CN109856409B - Application of hemoglobin control liquid for dry hemoglobin detection system - Google Patents
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Abstract
The invention relates to an application of a hemoglobin control solution in a dry hemoglobin detection system, which solves the technical problem that hemoglobin is gradually oxidized and directly dripped on dry hemoglobin detection test paper to penetrate the test paper to cause leakage pollution; introducing carbon monoxide gas into the hemoglobin liquid to prepare carboxyhemoglobin liquid; secondly, preparing thickening fluid; thirdly, mixing the carboxyhemoglobin liquid and the thickening liquid, and uniformly stirring to obtain the hemoglobin quality control liquid. The hemoglobin control solution can be stably stored for 30 days at room temperature, can be stably stored for more than 6 months in a refrigerator and can be stably stored for more than 1 year at the temperature of minus 20 ℃, so that the hemoglobin solution can be promoted to be transversely and uniformly diffused on the hemoglobin dry chemical detection test paper, and the leakage condition is greatly reduced.
Description
Technical Field
The invention relates to the technical field of medical examination, in particular to application of a hemoglobin control solution in a dry hemoglobin detection system.
Background
Hemoglobin (Hb) is a main component of erythrocytes, has a reversible binding function with oxygen molecules, and is a main carrier for transportation and exchange of oxygen in the human body. Pathophysiological changes in many diseases involve changes in Hb quality or quantity, such as physical examination of infants, training of athletes, health examination of women of childbearing age during pregnancy, and clinical examination for disease judgment related to hemoglobin reduction.
Hemoglobin analysis and detection systems, most of which use the reference hemoglobin method, the Cyania methemoglobin (HiCN) assay, recommended by the International Commission on standardization of hematology. The test process is that red blood cells in blood are firstly cracked by a hemolytic agent to release hemoglobin, then the hemoglobin reacts with the application liquid of the ferricyanide to generate stable ferricyanide Hemoglobin (HiCN), and the concentration of the hemoglobin in the blood is calculated by measuring the absorbance of the stable ferricyanide hemoglobin at 540 nm.
For a dry hemoglobin detection system, the following technical problems exist when red blood cells are used as quality control substances: 1. the cell membrane of the red blood cell is not broken, and the red blood cell can be cracked by a cracking agent during testing, so that the red blood cell curing technology cannot be adopted. At present, the average life of the blood bank red blood cells is 35 days, and the minimum requirement of the quality control substances sold in the market is half a year. 2. During the preservation of red blood cells, after the red blood cells are subjected to self-hemolysis, cell membrane fragments can form precipitates, the uniformity of a quality control substance is influenced, and the cell membrane fragments can influence the diffusion uniformity of hemoglobin on a dry hemoglobin test paper microporous filter membrane, so that the test result is influenced.
The existing hemoglobin liquid as a hemoglobin quality control substance has two technical problems: firstly, hemoglobin liquid is used as hemoglobin quality control liquid, and hemoglobin is gradually oxidized in the storage process, so that the detection value of the hemoglobin quality control liquid is unstable, and the real states of a reagent and an instrument cannot be monitored. Secondly, the hemoglobin liquid is directly dripped on the dry hemoglobin test paper, leakage can occur, the instrument is polluted, and the instrument detection is influenced.
Therefore, it is necessary to provide a method for preparing a hemoglobin-based control solution which can solve the above-mentioned problems.
Disclosure of Invention
The technical problem to be solved by the invention is that in the prior art, hemoglobin is gradually oxidized, and hemoglobin liquid is directly dripped on dry hemoglobin test paper to cause leakage. The application of the dry hemoglobin detection system has the characteristics of difficult oxidation and difficult leakage.
In order to solve the technical problems, the technical scheme is as follows:
use of a dry hemoglobin detection system, the use comprising a hemoglobin-based control fluid.
In the present invention, the preparation method of the hemoglobin quality control solution comprises:
step one, carrying out centrifugation, washing and cell lysis treatment on a blood sample, filtering to obtain a hemoglobin liquid, introducing carbon monoxide gas into the hemoglobin liquid, and preparing a carboxyhemoglobin liquid to prepare the carboxyhemoglobin liquid;
step two, preparing thickening fluid;
and step three, mixing the carboxyhemoglobin liquid and the thickening liquid, and uniformly stirring to obtain the hemoglobin quality control liquid.
