CN110243651B - Reticulocyte detection kit and application thereof - Google Patents
Reticulocyte detection kit and application thereof Download PDFInfo
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- CN110243651B CN110243651B CN201810185799.4A CN201810185799A CN110243651B CN 110243651 B CN110243651 B CN 110243651B CN 201810185799 A CN201810185799 A CN 201810185799A CN 110243651 B CN110243651 B CN 110243651B
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- 210000001995 reticulocyte Anatomy 0.000 title claims abstract description 65
- 238000001514 detection method Methods 0.000 title claims abstract description 21
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 27
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 20
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 20
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 20
- 239000000980 acid dye Substances 0.000 claims abstract description 18
- 238000010790 dilution Methods 0.000 claims abstract description 15
- 239000012895 dilution Substances 0.000 claims abstract description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000000243 solution Substances 0.000 claims abstract description 13
- 239000003085 diluting agent Substances 0.000 claims abstract description 12
- 239000002280 amphoteric surfactant Substances 0.000 claims abstract description 11
- 239000002736 nonionic surfactant Substances 0.000 claims abstract description 10
- 239000004094 surface-active agent Substances 0.000 claims abstract description 9
- 239000012192 staining solution Substances 0.000 claims abstract description 8
- 150000001875 compounds Chemical class 0.000 claims abstract description 6
- 239000003960 organic solvent Substances 0.000 claims abstract description 6
- 239000006172 buffering agent Substances 0.000 claims abstract description 4
- MTNDZQHUAFNZQY-UHFFFAOYSA-N imidazoline Chemical compound C1CN=CN1 MTNDZQHUAFNZQY-UHFFFAOYSA-N 0.000 claims abstract description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 50
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 30
- 239000007788 liquid Substances 0.000 claims description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- SEQKRHFRPICQDD-UHFFFAOYSA-N N-tris(hydroxymethyl)methylglycine Chemical compound OCC(CO)(CO)[NH2+]CC([O-])=O SEQKRHFRPICQDD-UHFFFAOYSA-N 0.000 claims description 7
- 238000003149 assay kit Methods 0.000 claims description 7
- 239000003755 preservative agent Substances 0.000 claims description 7
- 125000000217 alkyl group Chemical group 0.000 claims description 6
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 claims description 6
- 230000002335 preservative effect Effects 0.000 claims description 6
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 6
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 5
- 150000003839 salts Chemical class 0.000 claims description 5
- 239000007995 HEPES buffer Substances 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 4
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims description 4
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- 239000005711 Benzoic acid Substances 0.000 claims description 3
- 239000007993 MOPS buffer Substances 0.000 claims description 3
- UZMAPBJVXOGOFT-UHFFFAOYSA-N Syringetin Natural products COC1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UZMAPBJVXOGOFT-UHFFFAOYSA-N 0.000 claims description 3
- 239000007997 Tricine buffer Substances 0.000 claims description 3
- 239000007983 Tris buffer Substances 0.000 claims description 3
- 235000010233 benzoic acid Nutrition 0.000 claims description 3
- KCFYHBSOLOXZIF-UHFFFAOYSA-N dihydrochrysin Natural products COC1=C(O)C(OC)=CC(C2OC3=CC(O)=CC(O)=C3C(=O)C2)=C1 KCFYHBSOLOXZIF-UHFFFAOYSA-N 0.000 claims description 3
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 claims description 3
- 239000004334 sorbic acid Substances 0.000 claims description 3
- 235000010199 sorbic acid Nutrition 0.000 claims description 3
- 229940075582 sorbic acid Drugs 0.000 claims description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- 125000004432 carbon atom Chemical group C* 0.000 claims description 2
- 125000005843 halogen group Chemical group 0.000 claims description 2
- 229960005323 phenoxyethanol Drugs 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 229920000136 polysorbate Polymers 0.000 claims description 2
- 125000000542 sulfonic acid group Chemical group 0.000 claims description 2
- DVLFYONBTKHTER-UHFFFAOYSA-N 3-(N-morpholino)propanesulfonic acid Chemical compound OS(=O)(=O)CCCN1CCOCC1 DVLFYONBTKHTER-UHFFFAOYSA-N 0.000 claims 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 claims 1
- 239000000975 dye Substances 0.000 abstract description 37
- 238000005259 measurement Methods 0.000 abstract description 24
