CN115372350A - Single-detection double-judgment method for free heme - Google Patents
Single-detection double-judgment method for free heme Download PDFInfo
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Classifications
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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
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
Abstract
The invention discloses a single-detection double-judgment method for free heme, which is characterized in that a glass slide with a preset chromogenic reagent is used as a sample liquid bearing tool, sample liquid is dropwise added to the reagent of the glass slide to finish single-detection operation, if the free heme exists, the sample liquid reacts with the chromogenic reagent and then changes color, the color is combined with substances in cells to dye the cells, the color change condition of the sample liquid is visually observed, whether the cells are dyed or not is observed under a microscope, and whether the sample liquid contains the free heme or not is judged by a chemical chromogenic method and a pathological staining method. The state of the reactant is judged by two technologies in different fields through one operation, the two technologies are mutually verified and supplemented, and the result judgment is carried out by two medical means together.
Description
Technical Field
The invention relates to a detection method, in particular to a single-detection double-judgment method for free heme.
Background
A good screening reagent and detection method needs to have the following characteristics: fast, convenient, noninvasive, economical and accurate. With this standard control, there is currently no effective screening means. The method has the advantages of improving the detection sensitivity of the free heme, along with simple and convenient use, no pain, low price and easy acceptance, and can be popularized in the physical examination of large-area people and detected in numerous asymptomatic people.
In recent years, with the intensive research on the mutation of oncogenes and tumor suppressor genes induced by human epithelial cells, various growth factors and downstream signaling pathways, the important influence of the interaction between tumor cells and the microenvironment in which the tumor cells are located on the remodeling of tumor metabolism is gradually recognized. Numerous studies have shown that tumor cells initiate the pentose phosphate pathway of sugar metabolism, forming the "Warburg effect" of tumor cells. The main characteristic of the effect is abnormal carbohydrate metabolism of the mitochondria of cells, which is a pentose phosphate pathway. This pathway produces a large amount of reducing substances, which in turn activates the fenton's reaction in the cell, and the peroxidation reaction produces a large amount of free radicals, which destroy the enzyme with heme as the active group, thus producing Free Heme (FH), a marker of tumor cells. FH has the effect of peroxidases, activates the redox reaction of the combination of chemical substances containing hydrogen donor and oxygen donor, the final product of the reaction is blue, and the tissue cells in the specimen are stained blue, which is detectable. Therefore, FH can be through chemical color, histiocyte staining methods for qualitative detection. Currently, the clinical chemical color development method generally uses a test tube as a carrying tool, and the histocyte staining method uses a glass slide as a carrying tool, which are operated separately, and the clinical laboratory and the pathology laboratory operate separately, which brings great inconvenience to the clinic.
CN112485246A, a single epithelial tissue tumor cell exudate free heme chromogenic solution and a preparation method thereof, which provide a chromogenic solution required to be used by the invention, but do not indicate that a convenient and reliable detection method can be adopted.
Disclosure of Invention
In order to solve the technical problems and the difficulties, the invention provides a method for rapidly detecting free heme by taking a glass slide (reagent glass slide) with a preset specific reagent as a bearing body, dripping a specimen once, and combining two technical means of a chemical color development method and a pathological staining method into a whole.
A single-detection double-judgment method for free heme uses a glass slide with a preset color reagent as a sample liquid bearing tool, the sample liquid is dripped on the reagent of the glass slide to complete single-detection operation, if the free heme exists, the color is changed after reaction with the color reagent, the color is combined with substances in cells to dye the cells, the color change condition of the sample liquid is visually observed, whether the cells are dyed is observed under a microscope, and whether the sample liquid contains the free heme is determined by a chemical color development method and a pathological dyeing method.
Further, the color developing reagent comprises a reagent stock solution A containing a hydrogen donor and a reagent stock solution B containing an oxygen donor, and the color developing reagent is dried on the glass slide to form a film.
Furthermore, the specimen liquid is any one of nasopharyngeal effusion, rectal intestinal juice, sputum, cervical effusion, prostatic juice, uterine and uterine cavity effusion and vaginal body fluid.
Further, the dosage of the sample liquid is 50 microliters.
Further, the determination detection result is determined within 1 minute after the dripping of the sample solution.
Further, the chemical color development method is as follows: the color of the reactant generated by the specimen liquid can be directly observed by naked eyes, and the color can be changed into dark blue, light blue and colorless.
