysin and cell grouping counting for flow cytometry detection - Google Patents
Improved hemolysin, application thereof and method for special hemolysin and cell grouping counting for flow cytometry detection Download PDFInfo
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- CN115655829A CN115655829A CN202211359742.4A CN202211359742A CN115655829A CN 115655829 A CN115655829 A CN 115655829A CN 202211359742 A CN202211359742 A CN 202211359742A CN 115655829 A CN115655829 A CN 115655829A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- 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
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- 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
- G01N2015/1486—Counting the particles
Abstract
The invention discloses an improved hemolysin, application thereof and a method for special hemolysin and cell grouping counting for flow cytometry detection, wherein the hemolysin is prepared from saponin, paraformaldehyde and TritonX-100. In severe patients with ACLF, the proportion of the mononuclear cells after hemolysis of the improved hemolysin of the invention is obviously higher than that of BDLysing TM The ratio of solution hemolysis shows that the damage of the modified hemolysin of the invention to the mononuclear cell is less than BDlysine TM solution can obtain higher proportion of mononuclear cells, and is more beneficial to research of the mononuclear cells.
Description
The application is a divisional application with application date of 2018, 08 and 06 days, application number of 201810883430.0 and invention name of reagent and application thereof in a combined immune effect early-stage rapid evaluation method.
Technical Field
The invention belongs to the field of biomedicine, and relates to an improved hemolysin, application thereof and a method for special hemolysis and cell grouping counting for flow cytometry detection.
Background
Flow cytometry has been used in various fields of scientific research and medical clinical tests, such as examination of immune function, detection of tumor antigens, metastatic and tumor suppressor genes and other disease-related genes, apoptosis, aids diagnosis and prognosis, and the like. Flow Cytometry (FCM) is a technique that can perform one-by-one, multi-parameter, rapid qualitative, quantitative analysis or sorting of single-column cells or biological particles in a rapid linear flow state, is one of advanced techniques in the field of modern life science, and is widely applied in the fields of basic research, clinical detection and the like.
Leukocyte clustering and enumeration are important indicators in the analysis of human blood in medical diagnostics. There are five types of leukocytes in blood, lymphocytes, monocytes, neutrophils, eosinophils, basophils, and the latter three are collectively called granulocytes, and thus the leukocytes are classified into three subgroups, i.e., lymphocytes, monocytes, and granulocytes.
The effective separation of the mononuclear cells with high proportion is an important link in the early rapid evaluation method of the combined immune effect. Because bioterrorism agent infection is extremely dangerous and pathogenic, an immunization scheme based on a single immune preparation often cannot play an effective protection role, and a new combined immune technology is urgently required to be developed. In order to deal with the threat of bioterrorism agents and warfare agents, the United states issued the Joint immune ordinance in the 80 th century, with the emphasis on developing joint countermeasure including combination vaccines and broad-spectrum drugs. At present, china does not have a mature combined vaccine or combined immunization scheme of bioterrorism agents. Because of the uncertainty of the combined immunity, the promotion or inhibition of the immune response among the members, the interference degree, the efficiency of the double infection after the combined immunity is infected and the like all influence the effect of the combined immunity. In addition, under the condition that emergency combined immunization is needed, after a reasonable combined immunization scheme is formulated, how to quickly evaluate the effect of the combined immunization in high flux is a difficult problem to be solved urgently. The current criteria for the evaluation of the immunological effects include both the level of neutralizing antibodies and the intensity of the cellular immune response, especially the CD8+ T cell (Tc) response. However, it takes 10 days or more from immunization to detection of the production of antibodies and the like, and this detection method is disadvantageous for the evaluation of the effect in the case of emergency combined immunization, and a new evaluation method and technique are required for judging the effect of combined immunization at the initial stage of immunization. It is known to those skilled in the art that macrophages in innate immune cells play a key role in the early phagocytosis and clearance of pathogenic microorganisms, and that macrophages can differentiate into M1 type, which promotes anti-infection response, and M2 type, which suppresses immune response. The reports and researches in the literature show that the macrophage phenotype is related to the later cellular immune response capability, the M1 phenotype causes strong anti-infection immune response, and the M2 phenotype means low anti-infection cellular immune response, so that the combined immune effect of the vaccine can be quickly judged by analyzing the macrophage phenotype at an early stage. Macrophages are induced by monocytes, and in order to study whether the phenotype of macrophages can be studied in vitro to achieve the purpose of rapidly judging the combined immune effect of the vaccine, a large amount of monocytes need to be obtained, which requires efficient separation of leukocytes in blood.