- 238000004043 dyeing Methods 0.000 abstract description 13
- 210000004027 cell Anatomy 0.000 abstract description 9
- 239000000126 substance Substances 0.000 abstract description 2
- 231100000419 toxicity Toxicity 0.000 abstract description 2
- 230000001988 toxicity Effects 0.000 abstract description 2
- 210000000601 blood cell Anatomy 0.000 description 17
- 238000001914 filtration Methods 0.000 description 14
- 239000012528 membrane Substances 0.000 description 14
- 238000003756 stirring Methods 0.000 description 14
- 238000002156 mixing Methods 0.000 description 13
- 238000000034 method Methods 0.000 description 9
- 239000002253 acid Substances 0.000 description 7
- 239000003513 alkali Substances 0.000 description 7
- 239000008367 deionised water Substances 0.000 description 7
- 229910021641 deionized water Inorganic materials 0.000 description 7
- 238000007789 sealing Methods 0.000 description 7
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 6
- 210000003743 erythrocyte Anatomy 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 230000007547 defect Effects 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 4
- NCBISIFFSNXYQJ-UHFFFAOYSA-N 1-dodecyl-4,5-dihydroimidazole Chemical compound CCCCCCCCCCCCN1CCN=C1 NCBISIFFSNXYQJ-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 210000000805 cytoplasm Anatomy 0.000 description 3
- 238000003359 percent control normalization Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- QOFJQOYMHJAEFE-UHFFFAOYSA-N 1-tetradecyl-4,5-dihydroimidazole Chemical compound CCCCCCCCCCCCCCN1CCN=C1 QOFJQOYMHJAEFE-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 239000003093 cationic surfactant Substances 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- -1 polymethylene Polymers 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229920001214 Polysorbate 60 Polymers 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 229930002877 anthocyanin Natural products 0.000 description 1
- 235000010208 anthocyanin Nutrition 0.000 description 1
- 239000004410 anthocyanin Substances 0.000 description 1
- 150000004636 anthocyanins Chemical class 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000003651 basophil Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 238000004364 calculation method Methods 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- SQHOAFZGYFNDQX-UHFFFAOYSA-N ethyl-[7-(ethylamino)-2,8-dimethylphenothiazin-3-ylidene]azanium;chloride Chemical compound [Cl-].S1C2=CC(=[NH+]CC)C(C)=CC2=NC2=C1C=C(NCC)C(C)=C2 SQHOAFZGYFNDQX-UHFFFAOYSA-N 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- KCXFHTAICRTXLI-UHFFFAOYSA-N propane-1-sulfonic acid Chemical compound CCCS(O)(=O)=O KCXFHTAICRTXLI-UHFFFAOYSA-N 0.000 description 1
- 238000007430 reference method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
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- 229920002477 rna polymer Polymers 0.000 description 1
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- 150000003384 small molecules Chemical class 0.000 description 1
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Images
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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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N1/31—Apparatus therefor
-
- 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
- G01N1/38—Diluting, dispersing or mixing samples
-
- 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
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
-
- 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
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- 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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a reticulocyte detection kit, which comprises: a staining solution and a sufficient amount of a dilution solution, the staining solution comprising a specific nucleic acid dye and an organic solvent, the specific nucleic acid dye being a compound represented by formula 3: the diluent comprises a buffering agent, at least one surfactant and water, and the pH value of the detection kit is 7-11; the surfactant comprises an amphoteric surfactant, wherein the amphoteric surfactant is a compound shown in an imidazoline type 4; meanwhile, the application of the kit for preparing the reagent for measuring the reticulocyte content is disclosed. The dye used in the invention can easily enter the cells treated by the diluent, combines with nucleic acid substances in the molecules, has low dye safety and toxicity, uses the amphoteric surfactant and the nonionic surfactant to treat the cells, has obvious RET maturity degree distinction limit after dyeing by the dye, has accurate result measurement, and can also measure the content of the platelets.
