CN112415181A - Basophil degranulation detection kit, detection method and application thereof - Google Patents
Basophil degranulation detection kit, detection method and application thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
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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
- G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
Abstract
The invention discloses a basophil granulocytic degranulation detection kit, which comprises a stimulation buffer solution, a stimulation quality control A, a stimulation quality control B, a staining reagent, a dissolving reagent and a cleaning buffer solution; the stimulation buffer solution comprises calcium ions, heparin and interleukin 3; the stimulation quality control A is a monoclonal antibody of an anti-high-affinity IgE receptor; the stimulation quality control B is fMLP; the staining reagent is a mixture of anti-CD63-FITC and anti-CCR 3-PE. Also discloses application of the kit and a detection method thereof. The kit can be used for detecting perioperative anaphylaxis, provides a new method for improving perioperative anaphylaxis diagnosis and treatment, enables anaphylaxis to be diagnosed in time, and correctly identifies the allergen.
Description
Technical Field
The invention relates to the technical field of anaphylactic reaction detection, in particular to a basophil degranulation detection kit, a detection method and application thereof.
Background
In recent years, with the widespread application of a large amount of synthetic drugs in the process of anesthesia, it has been found in clinic that the serious allergic reaction (Anaphylaxis) induced by anesthetic drugs is increasing year by year. The data show that the perioperative anaphylaxis incidence rate is 1/20000-1/3500, and the incidence rate is reported to be 1/353, so the occurrence rate is not rare. Perioperative allergens are not clear in most patients and they still risk re-allergy when they are re-operated. At present, a standardized diagnosis system for drug-induced severe anaphylactic reaction in the anesthesia process is not available, and systematic research on high-risk factors of the drug-induced severe anaphylactic reaction is lacked at home and abroad. Many anesthesia department doctors have insufficient knowledge of perioperative anaphylaxis, so that diagnosis and treatment are not timely or wrong. Meanwhile, because the detection means of the allergen is limited, most of the allergens can be primarily judged only by virtue of medical history. The willingness of doctors and patients to improve perioperative anaphylaxis diagnosis and treatment is urgent.
An effective method for detecting perioperative severe anaphylactic reaction allergen is found, and the method has great significance for guiding patients to take medicine again for surgery and reducing perioperative risk. The methods for detecting allergens are classified into in vivo methods and in vitro methods. In vivo methods include Skin tests (Skin Test, ST) and challenge tests, and in vitro tests include Histamine (Histamine) and Tryptase (Tryptase) level detection, sIgE detection, and the like. However, no uniform and definite perioperative allergen detection method exists at present.
Because of the risk of inducing severe allergic reactions, perioperative allergens cannot be diagnosed clinically using the gold standard of challenge Test (PT), which is the most commonly used means for allergen detection in clinical settings, mainly including Skin Prick Test (SPT) and intradermal Test (IDT). Since ST can produce false positive results by directly stimulating histamine production by skin mast cells via non-IgE pathways, there is currently some controversy over its effectiveness in detecting allergens. In addition, ST has strict requirements on the concentration of operational and experimental drugs, and has certain risks of pain stimulation and induction of systemic anaphylaxis, which all limit the application of skin tests in perioperative allergen detection.
The in vitro detection means avoids the exposure risk of the patient and reduces other interference factors, thereby being an effective supplement for skin tests. The IgE detection is a well-known reliable method for detecting allergic diseases in vitro, and commonly used detection means comprise a radioactive allergen adsorption Test (RAST) and an immunocAP system and the like. The ImmunoCAP system is the most advanced quantitative detection system for IgE at present, and is a fluorescence enzyme immunoassay (RAST FEIA) developed on the basis of RAST, and the detection principle is that an antigen or an allergen is combined with a specific human antibody IgE on a stationary phase, the antigen in a sample and the antigen on the stationary phase are combined with IgE in a competitive manner, an enzyme-labeled anti-IgE secondary antibody is added into a stationary phase, and finally fluorescence is measured and compared with a standard curve. The ImmunoCAP can accurately and reliably detect the sIgE, and the result has good clinical relevance. ImmunoCAP is commonly used for detecting food and inhalant allergens, has higher diagnostic sensitivity and specificity, but has few related data for detecting perioperative allergen and has not been researched domestically.