Hemolysin is a key reagent for counting or classifying leucocytes, belongs to one of the most commonly used reagents in clinical examination, and is matched with diluent and the like to be used in a blood cell analyzer (such as a flow cytometer). Differential white blood cell counting requires dilution of the blood sample and a specific hemolysin, which on the one hand allows rapid lysis of Red Blood Cells (RBC) and on the other hand white blood cell counting after lysis of the RBC, in order not to interfere with the white blood cell detection (since the number of red blood cells in blood is much greater than the number of white blood cells). On the other hand, to ensure the detection of three subpopulations of leukocytes, the volume of leukocytes also needs to be changed to some extent, and the leukocyte membrane needs to be dissolved to some extent in order to release cytoplasm while maintaining the integrity of the cell nucleus and to maintain a certain volume for the grouping measurement.
In blood sample treatment, hemolysin has obvious influence on cell grouping of peripheral blood, and different hemolysins have obvious influence on experimental results of different diseases. At present, hemolysin used by a flow cytometer basically depends on import and cannot be supplied in time, which brings great inconvenience and waste to users.
Disclosure of Invention
The sample to be detected by the flow cytometry may be Peripheral Blood Mononuclear Cells (PBMC) obtained by density gradient centrifugation, or may be anticoagulated blood without centrifugation. The preparation of PBMC samples takes a certain time to separate, and some surface molecular markers are obviously reduced after PBMC is separated, so that in-vivo results cannot be truly reflected. And the flow type staining analysis is carried out after red blood cell lysis is carried out on the freshly collected peripheral blood, so that the operation is simpler and more convenient, the time is greatly shortened, and surface molecules originally influenced by the lymphocyte separating medium are basically not changed after hemolysis and are most similar to in-vivo results. There are many ways to lyse red blood cells, but either lysate will affect the viability of the white blood cells to a greater or lesser extent or cause loss of cells while lysing red blood cells. Therefore, different diseases and subjects should use different lysis methods to get the closest true results in vivo.
Improved hemolysin and BD lysis of the present invention TM Compared with solution, the erythrocyte lysis effect is better, and each cell group is very obvious in the FSC/SSC graph. The two hemolysins have no obvious influence on the proportion of neutrophils, lymphocytes and monocytes of HC and CHB patients. However, the results of this study show that the proportion of monocytes after hemolysis of the modified hemolysin is significantly higher than BDLysing in severe patients with ACLF TM The ratio of hemolysis shows that the damage of the modified hemolysin to the mononuclear cell is less than that of BD Lysing TM solution, and therefore a higher proportion of monocytes can be obtained. Applicants' previous practice also found that PBMCs from severe ACLF patients were far fewer in number than healthy persons; coupled with the low proportion of monocytes in peripheral blood, this makes the study of monocytes in peripheral blood of critically ill ACLF patients more difficult. The whole blood cell staining operation is simple and convenient, and the loss of the mononuclear cells during the separation of the lymphocytes is greatly reduced, so the whole blood cell staining method is more suitable for the research of the mononuclear cellsHas the advantages. Moreover, the results of the present invention show that the improved hemolysin can obtain a higher proportion of monocytes in severe ACLF patients, which is undoubtedly more advantageous for the study of monocytes.
According to one aspect of the present invention there is provided an improved hemolysin for blood cell analysis, said hemolysin comprising the following components:
saponin: saponins are very strong surfactants and form soaps even when highly diluted. Saponins have stimulating effect on heart and are strong hemolytic agent.
Paraformaldehyde: strong permeability, uniform fixation, small tissue shrinkage, good dyeing effect, and prevention and minimization of artificial dissolution and loss in the tabletting process.
0.001% of Triton X-100: triton X-100 cell lysate is a classical lysate that rapidly lyses cellular tissue and obtains proteins. And maintain the original protein-protein interaction.
Hemolysin preferably has a pH value of 5.0 to 8.0, and if the pH value is too low or too high, damage to leukocytes can occur. The EDTApH value of the invention is 7.4, which is relatively stable.