Description
Technical Field
The invention belongs to the field of blood cell analysis, and mainly relates to a reticulocyte detection reagent and application thereof. The reticulocyte detection kit adopts novel non-toxic, high-sensitivity and high-stability nucleic acid dye and diluent, and can accurately classify the reticulocyte.
Background
Reticulocytes are one type of red blood cells, the transition phase between late and mature red blood cells, in which small amounts of basophilic RNA remain in the cytoplasm, the higher the RNA content, the more immature the reticulocyte. The peripheral blood of normal people is mature red blood cells and a small amount of reticulocytes (adult: percent, 0.5% -1.5%, neonate: percent, 3.0% -6.0%), so that clinical examination can judge the information of patients according to the maturity and content of the reticulocytes, and the clinical examination is mainly used for judging the anemia type.
At present, two main methods for detecting reticulocytes are: one is manual and one is instrumental. And (3) manual method: as basophils such as ribosomes, ribonucleic acids and the like remain in the cytoplasm of the reticulocytes, blue or blue-green branch point-like or even reticulate structures can be seen in the cytoplasm after the living body is dyed with the brilliant tar blue or the new methylene blue, so that the reticulocytes are distinguished. The method comprises the following steps: the fluorescent dye is mainly used for combining with RNA in reticulocytes, when laser emitted by FCM irradiates on the cells, the cells emit fluorescence with specific color, and the RNA content is measured according to the intensity of the fluorescence, so that the percentage of Ret (RET%) in mature red blood cells is accurately measured. RET maturity can be classified into low fluorescence intensity reticulocytes (LFR), medium fluorescence intensity reticulocytes (MFR) and high fluorescence intensity reticulocytes (HFR) according to the proportion of the parameters such as the intensity of fluorescence emitted after laser irradiation, the light absorption amount, the light scattering amount and the like. The higher the fluorescence intensity, the more naive the reticulocytes. The manual method has the advantages of long time consumption, low efficiency and high cost. Thus, instrumentation is a major clinical need.
At present, a plurality of manufacturers research the instrument method, and corresponding progress is made, and the related technologies in the prior art are as follows:
the first prior art is: chinese patent publication No. CN 1172953A discloses an anthocyanin type dye for dyeing reticulocyte, which has the formula 1
A specific dye for staining leukocytes. The cationic surfactant is used as an accelerating agent, and the determination is carried out under a certain buffer system. The method has the defects that various dyes are harmful to the environment and human body, and the differentiation of leucocytes from reticulocytes in abnormal lymphocyte blood is not obvious. In addition, RET maturity differentiation limits are not readily apparent.
And the second prior art is as follows: chinese patent publication No. CN 101344472A discloses a fluorescent dye of formula 2
Buffers, accelerating agents and osmolality adjusting agents. The method has the defects that the dye has a certain harm to human bodies, and the RET maturity distinguishing limit is not obvious.
The third prior art is: US patent 5821127 discloses the use of polymethylene blue as a specific dye and cationic and amphoteric surfactants as an accelerating agent. The method has the defects that the secondary dye has nonspecific staining on red blood cells, the background intensity is high, and the RET maturity differentiation limit is not obvious.
In view of the above, there are many disadvantages in the prior art, such as damage to dye, stability, nonspecific staining of red blood cells, insignificant differentiation of RET maturation degree, high reagent cost, complex preparation, etc., and thus further improvement of the prior art is required.
Disclosure of Invention
The invention aims at: in view of the shortcomings of the prior art, the invention aims to provide a reticulocyte detection kit, and the nucleic acid dye in the kit has the characteristics of no toxicity, high sensitivity and high stability.