Disclosure of Invention
In view of the above, the present invention aims to provide a basophil degranulation detection kit, a detection method and an application thereof, which can be used for perioperative anaphylaxis detection, and provide a new method for improving perioperative anaphylaxis diagnosis and treatment, so that anaphylaxis can be diagnosed in time, and an allergen can be identified correctly.
In order to achieve the purpose, the invention adopts the following technical scheme:
a basophil granulocytic degranulation detection kit comprises a stimulation buffer solution, a stimulation quality control A, a stimulation quality control B, a staining reagent, a dissolving reagent and a cleaning buffer solution; the stimulation buffer solution comprises calcium ions, heparin and interleukin 3; the stimulation quality control A is a monoclonal antibody of an anti-high-affinity IgE receptor; the stimulation quality control B is fMLP; the staining reagent is a mixture of anti-CD63-FITC and anti-CCR 3-PE.
Preferably, the dissolving agent is NH4Cl、KHCO3Or Na2A 10-fold concentrate of EDTA.
Preferably, the stimulation buffer solution, the stimulation quality control A and the stimulation quality control B are all freeze-dried powder, the staining reagent is 2.2mL, and the dissolving reagent is 25 mL. The stimulus buffer was reconstituted with 50mL of heat source-removing water, and stimulus quality control A and stimulus quality control B were reconstituted with 1.5mL of heat source-removing water.
The application of basophil degranulation detection kit is used for in-vitro detection of immediate-type anaphylaxis and detection of suspicious allergen causing hypersensitivity.
A detection method of a basophil degranulation detection kit comprises the following steps:
(1) collecting a sample: inverting the venous blood collection tube for several times, and uniformly mixing the anticoagulation blood samples;
(2) labeling the test tube: for each patient, a label is made on each tube separately,
wherein: PB-patient background;
PC1 is a monoclonal antibody against high affinity IgE receptor for stimulating quality control;
stimulating quality control by using fMLP (polycarbonate-glycine-serine) 2;
ax-y ═ allergen;
(3) cell stimulation: adding the stimulus in the kit into each labeled test tube of each patient respectively;
adding 50 μ L of stimulation buffer solution into PB test tube as detection background,
50 μ L of stimulus quality control A was added to a PC1 test tube,
50 μ L of stimulus quality control B was added to a PC2 test tube,
adding 50 mu L of allergen solution into an Ax-y test tube;
then, 100. mu.L of stimulation buffer and 50. mu.L of patient whole blood sample were added to each tube and gently mixed;
(4) dyeing: adding 20 μ L staining reagent into the test tubes, mixing gently, covering the tube cover, and incubating in water bath at 37 deg.C for 15 min;
(5) dissolving: respectively adding 2mL of dissolving reagent into each test tube in the step (4), and gently mixing; centrifuging for 5 minutes in a centrifuge, removing supernatant, performing suction-drying treatment by using absorbent paper, then resuspending cells by using a cleaning buffer solution, and placing the cells in a vortex mixer to perform soft vortex to obtain a cell suspension;
(6) and (3) detection: and (5) detecting the cell suspension obtained in the step (5) by using a flow cytometer.
Preferably, the preservation of the blood sample in step (1): for the detection of protein allergens, the blood sample needs to be stimulated immediately after being collected or stored at 2-8 ℃ for 48 hours at most; for drug and chemical allergen testing, the collected blood samples were stored at 2-8 ℃ for up to 24 hours.
Preferably, the dissolving reagent in step (5) needs to be preheated to 18-28 ℃. Dissolving reagent at 2-8 deg.C
Crystals may form during storage and are dissolved by standing at 18-28 ℃ before dilution.
Preferably, the centrifugal force of the centrifuge in the step (5) is 500 Xg.
Preferably, the washing buffer in step (5) is 300. mu.L.
Preferably, the cell suspension in step (6) is stored under protection from light at 2-8 ℃.