The working principle of the invention is as follows: the thickening liquid is adopted in the hemoglobin control liquid prepared by the invention, and the thickening liquid can promote the transverse diffusion of the carboxyhemoglobin on the dry hemoglobin detection test paper and effectively reduce the leakage condition. The hemoglobin control solution prepared by the invention can highly simulate the diffusion behavior of blood, and is quite suitable for a dry hemoglobin detection system.
In the above scheme, for optimization, further, the preparation of the thickening fluid in the second step comprises: the method comprises the following steps of taking ultrapure water as a solvent, sequentially adding a buffer solution, a preservative, a surfactant, a cryoprotectant, an antioxidant and a thickening agent into the ultrapure water, and uniformly stirring in the adding process.
Furthermore, the concentration of the buffer solution in the thickening solution is 0-30 g/dL, the concentration of the preservative is 0-2 g/dL, the concentration of the surfactant is 0-10 g/dL, the concentration of the cryoprotectant is 0-90 g/dL, the concentration of the antioxidant is 0-20 g/dL, and the concentration of the thickening agent is 0-10 g/dL.
Further, the pH value of the buffer solution is 6-8, and the buffer solution is one or more of Tris (hydroxymethyl) aminomethane, hydrochloric acid, dibasic hydrogen phosphate, monobasic hydrogen phosphate and borate.
Further, the preservative is Proclin 150, proclin 300, sodium benzoate, potassium sorbate, naF, naN 3 And one or more of phenoxyethanol.
Further, the surfactant is one or more of sodium dodecyl sulfate, triton X-100, tween-20 and polyvinylpyrrolidone.
Further, the cryoprotectant is one or more of 1,2-propylene glycol, glycerol, glucose and sucrose.
Further, the antioxidant is one or more of Vc, ve, glycine, reduced glutathione, naDPH and nicotinamide.
Further, the thickening agent is one or more of carboxymethyl cellulose, sodium alginate, xanthan gum, polyacrylic acid, sodium polyacrylate, polyacrylamide and carbomer 940.
The invention adopts carboxyhemoglobin liquid as a quality control substance of a dry-type hemoglobin detection system, and the detection system adopts a cyanmethemoglobin (HiCN) determination method. The theoretical basis for substituting hemoglobin by carboxyhemoglobin is as follows: in the test process, the carboxyhemoglobin reacts with potassium ferricyanide, ferrous ions of porphyrin rings contained in the carboxyhemoglobin are oxidized into ferric ions, and the ferric ions are dissociated with carbon monoxide to generate the methemoglobin; when hemoglobin reacts with potassium ferricyanide, methemoglobin is also produced. The invention utilizes the characteristic that carboxyhemoglobin is not easily oxidized by oxygen, aims to obtain the high-stability hemoglobin quality control liquid, and is applied to a dry-type hemoglobin detection system.
The invention has the beneficial effects that: the hemoglobin control solution prepared by the invention can be stably stored for 30 days at room temperature (25 +/-5 ℃), and can be stably stored for 6 months at-20 ℃ for 1 year or even longer at refrigerator (2-8 ℃). The hemoglobin control solution prepared by the invention can highly simulate the diffusion behavior of blood, and is quite suitable for a dry hemoglobin detection system.
Drawings
The invention is further illustrated with reference to the following figures and examples.
FIG. 1 is a schematic flow chart of a method for preparing a hemoglobin-based control solution;
FIG. 2 is a comparison of the UV-VIS absorption spectra of the reaction products of carboxyhemoglobin and hemoglobin, respectively, with a Cyc application solution;
FIG. 3 is a field test chart of the application of the hemoglobin quality control solution to the URIT-12 hemoglobin detection system;
FIG. 4 is a liquid UV-VIS spectrum of the product of the reaction of hemoglobin-based control solution with a cyanide ferric application solution;
FIG. 5 is a graph showing a comparison of the diffusion of the hemoglobin control solution, blood and hemoglobin solution on the H12 hemoglobin reagent sheet.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
The present embodiment provides an application of a dry hemoglobin detection system, which uses a hemoglobin control solution, and a preparation method of the hemoglobin control solution, as shown in fig. 1, including the following steps:
step one, carrying out centrifugation, washing and cell lysis treatment on a blood sample, and filtering to obtain a hemoglobin liquid; introducing carbon monoxide gas into the hemoglobin liquid to prepare carboxyhemoglobin liquid;
the blood sample in the first step can be extracted from mammals such as human, pig, sheep, cattle and the like or other sources. In the prior art, the biological safety of a blood sample is detected, and the safety of the blood sample is ensured.