In order to achieve the above object, the present invention provides a reticulocyte detection kit, comprising a staining solution and a sufficient amount of a dilution solution, wherein the staining solution comprises a specific nucleic acid dye and an organic solvent for dissolving the specific nucleic acid dye, and the specific nucleic acid dye is a compound represented by formula 3:
wherein R1, R2 and R3 are alkyl groups with less than four carbons, R4 is a sulfonic acid group, and X is a halogen atom;
the diluent comprises a buffering agent, at least one surfactant and water, and the pH value of the detection kit is 7-11;
the surfactant comprises an amphoteric surfactant, and the amphoteric surfactant is a compound shown in an imidazoline type 4:
r represents an alkyl group or an alkyl group having 8 to 16 carbon atoms.
The solvent of the specific nucleic acid dye solution is one or a mixture of two of glycol, methanol, ethanol and propanol;
the buffer is selected from one or two of Tris (2-amino-2- (hydroxymethyl) -1, 3-propanediol), HEPES (4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid), MOPS (3- (N-morpholine) propane sulfonic acid), tricine (N-Tris (hydroxymethyl) methyl glycine) and phosphate buffer;
the reticulocyte detection kit comprises:
dyeing liquid: specific nucleic acid dye 0.20-1.00mmol
Organic solvent 1L
Dilution liquid:
further, the reticulocyte detection kit comprises:
dyeing liquid: 0.50-0.80mmol of specific nucleic acid dye
Organic solvent 1L
Dilution liquid:
the pH value adjusting agent mainly comprises hydrochloric acid, formic acid, sodium hydroxide and potassium hydroxide.
The osmolality is between 180 and 280mOsm/kg, preferably between 200 and 250 mOsm/kg.
Further, the diluent in the reticulocyte detection kit can also comprise a preservative, salt and the like. Typical preservatives are benzoic acid and its salts, sorbic acid and its salts, phenoxyethanol, formaldehyde and Proclin300, etc. The usage amount of the preservative is usually 0.05-3g/L, different concentrations of the preservative can be selected, and the normal function of the reagent in the effective period is ensured.
Preferably, the preservative is added to the diluent at a concentration of 0.2-1.0g/L.
In addition, in order to enhance the dispersion and solubilization of the reagent, a nonionic surfactant may be added to the diluent as appropriate;
the nonionic surfactant is a polyethylene glycol type nonionic surfactant or a Tween series milder surfactant, and the concentration of the nonionic surfactant is 0.3-0.5g/L.
Optimally, a reticulocyte assay kit comprising:
dyeing liquid: specific nucleic acid dye 0.70mmol
Dilution liquid:
use of a reticulocyte detection kit wherein the specific nucleic acid dye is used for reticulocyte detection.
The beneficial effects are that: as the dye used in the invention is a novel small-molecule dye, the dye can easily enter cells treated by the diluent and combine with nucleic acid substances in the molecules. Solves the technical difficulty that macromolecular dye is difficult to enter the interior of a molecule so that the dyeing effect is poor. Meanwhile, the dye used in the invention has low safety toxicity, and solves the defect of high toxicity of the common nucleic acid dye. The cells are treated by using the amphoteric surfactant and the nonionic surfactant, and after the cells are dyed by the dye, RET maturity is obviously differentiated and the result is accurate. And simultaneously, the content of the platelet can be measured.
The invention relates to a matched reagent of a blood cell analyzer, and the treated cells emit fluorescence with specific wavelength under the irradiation of laser, and the cells are distinguished and counted through the difference of scattered light and fluorescence intensity to obtain the proportion of reticulocytes with different light intensities. Thereby obtaining the test result.