The invention is further explained below:
the principle is as follows: the two types of severe allergic reactions of IgE mediation and non-immune mediation can generate degranulation of basophils, the marker molecule CD63 expressed on the resting basophils is obviously increased, the activation degree of the basophils can be directly reflected, and the method is the optimal observation index of the activation of the basophils. The Basophils Activation Test (BAT) utilizes the principle, after Basophils are stimulated by allergen (antigen or quality control), a flow cytometer is used for observing the increase of the expression of a marker molecule CD63, the specific Activation of the Basophils is detected, and then drugs inducing severe anaphylactic reaction are effectively identified.
Principle explanation of the detection process: the stimulation buffer and allergen are added to EDTA anticoagulated whole blood samples from patients suspected of having allergy/hypersensitivity. The process of allergen-mimetic in vivo reactions, i.e. specific IgE antibodies bind to the cell surface via bridging with the corresponding allergen, activating intracellular signaling cascades leading to the activation of degranulation of basophils. During this degranulation, intracellular complexes affect the expression of the transmembrane protein CD63 on the cell surface and exposure to the extracellular matrix.
After the cell stimulation was complete, staining reagents were added, CCR3 was expressed continuously on eosinophils and basophils, residual red blood cells were removed by lysis, the cells were resuspended in wash buffer and examined by flow cytometry after a brief centrifugation step.
Storage method and expiration date of reagents in the kit:
(1) the unopened kit needs to be stored at 2-8 ℃.
(2) For unsealed or reconstituted reagents: the stimulation buffer solution, the stimulation quality control A and the stimulation quality control B are re-dissolved and then stored at the temperature of minus 20 ℃ for 6 months, and if the stimulation buffer solution, the stimulation quality control A and the stimulation quality control B are required to be used for multiple times, the storage is required to be separately packaged; the dissolving reagent needs to be stored at 2-8 ℃ and is stable for 6 months; the staining reagent and the washing buffer solution need to be stored at 2-8 ℃, and can be stably stored until the validity period marked by the bottle label.
Allergen(s):
protein allergen: where the protein allergen is in a liquid concentrated form for quality control and transport (1 μ L/bottle), the protein allergen needs to be stored refrigerated and diluted before use.
Drugs and chemical allergens: the low molecular weight allergens provided herein are shipped as a lyophilized powder, which requires refrigeration storage and reconstitution prior to use.
If allergens from other sources are used for the detection of the kit, the following conditions need to be met:
(1) non-matrix bound allergen (solid or liquid phase);
(2) it is prepared without cytotoxic compounds (stabilizer, antiseptic), such as glycerol, phenol, sodium azide or thimerosal (thimerosal) allergen.
Collecting and storing samples:
before collecting the sample, the patient should stop taking systemic immunosuppressive drugs such as corticosteroid and disodium cromoglycate for more than 24 hours.
And (3) collecting enough whole blood by using a K-EDTA (ethylene diamine tetraacetic acid) anticoagulation tube, and filling the blood in the blood collection tube as full as possible when a blood sample is collected. When the blood sample fails to fill the blood collection tube (< 50% volume), the EDTA concentration is too high, possibly leading to false negative results.
A1 mL whole blood sample was available for 18 test tubes. Note that the blood samples were not centrifuged or frozen.
The operation is as follows:
(1) water suggested for the test of the present kit: the use of sterile, ultra-pure, pyrogen-free water to reconstitute the cell stimulation buffer is important to obtain good and reproducible basophil stimulation results. The following water is also contemplated: cell culture grade ultrapure water, infusion grade ultrapure water or deionized water or double distilled water filtered through a 10kDa filter.
The lysis reagent may be reconstituted with deionized water, double distilled water, or water of the same grade as used for cell stimulation.
(2) Avoiding allergen contamination during cell stimulation:
inhaled allergens in the laboratory can contaminate open-set patient blood samples and isolated cells, potentially leading to an increase in the detection background. Therefore, care should be taken to cover the tube during blood sample separation and cell stimulation. Cell stimulation should avoid the presence of interfering factors such as fenestrations, dust mites, pollen, rubber gloves and potentially rubber contained by the instrument. Therefore, it is recommended that both the cell preparation and stimulation steps be performed in a sterile operating station.
(3) Tissue culture grade microplates may be used in performing the cell stimulation and labeling reactions.