In this example, the blood sample was washed 3 times and centrifuged 3 times. It should be noted that the number of washing and the number of centrifugation may be set separately, and the number of washing and centrifugation is not limited to the number of times of the embodiment. The cell lysis treatment mode can adopt cracking agent, swelling, repeated freeze thawing and other modes.
Step two, preparing thickening liquid; adding buffer solution, antiseptic, surfactant, cryoprotectant, antioxidant and thickener into ultrapure water under stirring.
And step three, mixing the carboxyhemoglobin liquid and the thickening liquid, and uniformly stirring to obtain the hemoglobin quality control liquid.
The specific components and concentrations of the thickening fluid are shown in table 1 below:
TABLE 1 compositions and concentrations of thickening fluids
The solvent of the thickening solution is ultrapure water. In the embodiment of the invention, the amount of the carboxymethyl cellulose is added according to the viscosity of the thickening fluid required actually. The use amount of the carboxymethyl cellulose can influence the viscosity of the thickening solution, and finally influences the diffusion speed of the hemoglobin quality control solution on the surface of the microporous filter membrane of the dry hemoglobin test paper, thereby influencing the test result.
Preferably, the components and concentrations of the thickening fluid can be as shown in table 2:
TABLE 2 ingredients and concentrations of thickening fluids
| Composition of thickening fluid | Concentration (g/dL) |
| Borate salt | 15％ |
| Dihydrogen phosphate salt | 5 |
| Proclin | |
| 300 | 1‰ |
| Potassium sorbate | 1‰ |
| Polyvinylpyrrolidone | 1 |
| Glucose | |
| 2％ | |
| Glycerol | 5％ |
| Vc | 5‰ |
| Carboxymethyl cellulose | 0.5％ |
The hemoglobin control solution prepared in this example can be completely applied to a hemoglobin analysis system using a cyanized ferric iron (HiCN) method. Firstly, blood and the hemoglobin quality control solution of the invention are respectively reacted with cyanide ferric application solution, and then an ultraviolet-visible spectrophotometer is adopted to detect the absorbance of the product in the range of 200-800nm to obtain a light absorption spectrum contrast chart, as shown in figure 2. It can be observed from FIG. 2 that at 540nm, the absorbance curves of blood and the final product of the inventive hemoglobin-based control solution coincide. Therefore, experiments prove that the hemoglobin control solution provided by the invention can be completely suitable for a hemoglobin analysis system adopting a cyanamide ferric iron (HiCN) method.
Comparative example 1: the currently commercially available hemoglobin control solution is not suitable for URIT-12 hemoglobin analyzer, so step two in example 1 is omitted, no carbon monoxide is introduced into the hemoglobin solution, and the other steps are performed in the same manner. Finally, the hemoglobin liquid is mixed with the thickening liquid to obtain the hemoglobin quality control liquid of the comparison product 1.
Example 2:
the procedure for preparing the hemoglobin-based control solution of this example was the same as in example 1. The difference lies in the specific components and concentrations of the thickening liquid, as shown in table 3:
TABLE 3 ingredients and concentrations of thickening fluids
| Composition of thickening fluid | Concentration (g/dL) |
| Dibasic salts of hydrogen phosphate | 0-20％ |
| Dihydrogen phosphate salt | 0-20％ |
| Proclin 150 | 0-1％ |
| Potassium sorbate | 0-1％ |
| Tween-20 | 0-5％ |
| Sucrose | 0-5％ |
| Glycerol | 0-10％ |
| Nicotinamide | 0-10％ |
| Xanthan gum | 0-10％ |
The solvent is ultrapure water. The xanthan gum used in the embodiment of the invention needs to be added according to actual needs. The usage amount of xanthan gum influences the viscosity of the thickening solution, and finally influences the diffusion speed of the hemoglobin quality control solution on the surface of the microporous filter membrane of the dry hemoglobin test paper, thereby influencing the test result.