The invention provides an improved hemolysin, which consists of the following components: saponin, paraformaldehyde, and triton x-100.
Preferably, the hemolysin consists of: 5% (w/v) of saponin, 0.001% (v/v) of Triton X-100 and 1.5% (v/v) of paraformaldehyde.
According to another aspect of the present invention there is provided a method of formulating hemolysin as hereinbefore described, said method comprising the steps of:
(1) Preparing a paraformaldehyde solution;
(2) Adding TritonX-100 into the prepared polyformaldehyde solution;
(3) And (3) adding saponin into the solution in the step (2) and fully dissolving.
Preferably, the volume percentage content of the paraformaldehyde in the paraformaldehyde solution is 1.5%; adding Triton X-100 into the paraformaldehyde solution to obtain a mixed solution, wherein the volume percentage of Triton X-100 in the mixed solution is 0.001%; adding 0.05g of saponin into the mixed solution obtained by adding Triton X-100 into the paraformaldehyde solution; when the volume of the paraformaldehyde solution in the step (1) is 100ml, the mass of the saponin added in the step (3) is 0.05g.
Preferably, the method comprises the steps of:
(1) Preparing 100ml of 1.5% paraformaldehyde;
(2) Adding Triton X-100 into 100ml 1.5% paraformaldehyde to make the concentration of Triton X-100 0.001%;
(3) Adding 0.05g of saponin into the prepared solution, and fully dissolving.
The hemolysin obtained by the preparation method comprises the following components: 5% (w/v) saponin, 0.001% (v/v) TritonX-100,1.5% (v/v) paraformaldehyde.
The invention provides application of the improved hemolysin in the calculation scheme in preparing and separating products of neutrophils, lymphocytes and monocytes in blood.
The invention provides application of the improved hemolysin in the calculation scheme in special hemolysis and cell grouping counting for flow cytometry detection.
According to another aspect of the present invention, the present invention provides a method for detecting hemolysis specifically by a flow cytometer, the method comprising the following steps:
and (2) adding the diluted hemolysin into anticoagulated peripheral blood according to the volume ratio of 10.
Further, the method for detecting hemolysis by using a flow cytometer provided by the present invention comprises the following steps:
(1) Taking a peripheral blood sample, adding an anticoagulant, storing at room temperature, and using within 24 hours;
(2) Taking 100 mul of anticoagulated peripheral blood and putting the anticoagulated peripheral blood into a flow tube;
(3) Adding corresponding fluorescent antibody, mixing evenly, and incubating for 20-30 minutes at room temperature in a dark place;
(4) 1ml of hemolysin which is 1 times of the hemolysin in the technical scheme is added and is rapidly and uniformly shaken, and the mixture is placed for 5 to 10 minutes at room temperature.
Preferably, the anticoagulant comprises heparin, edetate, citrate, or oxalate.
According to yet another aspect of the present invention, there is provided a method for counting white blood cells in a group, the method comprising the steps of:
(1) Hemolysis is carried out according to the hemolysis method described in the above technical scheme;
(2) Centrifuging and removing supernatant;
(3) Adding a cell staining buffer solution for washing, centrifuging, and removing a supernatant;
(4) Fixing with fixing liquid, storing in dark at 2-8 deg.C, and counting and analyzing by flow cytometry leukocyte group in 48 h.
Preferably, the rotation speed of the centrifugation is 1500r/min, and the time is 5min; the volume of the cell staining buffer solution is 1ml; the cell staining buffer solution is a PBS cell staining buffer solution; the volume of the fixing solution is 400 mu l; the fixing liquid is paraformaldehyde.
Preferably, the method comprises the steps of:
(1) Hemolysis was performed according to the hemolysis method described above;
(2) Centrifuging at 1500r/min for 5min, and removing supernatant;
(3) Adding 1ml cell staining buffer solution (PBS) for washing, centrifuging at 1500r/min for 5min, and removing supernatant;
(4) Fixing with 400 μ l of fixative, storing at 2-8 deg.C in dark place, and performing computer analysis within 48 h; the fixing liquid is paraformaldehyde.
Anticoagulants useful in the present invention include, but are not limited to, 4 of heparin, edetate (EDTA salt), citrate, oxalate, and the like.