Flow cytometry: argon ion laser (blue-green exciting light) with 488nm wavelength and matched software. Flow cytometry requires the detection of several indicators: forward angle light scattering (FSC), opposite side angle light scattering (SSC) and two fluorescent dyes FITC, PE.
The invention has the beneficial effects that:
the kit disclosed by the invention is wide in application range, can be used for detecting food and inhalant allergens, can also be used for predicting perioperative anaphylaxis and screening anesthetic suitable for patients, so that severe anaphylactic reaction caused by the patients is effectively avoided; the detection of the allergen by adopting the kit only needs to take a venous blood sample once, so that the pain of a patient is reduced. The kit is used for diagnosing severe anaphylactic reaction induced by muscle relaxant, the sensitivity and specificity of the kit are respectively 89.7% and 93.3%, and the specificity for the severe anaphylactic reaction induced by rocuronium bromide is up to 100%.
Drawings
FIG. 1 is a schematic representation of a leukocyte dispersion population on a FSC/SSC histogram;
FIG. 2 is the basophil CCR3posA schematic of the population;
FIG. 3 is a background basophil profile of a patient;
FIG. 4 is a profile of the patient's stimulated quality control PC1 basophils;
FIG. 5 is a profile of the patient's stimulated quality control PC2 basophils;
FIG. 6 is a basophil profile of allergen A1;
figure 7 is a basophil profile of allergen a 2.
Detailed Description
The present invention will be further described with reference to the following examples.
A basophil granulocytic degranulation detection kit comprises a stimulation buffer solution, a stimulation quality control A, a stimulation quality control B, a staining reagent, a dissolving reagent and a cleaning buffer solution; the stimulation buffer solution comprises calcium ions, heparin and interleukin 3; the stimulation quality control A is a monoclonal antibody of an anti-high-affinity IgE receptor; stimulation quality control B is fMLP; the staining reagent is a mixture of anti-CD63-FITC and anti-CCR 3-PE; dissolutionThe reagent is NH4Cl、KHCO3Or Na2A 10-fold concentrate of EDTA.
Wherein, the stimulation buffer solution, the stimulation quality control A and the stimulation quality control B in each kit are all freeze-dried powder, the stimulation buffer solution is redissolved by 50mL of heat source removing water, and the stimulation quality control A and the stimulation quality control B are redissolved by 1.5mL of heat source removing water. The staining reagent was 2.2mL and the lysis reagent was 25 mL.
The application of basophil degranulation detection kit is used for in-vitro detection of immediate-type anaphylaxis and detection of suspicious allergen causing hypersensitivity.
A detection method of a basophil degranulation detection kit comprises the following steps:
(1) collecting a sample: the venous blood collection tube is inverted for several times, and the anticoagulated blood sample is mixed evenly.
Preservation of blood samples: for the detection of protein allergens, the blood sample needs to be stimulated immediately after being collected or stored at 2-8 ℃ for 48 hours at most; for drug and chemical allergen testing, the collected blood samples were stored at 2-8 ℃ for up to 24 hours.
(2) Labeling the test tube: for each patient, a label is made on each tube separately,
wherein: PB-patient background;
PC1 is a monoclonal antibody against high affinity IgE receptor for stimulating quality control;
stimulating quality control by using fMLP (polycarbonate-glycine-serine) 2;
ax-y ═ allergen.
(3) Cell stimulation: adding the stimulus in the kit into each labeled test tube of each patient respectively;
adding 50 μ L of stimulation buffer solution into PB test tube as detection background,
50 μ L of stimulus quality control A was added to a PC1 test tube,
50 μ L of stimulus quality control B was added to a PC2 test tube,
adding 50 mu L of allergen solution into an Ax-y test tube;
then, 100. mu.L of stimulation buffer and 50. mu.L of patient whole blood sample were added to each tube and gently mixed.
(4) Dyeing: mu.L of each staining reagent was added to the tube, mixed gently, the tube cap was closed, and incubated in water bath at 37 ℃ for 15 minutes.