Drawings
The invention relates to a reticulocyte detection kit, which is described in detail below with reference to the attached drawing figures and detailed description, wherein:
FIG. 1 is a schematic diagram of a scatter plot of a kit of the present invention on a companion blood cell analyzer;
FIG. 2 shows the results of the reagent of example 4 and the manual RET% control 1;
FIG. 3 shows the results of the reagent of example 4 and the manual RET# control 1;
FIG. 4 shows the results of the reagent of example 4 and the manual RET% control 2;
FIG. 5 shows the results of the reagent of example 4 and the manual RET# control 2;
FIG. 6 shows the results of the reagent of example 4 and the manual RET% control 3;
FIG. 7 shows the results of the reagent of example 4 and the manual RET# control 3.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and detailed description. It should be understood that the detailed description is presented herein for purposes of illustration only and is not intended to limit the invention.
Example 1:
a reticulocyte detection kit, a solution is prepared according to the following scheme:
dyeing liquid:
0.50mmol/L of dye of formula 5;
930mL of ethylene glycol;
70mL of ethanol;
dilution liquid:
mixing the dye of formula 5 with ethylene glycol and ethanol at the above ratio, stirring at room temperature until the dye is completely dissolved, filtering with 0.2 μm filter membrane, and sealing and keeping in dark place.
Mixing dodecyl imidazoline, tween 20, tris and Proclin300 according to the proportion, adding deionized water to 1L, stirring at room temperature until the mixture is completely dissolved, and adjusting the pH value to 8 by using acid or alkali. Filtering with 0.2 μm filter membrane, and storing.
The two reagents of this example were tested on a matched blood cell analyzer, the blank values met the requirements, and the results of measuring parameters related to reticulocytes are shown in table 1.
Table 1 example 1 measurement results
The results in Table 1 show that the RET% (percentage of reticulocytes) and RET# (total number of reticulocytes) of the reagent in this example on the matched blood cell analyzer deviate from the measurement results of the XN-1000 instrument within the allowable range, and the measurement results are accurate.
Example 2
The solution was prepared according to the following protocol:
dyeing liquid:
0.80mmol/L dye of formula 6;
930mL of ethylene glycol;
70mL of propanol;
dilution liquid:
mixing the dye of formula 6 with ethylene glycol and propanol at the above ratio, stirring at room temperature until the dye is completely dissolved, filtering with 0.2 μm filter membrane, and sealing and keeping in dark place.
Mixing tetradecyl imidazoline, tween 60, HEPES and ethylene glycol phenyl ether according to the proportion, adding deionized water to 1L, stirring at room temperature until the mixture is completely dissolved, and adjusting the pH value to 10 by using acid or alkali. Filtering with 0.2 μm filter membrane, and storing.
The two reagents of this example were tested on a matched blood cell analyzer, the blank values met the requirements, and the results of measuring parameters related to reticulocytes are shown in table 2.
Table 2 example 2 measurement results
The results in Table 2 show that the RET% (percentage of reticulocytes) and RET# (total number of reticulocytes) of the reagent in this example on the matched blood cell analyzer deviate from the measurement results of the XN-1000 instrument within the allowable range, and the measurement results are accurate.
Example 3:
the solution was prepared according to the following protocol:
dyeing liquid:
0.79mmol/L dye of formula 7;
930mL of ethylene glycol;
70mL of methanol;
dilution liquid:
mixing the dye of formula 7 with ethylene glycol and methanol at the above ratio, stirring at room temperature until the dye is completely dissolved, filtering with 0.2 μm filter membrane, and sealing and keeping in dark place.
The tetradecyl imidazoline, the glycol 400, the MOPS and the Proclin300 are mixed according to the proportion, deionized water is added to 1L, stirring is carried out at room temperature until the solution is complete, and the pH value is regulated to 8.5 by acid or alkali. Filtering with 0.2 μm filter membrane, and storing.
The two reagents of this example were tested on a matched blood cell analyzer, the blank values met the requirements, and the results of measuring parameters related to reticulocytes are shown in table 3.