Heparin is a mucopolysaccharide containing sulfate groups and has an average molecular mass of 15000 as the dispersed phase material. The anticoagulation principle is that the combination with antithrombin III results in the change of antithrombin III configuration and accelerates the formation of thrombin-thrombin complex to produce anticoagulation effect. In addition, heparin inhibits thrombin by means of a plasma cofactor (heparin cofactor ii). Common heparin anticoagulants are the sodium, potassium, lithium, and ammonium salts of heparin, with lithium heparin being the best but more expensive, the sodium and potassium salts increasing the sodium and potassium content of the blood, and the ammonium salts increasing the urea nitrogen content. The dosage of heparin anticoagulation is usually 10.0-12.5 IU/ml blood.
EDTA can react with Ca in blood 2+ The chelate is combined, the coagulation process is blocked, and the blood can not be coagulated. The EDTA salt comprises potassium, sodium and lithium salts, and EDTA-K2 is recommended by the International Commission on standardization of hematology, and has the highest solubility and the highest anticoagulation speed. EDTA salt is usually prepared as a 15% by weight aqueous solution, 1.2mg EDTA per ml blood, i.e. 0.04ml 15% EDTA solution per 5ml blood. EDTA salt can be dried at 100 deg.C without change of anticoagulation effect.
The citrate is mainly sodium citrate, and its anticoagulation principle is Ca in blood 2+ Binding to form chelate to make Ca 2+ The blood coagulation function is lost and the coagulation process is blocked, thereby preventing blood coagulation. Sodium citrate has Na 3 C 6 H 5 O 7 ·2H 2 O and 2Na 3 C 6 H 5 O 7 ·11H 2 Two crystals, typically 3.8% or 3.2% aqueous solutions prepared with the former, were mixed with blood at 1.
Oxalate is also a common anticoagulant, has the advantage of high solubility and has the action principle that oxalate dissociated after dissolution and Ca in a specimen 2+ Forming calcium oxalate precipitate to make Ca 2+ The coagulation function is lost and the coagulation process is blocked. Commonly used oxalate anticoagulants include sodium oxalate, potassium oxalate and ammonium oxalate, the common concentration of sodium oxalate is 0.1mol/L, and the ratio of sodium oxalate to blood is 1: and 9 parts.
In a specific embodiment of the invention, the cell staining buffer is PBS. The pH value of PBS is 7.2-7.4, which is close to the pH value of human blood, is the most suitable pH value for the survival of leucocytes, and can protect leucocytes from being damaged and antigen change.
According to another aspect of the invention, the invention provides the application of the hemolysin in the technical scheme in the preparation of products for quickly evaluating the combined immune effect in an early stage.
The product of the invention comprises the hemolysin described hereinbefore.
In an embodiment of the invention, the cell fixative is paraformaldehyde. Paraformaldehyde plays a role in fixing cell membranes, protects leukocyte membranes from being damaged to the greatest extent, and keeps the antigenicity of the leukocyte membranes unaffected.
The invention has the advantages and beneficial effects that:
the hemolysin improved by the method can obtain mononuclear cells with higher proportion in ACLF patients, and can obviously group three subsets of white blood cells.
The improved hemolysin has the advantages of low price of each component, convenient supply and easy acquisition, and reduces the product cost and the manufacturing cost.
The improved hemolysin has simple preparation process, needs no expensive instrument and equipment in the preparation process, and is simple and easy to produce.
The improved hemolysin of the present invention is non-toxic and non-hazardous to users and the environment.
Drawings
Figure 1 shows a flow cytogram of the morphology and proportion of monocyte and lymphocyte populations circled with FSC and SSC, where a: scatter plots circled by FSC and SSC; b: CD14 + And scatter plots circled by SSC; mono: a monocyte; lym: lymphocytes;
FIG. 2 shows a statistical graph of the proportion of monocytes in different treatment groups, wherein FIG. 2 is the left side of the graph showing the proportion of monocytes in 3 groups of people circled with FSC and SSC using different hemolysins; FIG. 2 shows a CD14 on the right side + The proportion of the mononuclear cells when different hemolysins are used by 3 groups of different people circled by the specific marker;
FIG. 3 shows a statistical plot of lymphocyte ratios in groups treated with different treatments, wherein FIG. 3 shows on the left the lymphocyte ratios in groups 3 circled with FSC and SSC when different hemolysins are used; on the right side of FIG. 3, a CD45 is used + Stranguria caused by different hemolysins in 3 different groups of people circled by specific markerProportion of the bar cells.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring harbor laboratory Press, 1989), or according to the manufacturer's recommendations.