(5) Dissolving: respectively adding 2mL of dissolving reagent preheated to 18-28 ℃ into each test tube in the step (4), and gently mixing; centrifuging for 5 minutes in a centrifuge with the centrifugal force of 500Xg, removing supernatant, performing suction drying treatment by using absorbent paper, then resuspending cells by using 300 mu L of cleaning buffer solution, placing the cells in a vortex mixer, and gently vortexing to obtain cell suspension; if the detection is not carried out timely, the cell suspension needs to be stored at the temperature of 2-8 ℃ in a dark place.
(6) And (3) detection: and (5) detecting the cell suspension obtained in the step (5) by using a flow cytometer.
Example 1
A detection method of a basophil degranulation detection kit is used for detecting whether a patient is allergic to allergen 1, and comprises the following steps:
(1) collecting a sample: blood collection was performed as described above, the tubes were inverted several times and the anticoagulated blood samples were mixed well.
(2) Labeling the test tube: for the patient, a label is made on each tube separately,
wherein: PB-patient background;
PC1 is a monoclonal antibody against high affinity IgE receptor for stimulating quality control;
stimulating quality control by using fMLP (polycarbonate-glycine-serine) 2;
a1 is allergen 1;
a2 is allergen 2.
(3) Cell stimulation: adding the stimulators in the kit into the labeled test tubes of the patient respectively;
adding 50 μ L of stimulation buffer solution into PB test tube as detection background,
50 μ L of stimulus quality control A was added to a PC1 test tube,
50 μ L of stimulus quality control B was added to a PC2 test tube,
adding 50 μ L allergen solution into test tubes A1 and A2;
then, 100. mu.L of stimulation buffer and 50. mu.L of patient whole blood sample were added to each tube and gently mixed.
(4) Dyeing: mu.L of each staining reagent was added to the tube, mixed gently, the tube cap was closed, and incubated in water bath at 37 ℃ for 15 minutes.
(5) Dissolving: respectively adding 2mL of dissolving reagent preheated to 18-28 ℃ into each test tube in the step (4), and gently mixing; centrifuging for 5 minutes in a centrifuge with the centrifugal force of 500Xg, removing supernatant, performing suction drying treatment by using absorbent paper, then resuspending cells by using 300 mu L of cleaning buffer solution, placing the cells in a vortex mixer, and gently vortexing to obtain cell suspension; if the detection is not carried out timely, the cell suspension needs to be stored at the temperature of 2-8 ℃ in a dark place.
(6) And (3) detection: and (5) detecting the cell suspension obtained in the step (5) by using a flow cytometer.
The detection of the flow cytometer includes the collection of sample information and the analysis of the information:
the flow cytometer is adjusted to the appropriate color setting prior to detection and then sample collection is performed for each tube of the patient.
During the data collection process of the sample, it is necessary to ensure that the white blood cells on the FSC/SSC histogram are divided into three discrete groups, and the amplified FSC and SSC signals are adjusted to obtain the sample graph shown in FIG. 1.
Typically, collection is stopped after 500-600 basophil signals are collected. At least 200 basophils were analyzed, which required a total of 50,000 and 100,000 leukocytes for analysis.
Respectively analyzing the acquired data; the data collected can be performed using any flow cytometer analysis software, such as FlowJo, flowmax, CellQuest, or others.
Flow cytometry analysis is based on 2 steps:
1. setting door 1(R1) first, at a low value (SSC) for side angle light scatteringlow) Region finding Total basophil CCR3posClusters are shown in FIG. 2.
2. The percentage of CD63 positive cells (FITC highlight fluorescence; Q1-UR) in basophils obtained in the R1 setting was calculated.
As shown in fig. 3, the basophil profile of the PB tube in the R1 setting is shown as a patient background for reference for other tube tests. According to CD63posAnd C63negThe distribution of the two forms of presentation is divided into four parts in FIG. 3, denoted Q1-UR and Q1-LR, where the number of basophils present in each quadrant is given in Table 1.
TABLE 1
As shown in FIGS. 4 and 5, when the activity of only one of the cells tested in the PC1 tube and the PC2 tube is greater than 10%, the basophil distribution diagram of the PC1 tube in the R1 setting indicates that the cells have the corresponding activity, and the test result is acceptable.
Wherein the cell activity N ═ NQ1-UR/(nQ1-UR+nQ1-LR). Wherein n isQ1-URThe number of cells in the Q1-UR region, nQ1-LRThe number of cells in the Q1-LR region.