Preferably, the concentrations of the components of the thickening fluid are as shown in table 4:
table 4 example of concentration of ingredients of thickener
| Components of the thickening fluid | Concentration (g/dL) |
| Dibasic salts of |
10％ |
| |
2％ |
| Proclin 150 | 1‰ |
| Sodium benzoate | 5‰ |
| Tween-20 | 5‰ |
| |
2％ |
| Glycerol | 5 |
| Nicotinamide | |
| 2％ | |
| Xanthan gum | 0.6％ |
Comparative example 2: step two in example 2 was omitted, carbon monoxide was not introduced into the hemoglobin solution, and the other steps were performed in the same manner. Finally, mixing the hemoglobin liquid with the thickening liquid to obtain a reference substance 2, namely the hemoglobin control liquid.
Example 3:
the procedure for preparing the hemoglobin-based control solution of this example was the same as in example 1. The difference lies in the specific components and concentrations of the thickening liquid, as shown in table 5:
TABLE 5 ingredients and concentrations of thickening fluids
The solvent is ultrapure water. The amount of carbomer 940 used in the embodiment of the invention needs to be added according to actual needs. The use amount of the carbomer 940 influences the viscosity of the thickening solution, and finally influences the diffusion speed of the hemoglobin quality control solution on the surface of the microporous filter membrane of the dry hemoglobin test paper, so that the test result is influenced.
Preferably, the concentrations of the components of the thickening fluid are as shown in table 6:
table 6 example of concentration of ingredients of thickener
Comparative example 3: step two in example 3 was omitted, and the same procedure was followed for the other steps without introducing carbon monoxide into the hemoglobin solution. Finally, mixing the hemoglobin liquid with the thickening liquid to obtain a contrast 3 hemoglobin quality control liquid.
The 6 hemoglobin control solutions of example 1, comparative example 2, and comparative example 3 were applied to a dry hemoglobin detection system.
The dry hemoglobin detection system is URIT-12 hemoglobin analyzer and H12 hemoglobin reagent sheet. As shown in FIG. 3, the left side of FIG. 3 is H12 hemoglobin reagent sheet, the right side is URIT-12 hemoglobin analyzer, the hemoglobin quality control liquid of the present invention is detected, and the value displayed on the liquid crystal display screen is 8.0g/dL.
In order to monitor the use state of the URIT-12 hemoglobin analyzer and the matched reagent sheet thereof, the high-value, the medium-value and the low-value hemoglobin control solutions are prepared according to the requirements of general quality control substances, and the detection concentrations are respectively 20.0g/dL, 14.0g/dL and 8.0g/dL.
The hemoglobin control solution test value is further traced, and the actual concentration of hemoglobin in the high-value, medium-value and low-value hemoglobin quality control solution is detected by adopting a cyaniding ferric iron (HiCN) method. The liquid ultraviolet visible absorption spectrum of the product of the reaction of the hemoglobin control solution and the cyanide ferric application solution is shown in figure 4. Fig. 4 shows that the concentration gradient of the hemoglobin control solution with high value, medium value and low value is very obvious, and the hemoglobin control solution can play a good quality control effect on a URIT-12 hemoglobin detection system.
In order to observe the stability of the hemoglobin-based control solutions of the present invention, the hemoglobin-based control solutions of example 1 and comparative example 1 were stored at room temperature (25. + -. 5 ℃), refrigerator (2-8 ℃), -20 ℃ respectively, and tested under the same conditions using URIT-12 hemoglobin analyzer and a kit, and the results of the stability test are shown in Table 7.
Table 7 shows the results of the condition monitoring test of the URIT-12 hemoglobin test system using the hemoglobin control solution of example 1 and comparative example 1.
TABLE 7 State monitoring test results for example 1 and comparative example 1
Note: "+" indicates a normal result and "-" indicates an abnormal result.
Table 8 shows the results of the condition monitoring test of the URIT-12 hemoglobin test system using the hemoglobin control solution of example 2 and comparative example 2.
TABLE 8 State monitoring test results for example 2 and comparative example 2
Table 9 shows the results of the monitoring test of the condition of the URIT-12 hemoglobin test system using the hemoglobin control solution of example 3 and comparative example 3.