TABLE 3 example 3 measurement results
The results in Table 3 show that the RET% (percentage of reticulocytes) and RET# (total number of reticulocytes) of the reagent in this example on the matched blood cell analyzer deviate from the measurement results of the XN-1000 instrument within the allowable range, and the measurement results are accurate.
Example 4:
the solution was prepared according to the following protocol:
dyeing liquid:
0.70mmol/L dye of formula 8;
930mL of ethylene glycol;
70mL of methanol;
dilution liquid:
mixing the dye of formula 8 with ethylene glycol and methanol at the above ratio, stirring at room temperature until the dye is completely dissolved, filtering with 0.2 μm filter membrane, and sealing and keeping in dark place.
Mixing dodecyl imidazoline, tween 20 and HEPES, proclin300 according to the above proportion, adding deionized water to 1L, stirring at room temperature until dissolution is complete, and adjusting pH to 8.5 with acid or alkali. Filtering with 0.2 μm filter membrane, and storing.
The two reagents of this example were tested on a matched blood cell analyzer, the blank values met the requirements, and the results of measuring parameters related to reticulocytes are shown in table 4.
Table 4 example 4 measurement results
The results in Table 4 show that the RET% (percentage of reticulocytes) and RET# (total number of reticulocytes) of the reagent in this example on the matched blood cell analyzer deviate from the measurement results of the XN-1000 instrument within the allowable range, and the measurement results are accurate.
Example 5:
the solution was prepared according to the following protocol:
dyeing liquid:
0.60mmol/L dye of formula 8;
ethylene glycol 900mL;
100mL of methanol;
dilution liquid:
mixing the dye of formula 8 with ethylene glycol and methanol at the above ratio, stirring at room temperature until the dye is completely dissolved, filtering with 0.2 μm filter membrane, and sealing and keeping in dark place.
Mixing decanyl imidazoline, tween 80, disodium hydrogen phosphate and sorbic acid according to the proportion, adding deionized water to 1L, stirring at room temperature until the mixture is completely dissolved, and adjusting the pH value to 8.5 by using acid or alkali. Filtering with 0.2 μm filter membrane, and storing.
The two reagents of this example were tested on a matched blood cell analyzer, the blank values met the requirements, and the results of measuring parameters related to reticulocytes are shown in table 5.
TABLE 5 example 5 measurement results
The results in Table 5 show that the RET% (percentage of reticulocytes) and RET# (total number of reticulocytes) of the reagent in this example on the matched blood cell analyzer deviate from the measurement results of the XN-1000 instrument within the allowable range, and the measurement results are accurate.
Example 6:
the solution was prepared according to the following protocol:
dyeing liquid:
0.20mmol/L dye of formula 8;
ethylene glycol 900mL;
100mL of methanol;
dilution liquid:
mixing the dye of formula 8 with ethylene glycol and methanol at the above ratio, stirring at room temperature until the dye is completely dissolved, filtering with 0.2 μm filter membrane, and sealing and keeping in dark place.
Mixing octaalkyl imidazoline, tween 80, tricine and formaldehyde according to the above proportion, adding deionized water to 1L, stirring at room temperature until dissolution is complete, and adjusting pH to 7 with acid or alkali. Filtering with 0.2 μm filter membrane, and storing.
The two reagents of the embodiment are tested on a matched blood cell analyzer, blank values meet requirements, and the measurement results of RET (percentage of reticulocytes) and RET# (total number of reticulocytes) of the reagents on the matched blood cell analyzer in the embodiment deviate from the measurement results of an XN-1000 instrument within an allowable range, and the measurement results are accurate.
Example 7:
the solution was prepared according to the following protocol:
dyeing liquid:
1.00mmol/L dye of formula 8;
ethylene glycol 900mL;
100mL of propanol;
dilution liquid:
mixing the dye of formula 8 with ethylene glycol and propanol at the above ratio, stirring at room temperature until the dye is completely dissolved, filtering with 0.2 μm filter membrane, and sealing and keeping in dark place.