The number of basophils in Q1-UR and Q1-LR in FIG. 4 is presented as shown in Table 2:
TABLE 2
| Acquisition area | Quantity (n ═) | Percent by weight% |
| All are | 72916 | 100.0 |
| R1 | 606 | 0.8 |
| Q1-UR(CD63pos) | 467 | 77.1 |
| Q1-LR(CD63neg) | 139 | 22.9 |
As can be calculated from the data in the table, the cell activity is more than 10%, and therefore, the result of detecting the allergen in the blood sample of the patient is acceptable.
As shown in fig. 6, the basophil profile for the a1 tube in the R1 setting, presented as the number of basophils in Q1-UR and Q1-LR as shown in table 3:
TABLE 3
| Acquisition area | Quantity (n ═) | Percent by weight% |
| All are | 63074 | 100.0 |
| R1 | 500 | 0.79 |
| Q1-UR(CD63pos) | 7 | 1.40 |
| Q1-LR(CD63neg) | 493 | 98.6 |
As shown in fig. 7, the basophil profile for the a2 tube in the R1 setting, with the number of basophils present in Q1-UR and Q1-LR as shown in table 4:
TABLE 4
| Acquisition area | Quantity (n ═) | Percent by weight% |
| All are | 80039 | 100.0 |
| R1 | 419 | 0.5 |
| Q1-UR(CD63pos) | 44 | 10.5 |
| Q1-LR(CD63neg) | 375 | 89.5 |
The critical value for interpretation of different allergen results is:
when the cell activity N and the stimulation index SI satisfy the above values, the result is judged to be positive, otherwise, the result is negative.
Stimulation index SI ═ AnQ1-UR+AnQ1-LR)/(PBnQ1-UR+PBnQ1-LR)，AnQ1-URThe number of cells in the Q1-UR region during the A cuvette test, AnQ1-LRPBn is the number of cells in the Q1-LR region during the A tube assayQ1-URThe number of cells in the Q1-UR region, PBn, during PB tube assayQ1-LRThe number of cells in the Q1-LR region during PB tube assay.
A1 and a2 are drug allergens, which can be calculated from tables 3 and 4: n-10.5%, indicating that the patient is allergic to allergen a 2.
Precision of the kit:
precision (patient background): the test was repeated 20 times for one blood sample, stimulated with stimulation buffer (patient background PB) and then analyzed on a flow cytometer in series. The results are shown in Table 5, expressed as percentage basophil activation and Mean Fluorescence Intensity (MFI) of CD 63-FITC. Precision was 16.2% CV.
Precision (positive quality control): one blood sample was assayed 20 times in duplicate, stimulated using positive control (STCON), and then serially analyzed on a flow cytometer. The results are shown in Table 5, expressed as percentage basophil activation and Mean Fluorescence Intensity (MFI) of CD 63-FITC. Precision was 5.4% CV.
TABLE 5
Difference in different personnel operations (positive quality control): 3.7-8.1% CV. Two blood samples of normal blood donors are respectively detected by 5 operators in the same day by using the kit, two positive quality controls STCON and fMLP in the kit are used for stimulation, and duplicate detection is carried out on each positive quality control.
The results are shown in Table 6, as the mean percentage of basophil activation (CD63+) for each operator analyzed with two positive control stimuli. It can be seen that the operational error of the kit is also within a reasonable range.
TABLE 6
Finally, the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting, and other modifications or equivalent substitutions made by the technical solutions of the present invention by those of ordinary skill in the art should be covered within the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A basophil granulocytic degranulation detection kit is characterized by comprising a stimulation buffer solution, a stimulation quality control A, a stimulation quality control B, a staining reagent, a dissolving reagent and a cleaning buffer solution; the stimulation buffer solution comprises calcium ions, heparin and interleukin 3; the stimulation quality control A is a monoclonal antibody of an anti-high-affinity IgE receptor; the stimulation quality control B is fMLP; the staining reagent is a mixture of anti-CD63-FITC and anti-CCR 3-PE.
2. The basophil degranulation detection kit according to claim 1, wherein the lysis reagent is NH4Cl、KHCO3Or Na2EDTA is 10 times of concentrated solution.