EXAMPLE 1 preparation of modified hemolysin
The preparation method comprises the following steps:
(1) 100ml of 1.5 percent paraformaldehyde is prepared;
(2) Adding Triton X-100 into 100ml of prepared 1.5% paraformaldehyde to make the concentration of Triton X-100 be 0.001%;
(3) Adding 0.05g of saponin into the prepared solution, and fully dissolving.
Example 2 hemolysin Effect testing
1. Clinical patient collection (including HC, CHB, ACLF)
Healthy Controls (HC) 15, 10 men, 5 women, age (36 ± 3) years; 15 chronic hepatitis B patients (CHB) are treated, wherein 10 men and 5 women are aged (40 +/-8), the diagnosis of chronic hepatitis B conforms to the guidance for preventing and treating chronic hepatitis B in 2010, and the diagnosis standard is as follows: the HBsAg and/or HBVDNA are still positive after the past history of hepatitis B or the HBsAg is positive for more than 6 months; 15 patients with chronic plus acute liver failure (ACLF), wherein 10 men and 5 women are aged (53 +/-6), the ACLF diagnosis conforms to the liver failure diagnosis and treatment guideline of 2006, and the diagnosis standard is as follows: the serum total bilirubin (TBil) level of a CHB patient is increased by more than 10 times of a normal value (more than 171 mu mol/L), the prothrombin activity (PTA) is less than 40 percent, and hepatic encephalopathy (grade more than or equal to 2) appears recently, or ascites or hepatorenal syndrome is combined; patients with combined HCV, HDV, HIV and autoimmune liver disease were excluded. Collecting 5ml of peripheral venous blood, and placing the peripheral venous blood into a lithium heparin anticoagulation vacuum blood collection tube. The CHB and ACLF patients are outpatients and inpatients in the third 0-second hospital 2015 of the liberation military of Chinese people from 12 months to 2018 months in 7 months.
2. Measurement of Effect of hemolysin
(1) The whole blood sample is stored at room temperature after EDTA (ethylene diamine tetraacetic acid) anticoagulation and used within 24 hours;
(2) Taking 100 mul of anticoagulated whole blood and putting the anticoagulated whole blood into a flow type tube;
(3) Adding corresponding fluorescent antibody CD45-FITC/CD14-PE, mixing uniformly, and incubating for 20-30 minutes at room temperature in a dark place;
(4) 1ml of lysis solution is added and rapidly and evenly oscillated, and the mixture is placed for 5 to 10 minutes at room temperature;
(5) Centrifuging at 1500r/min for 5min, and removing supernatant;
(6) Adding 1ml cell staining buffer solution (PBS) for washing, centrifuging at 1500r/min for 5min, and removing supernatant;
(7) Fixing with 400. Mu.l paraformaldehyde fixing solution, storing at 2-8 deg.C in dark place, and performing mechanical analysis within 48 hr.
The experiment was divided into two groups, one group of blood was treated with lysate, the hemolysin prepared in example 1, and one group of blood was treated with lysate, BD lysis TM Solution (Invitrogen).
3. Results of the experiment
Lymphocytes and monocytes (denoted by forward scattered light (FSC/SSC)) were gated out by forward scattered light (FSC) and side scattered light (SSC). From the FSC/SSC graphs, it can be seen that both the modified hemolysin (DIY) and BDlysine (TM) Solution (BD) prepared in example 1 are capable of lysing erythrocytes completely without destroying leukocytes, and that the neutrophil, lymphocyte and monocyte groupings are clear (see FIG. 1A, where the values are percentages). Using CD14 + And side scatter light (SSC) gated lymphocytes and monocytes (CD 14) + in/SSC representation). From CD14 + The graphs for/SSC show that the modified hemolysin (DIY) and BD lysine TM Solution (BD) can completely dissolve erythrocytes without destroying leukocytes, and the groups of neutrophils, lymphocytes and monocytes are clear (see FIG. 1B, wherein the values are percentages). Hemolysin and BD lysis prepared in example 1 in HC and CHB patients TM After Solution hemolysis, statistics were carried out on lymphocytes (see FIG. 3), monocytesThe proportion of cells (see FIG. 2) was not statistically different; the post-hemolytic ratio BDLysing of hemolysin prepared in example 1 in critically ill patients with ACLF TM Monocyte clustering after Solution hemolysis was clear and high in proportion (see FIG. 2). Ns in the graph represents no statistical difference between the two groups; * Represents < 0.01, and has significant difference between two groups and statistical significance.
