CN110243651B - Reticulocyte detection kit and application thereof - Google Patents

Reticulocyte detection kit and application thereof Download PDF

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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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reticulocyte
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请求不公布姓名
余晓尘
舒家发
李春晖
伍耀婵
崔晓磊
辛佩轩
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Shenzhen Dymind Biotechnology Co Ltd
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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
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    • G01—MEASURING; TESTING
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    • G01—MEASURING; TESTING
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    • G—PHYSICS
    • G01—MEASURING; TESTING
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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

Reticulocyte detection kit and application thereof
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
Figure BDA0001590208290000021
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
Figure BDA0001590208290000022
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:
Figure BDA0001590208290000031
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:
Figure BDA0001590208290000041
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:
Figure BDA0001590208290000042
further, the reticulocyte detection kit comprises:
dyeing liquid: 0.50-0.80mmol of specific nucleic acid dye
Organic solvent 1L
Dilution liquid:
Figure BDA0001590208290000043
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:
Figure BDA0001590208290000051
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:
Figure BDA0001590208290000071
Figure BDA0001590208290000072
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
Figure BDA0001590208290000081
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:
Figure BDA0001590208290000082
Figure BDA0001590208290000091
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
Figure BDA0001590208290000092
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:
Figure BDA0001590208290000101
Figure BDA0001590208290000102
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
Figure BDA0001590208290000111
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:
Figure BDA0001590208290000112
Figure BDA0001590208290000121
Figure BDA0001590208290000122
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
Figure BDA0001590208290000131
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:
Figure BDA0001590208290000132
Figure BDA0001590208290000141
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
Figure BDA0001590208290000142
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:
Figure BDA0001590208290000151
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:
Figure BDA0001590208290000152
Figure BDA0001590208290000161
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
Figure BDA0001590208290000162
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:
Figure FDA0004134214600000011
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:
Figure FDA0004134214600000012
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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