3. The basophil degranulation detection kit according to claim 1, wherein the stimulus buffer solution, the stimulus quality control A and the stimulus quality control B are all lyophilized powder, the staining reagent is 2.2mL, and the lysis reagent is 25 mL.
4. Use of the basophil degranulation test kit of claim 1 for the in vitro detection of immediate hypersensitivity reactions and the detection of suspected allergens causing hypersensitivity reactions.
5. The method for detecting the basophil degranulation detection kit according to claim 1, which comprises the following steps:
(1) collecting a sample: inverting the venous blood collection tube for several times, and uniformly mixing the anticoagulation blood samples;
(2) labeling the test tube: for each patient, a label is made on each tube separately,
wherein: PB = patient background;
PC1 = monoclonal antibody stimulation quality control against high affinity IgE receptor;
PC2 = fMLP stimulation quality control;
ax-y = allergen;
(3) cell stimulation: adding the stimulus in the kit into each labeled test tube of each patient respectively;
adding 50 μ L of stimulation buffer solution into PB test tube as detection background,
50 μ L of stimulus quality control A was added to a PC1 test tube,
50 μ L of stimulus quality control B was added to a PC2 test tube,
adding 50 mu L of allergen solution into an Ax-y test tube;
then, 100. mu.L of stimulation buffer and 50. mu.L of patient whole blood sample were added to each tube and gently mixed;
(4) dyeing: adding 20 μ L staining reagent into the test tubes, mixing gently, covering the tube cover, and incubating in water bath at 37 deg.C for 15 min;
(5) dissolving: respectively adding 2mL of dissolving reagent into each test tube in the step (4), and gently mixing; centrifuging for 5 minutes in a centrifuge, removing supernatant, performing suction-drying treatment by using absorbent paper, then resuspending cells by using a cleaning buffer solution, and gently vortexing to obtain a cell suspension;
(6) and (3) detection: and (5) detecting the cell suspension obtained in the step (5) by using a flow cytometer.
6. The method for detecting a basophil degranulation detection kit according to claim 5, wherein the preservation of the blood sample in step (1): for the detection of protein allergens, the blood sample needs to be stimulated immediately after being collected or stored at 2-8 ℃ for 48 hours at most; for drug and chemical allergen testing, the collected blood samples were stored at 2-8 ℃ for up to 24 hours.
7. The method of detecting a basophil degranulation detection kit according to claim 5, wherein the lysis reagent in step (5) is preheated to 18-28 ℃.
8. The method of detecting a basophil degranulation detection kit according to claim 5, wherein the centrifugal force of the centrifuge in step (5) is 500 Xg.
9. The method of detecting a basophil degranulation detection kit according to claim 5, wherein the washing buffer in step (5) is 300. mu.L.
10. The method for detecting the basophil degranulation detection kit according to claim 5, wherein the cell suspension in the step (6) is preserved under the condition of being protected from light at 2-8 ℃.
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| US20100221756A1 (en) * | 2007-09-11 | 2010-09-02 | Buhlmann Laboratories Ag | Allergy test based on flow cytometric analysis |
| CN104677810A (en) * | 2015-01-30 | 2015-06-03 | 广东医学院附属医院 | Kit for detecting basophil activation and using method of kit |
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| CN107860907A (en) * | 2017-11-02 | 2018-03-30 | 中国医学科学院北京协和医院 | Application of the albumen of Pru p 3 in wormwood artemisia pollen correlation peach allergy detection kit is prepared |
| CN111549091A (en) * | 2020-05-21 | 2020-08-18 | 核工业总医院 | Test method for basophil activation and degranulation |
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| US20100221756A1 (en) * | 2007-09-11 | 2010-09-02 | Buhlmann Laboratories Ag | Allergy test based on flow cytometric analysis |
| CN104677810A (en) * | 2015-01-30 | 2015-06-03 | 广东医学院附属医院 | Kit for detecting basophil activation and using method of kit |
| CN107621548A (en) * | 2017-09-15 | 2018-01-23 | 深圳市因诺赛生物科技有限公司 | Sequestered and the total IgE measure diagnostic kits of cell mating type and preparation method in people's whole blood |
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