Mixing dodecyl imidazoline, HEPES and benzoic acid according to the proportion, adding deionized water to 1L, stirring at room temperature until the mixture is completely dissolved, and adjusting the pH value to 11 by using acid or alkali. Filtering with 0.2 μm filter membrane, and storing.
The two reagents of the embodiment are tested on a matched blood cell analyzer, blank values meet requirements, and the measurement results of RET (percentage of reticulocytes) and RET# (total number of reticulocytes) of the reagents on the matched blood cell analyzer in the embodiment deviate from the measurement results of an XN-1000 instrument within an allowable range, and the measurement results are accurate.
Comparing with the manual method:
the kit in embodiment 4 of the present invention has a high correlation between RET (percentage of reticulocytes) and RET# (total number of reticulocytes) measurement results and manual (reference method WS/T346-2011 for reticulocyte count) measurement results on a matched instrument, wherein the sample size per test is more than 100 parts (more than half of samples are RET# is 0.02X10) 6 /μl). Specific comparison results are shown in fig. 2-7, and calculation results are shown in table 6.
TABLE 6 comparative correlation of the example 4 kit to the results of the manual assay
The embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present invention and the scope of the claims, which are to be protected by the present invention. In addition, although specific terms are used in the present specification, these terms are for convenience of description only and do not limit the present invention in any way.
Claims (10)
1. A reticulocyte assay kit comprising: a staining solution and a dilution solution, wherein the staining solution comprises a specific nucleic acid dye and an organic solvent for dissolving the specific nucleic acid dye, and the specific nucleic acid dye is a compound shown in a formula 3:
wherein R1, R2 and R3 are alkyl groups with less than four carbons, R4 is a sulfonic acid group, and X is a halogen atom;
the diluent comprises a buffering agent, at least one surfactant and water, and the pH value of the detection kit is 7-11;
the surfactant comprises an amphoteric surfactant, and the amphoteric surfactant is a compound shown in an imidazoline type 4:
r represents an alkyl group or an alkyl group having 8 to 16 carbon atoms.
2. The reticulocyte assay kit according to claim 1, wherein: the organic solvent is one or two of glycol, methanol, ethanol and propanol.
3. The reticulocyte assay kit according to claim 1, wherein: the buffer is selected from one or two of Tris, HEPES, MOPS, tricine and phosphate buffer.
4. The reticulocyte detection kit according to claim 1, wherein said staining solution: wherein the concentration of the specific nucleic acid dye is 0.20-1.00mmol/L; the dilution liquid: wherein each 1L of water contains 0.2-1.5g of amphoteric surfactant and 0.5-8.0g of buffering agent.
5. The reticulocyte detection kit according to claim 1, wherein said staining solution: wherein the concentration of the specific nucleic acid dye is 0.50-0.80mmol/L; the dilution liquid: wherein, each 1L of water contains 0.3-1.0g of amphoteric surfactant and 4.0-6.0g of buffer; the pH value is 8-10.
6. The reticulocyte detection kit according to any of claims 1-5, wherein said diluent further comprises a preservative comprising at least one of benzoic acid and salts thereof, sorbic acid and salts thereof, phenoxyethanol, formaldehyde, and Proclin 300.
7. The reticulocyte assay kit according to claim 6, wherein said preservative is present in said diluent at 0.2-4.0g per 1L of water.
8. The reticulocyte assay kit according to any one of claims 1-5, wherein said surfactant further comprises a nonionic surfactant, said nonionic surfactant being a glycol type nonionic surfactant or a tween series surfactant.
9. The reticulocyte assay kit according to claim 8, wherein said diluent comprises 0.3-0.5g nonionic surfactant per 1L water.
10. The use of the reticulocyte detection kit according to any one of claims 1 to 5 for preparing a reagent for measuring reticulocyte content.
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US20080102526A1 (en) * | 2006-10-30 | 2008-05-01 | Sysmex Corporation | Reagent, reagent kit and analyzing method |
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