Table 9, example 3 and comparative example 3 State monitoring test results
The results in tables 7, 8 and 9 show that the hemoglobin control solution prepared in example 1 was stably preserved at room temperature for 30 days, at 2 to 8 ℃ for 6 months, and at-20 ℃ for 12 months, in the URIT-12 hemoglobin assay system; the stability of the hemoglobin control solution prepared by the reference 1 is obviously inferior to that of the hemoglobin control solution prepared in the example 1.
In order to observe the advantages of uniform diffusion and leakage prevention of the hemoglobin control solution of the present invention, the hemoglobin control solution, blood and hemoglobin solution of examples 1-3 were respectively dropped on the URIT-12 hemoglobin reagent sheet. As shown in fig. 5, the hemoglobin control fluid diffusion radius is close to that of blood, indicating that the diffusion speed is close to that of blood; whereas the hemoglobin fluid has a larger diffusion radius, indicating faster diffusion and partial fluid leakage is observed. Therefore, the hemoglobin control solution has the advantages of uniform diffusion and leakage prevention.
Although the illustrative embodiments of the present invention have been described above to enable those skilled in the art to understand the present invention, the present invention is not limited to the scope of the embodiments, and it is to be understood that all the inventions utilizing the inventive concept can be protected by those skilled in the art as long as various changes are within the spirit and scope of the present invention as defined and defined in the appended claims.
Claims (3)
1. The application of the hemoglobin quality control liquid in a dry hemoglobin detection system;
the method is characterized in that: the hemoglobin quality control solution is prepared by the following method:
step one, carrying out centrifugation, washing and cell lysis treatment on a blood sample, filtering to obtain a hemoglobin liquid, and introducing carbon monoxide gas into the hemoglobin liquid to prepare a carboxyhemoglobin liquid;
step two, preparing thickening fluid;
step three, mixing the carboxyhemoglobin liquid and the thickening liquid, and uniformly stirring to obtain a hemoglobin quality control liquid;
the method for preparing the thickening fluid in the second step comprises the following specific steps: adding a buffer solution, a preservative, a surfactant, a cryoprotectant, an antioxidant and a thickening agent into ultrapure water serving as a solvent in sequence, wherein the adding process needs to be uniformly stirred;
the concentration of a buffer solution in the thickening solution is 12-20g/dL, the concentration of a preservative is 0.2-0.6g/dL, the concentration of a surfactant is 0.5-1g/dL, the concentration of a cryoprotectant is 7-12g/dL, the concentration of an antioxidant is 0.5-2g/dL, and the concentration of a thickening agent is 0.5-1g/dL; the pH value of the buffer solution is 6-8, and the buffer solution is one or more of Tris (hydroxymethyl) aminomethane, hydrochloric acid, dibasic hydrogen phosphate, monobasic hydrogen phosphate and borate; the preservative is one or more of Proclin 150, proclin 300, sodium benzoate, potassium sorbate, naF, naN3 and phenoxyethanol; the surfactant is one or more of sodium dodecyl sulfate, triton X-100, tween-20 and polyvinylpyrrolidone; the cryoprotectant is one or more of 1,2-propylene glycol, glycerol, glucose and sucrose.
2. Use according to claim 1, characterized in that: the antioxidant is one or more of Vc, ve, glycine, reduced glutathione, naDPH and nicotinamide.
3. Use according to claim 1, characterized in that: the thickener is one or more of carboxymethyl cellulose, sodium alginate, xanthan gum, polyacrylic acid, sodium polyacrylate, polyacrylamide and carbomer 940.
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| CN111066776A (en) * | 2019-11-09 | 2020-04-28 | 无锡市人民医院 | Ovum freezing protective agent and application thereof |
| CN111024959A (en) * | 2019-12-20 | 2020-04-17 | 深圳市蔚景生物科技有限公司 | Stable protein solution, preparation method thereof and detection kit |
| CN111929446B (en) * | 2020-08-25 | 2022-05-03 | 郑州安图生物工程股份有限公司 | Kit for quantitatively and qualitatively detecting anti-LKM-1 antibody and preparation method thereof |
| CN117705541B (en) * | 2024-02-05 | 2024-04-26 | 江西赛基生物技术有限公司 | Quality control product for detecting PD-1 of blood sample and preparation and quality control method thereof |
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