The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.
Claims (10)
1. An improved hemolysin, comprising the following components: saponin, paraformaldehyde, and triton x-100.
2. The modified hemolysin according to claim 1, characterized in that said hemolysin consists of: 5% saponin, 0.001% Triton X-100 and 1.5% paraformaldehyde; the saponin content is mass volume percentage; the TritonX-100 and the paraformaldehyde are in volume content.
3. A process for the preparation of an improved hemolysin according to claim 1 or 2, comprising the steps of:
(1) Preparing a paraformaldehyde solution;
(2) Adding TritonX-100 into the prepared polyformaldehyde solution;
(3) And (3) adding saponin into the solution obtained in the step (2) and fully dissolving.
4. The method according to claim 3, wherein the paraformaldehyde solution contains 1.5% by volume of paraformaldehyde; adding Triton X-100 into the paraformaldehyde solution to obtain a mixed solution with the Triton X-100 volume percent of 0.001%; when the volume of the paraformaldehyde solution in the step (1) is 100ml, the mass of the saponin added in the step (3) is 0.05g.
5. Use of the modified hemolysin of claim 1 or 2 for the preparation of a product for the isolation of neutrophils, lymphocytes, monocytes in blood.
6. Use of the modified hemolysin of claim 1 or 2 for flow cytometry for dedicated hemolysis and cell population counting.
7. A special hemolysis method for flow cytometry detection is characterized by comprising the following steps:
adding the diluted hemolysin of claim 1 or 2 into anticoagulated peripheral blood according to the volume ratio of 10;
(a) Taking a peripheral blood sample, adding an anticoagulant, and storing at room temperature for use; the anticoagulant comprises heparin, edetate, citrate or oxalate;
(b) Taking 100 mul of anticoagulated peripheral blood and putting the anticoagulated peripheral blood into a flow tube;
(c) Adding corresponding fluorescent antibody and mixing evenly, and incubating for 20-30 minutes at room temperature in a dark place;
(d) 1ml of 1 x the hemolysin is added and is quickly and uniformly shaken, and the mixture is placed for 5 to 10 minutes at room temperature.
8. A method for counting white blood cell clusters, comprising the steps of:
hemolysis according to the hemolysis method of claim 7;
(II) centrifuging and removing supernatant;
(III) adding a cell staining buffer solution for washing, centrifuging and removing a supernatant;
(IV) fixing with a fixing solution, storing at 2-8 ℃ in a dark place, and performing flow cytometry leukocyte grouping counting analysis within 48 h.
9. The method according to claim 8, characterized in that the rotation speed of the centrifugation is 1500r/min for 5min; the volume of the cell staining buffer solution is 1ml; the cell staining buffer solution is PBS cell staining buffer solution; the volume of the fixing solution is 400 mu l; the fixing liquid is paraformaldehyde.
10. Use of the modified hemolysin according to claim 1 or 2 for the preparation of a product for the early and rapid assessment of the combined immune response.
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| CN201810883430.0A CN109187121A (en) | 2018-08-06 | 2018-08-06 | Reagent and its application in combined immunization effect early stage fast appraisement method |
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| US6485923B1 (en) * | 2000-02-02 | 2002-11-26 | Lifescan, Inc. | Reagent test strip for analyte determination having hemolyzing agent |
| GB2372319A (en) * | 2000-12-12 | 2002-08-21 | R A Lab Ltd | A solid phase immunoassay involving blood cells which assay excludes washing steps |
| CN1287151C (en) * | 2005-01-04 | 2006-11-29 | 济南市中心医院 | Dedicated Hemolysin for flow cytometer and preparing method and application thereof |
| CN101620220A (en) * | 2008-06-30 | 2010-01-06 | 张晖 | Cytoperm capable of being used as hemolytic agent and using method thereof |
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