Further, the pathological staining method is to observe the staining of cells by reading the cells under a microscope, and 2 kinds of staining changes, namely blue staining and colorless staining can be presented.
Further, the double judgment means that the condition that blue appears by visual observation at the position of dripping the specimen liquid is positive by a chemical color development method; the pathological staining method is positive when the cell staining in the specimen liquid is blue under the microscope, and the test is judged to be positive when one of the chemical coloration method and the pathological staining method is positive.
Advantageous effects
1. The technology uses a reagent glass slide as a bearing tool, the sample to be detected is dripped at one time, the color development condition of the reactant is judged by naked eyes, and the staining condition of the histiocyte of the same reactant can be observed under a microscope.
2. The state of the reactant is judged by two technologies in different fields through one operation, the two technologies are mutually verified and supplemented, and the result judgment is carried out by two medical means together.
3. The technology is a bedside diagnosis, is rapid and convenient, can obtain a detection result in one minute, improves the working efficiency, saves the clinical cost, and is a scheme with high cost performance.
Detailed Description
The invention provides a single-detection double-judgment method for free heme, which is used for detecting a marker FH.
1. And (4) manufacturing a reagent glass slide.
The reagent glass slide is prepared by presetting quantitative reagent stock solution A and reagent stock solution B on a glass slide, and drying to form a film.
Further, the reagent stock solution a was prepared as follows (taking 100mL as an example):
step 1: 15g of anhydrous sodium dihydrogen phosphate is dissolved in 90mL of purified water; 0.8-1.2 g of anhydrous disodium hydrogen phosphate is dissolved in the supernatant to prepare 90mL of phosphate buffer.
Step 2: adding 0.08-0.12 g of 3,3', 5' -tetramethyl benzidine into 10mL of absolute ethyl alcohol for dissolving, adding into the buffer solution prepared in the step 1, and stirring until the mixture is completely dissolved.
Further, the reagent stock solution B was prepared as follows:
step 1: adding 0.08-0.12 mL of cumyl hydroperoxide into 100mL of purified water, and stirring until the cumyl hydroperoxide is completely dissolved;
step 2: 0.08mL to 0.12mL of 6-methoxyquinoline was added to the above solution, and the mixture was stirred until it was completely dissolved.
Further, the reagent slide is prepared as follows:
step 1: transversely placing a glass slide, and dripping the reagent stock solution A:50 microliter, gently shaking the glass slide to make the coating uniform, and drying in a constant temperature drying oven at 60 ℃ to form a reagent film.
Step 2: and dripping the reagent stock solution B:50 microliter, gently shaking the glass slide to make the coating uniform, and drying in a constant-temperature drying oven at 60 ℃ to form a reagent film.
And step 3: and hermetically packaging the slices by using a separated pathological section storage box.
Further, the packaged reagent slide is irradiated and sterilized outside and stored for later use.
2. Collecting a specimen:
the specimen detected by the technology is any one of nasopharyngeal effusion, rectal intestinal juice, sputum, cervical effusion, prostatic juice, uterine cavity effusion and vaginal body fluid.
Taking nasopharyngeal liquid as an example, the method comprises the following steps: selecting a proper nasopharynx swab to be put into the nasopharynx from the nasal cavity, enabling the nasopharynx swab to reach the nasopharynx along the inferior nasal passage, rotating the nasopharynx and the nasopharynx by 180 degrees from left to right, and wiping the nasopharynx and the nasopharynx, the eustachian tube and the pharyngeal orifice, the eustachian tube round pillow and the pharyngeal hidden tube in sequence to obtain nasopharynx seepage; the liquid sampling amount is 0.5 to 1mL, and the degree of wetting of the whole sampling swab is generally used. If the subject is too sensitive, local surface anesthesia may be appropriate, or the specimen may be collected under indirect laryngoscope.
The nasopharyngeal swab consists of a sampler head and a handle. The head of the sampler adopts nylon flocking technology, and has strong water absorption and hydrophobicity. Can easily suck the nasopharyngeal liquid after reaching the nasopharyngeal part.
Whether the tissue exudate of the part where the nasopharyngeal carcinoma is located can be obtained is the key of the accuracy of the detection result. Because the tumor is deep, and blind extraction is added, accurate extraction is not possible, and false negative results. Therefore, it is very important to obtain the materials comprehensively, accurately and even repeatedly.
Other standard solutions may be obtained by similar methods.
And (3) putting the sampling swab into 1mL of purified water in a test tube, stirring to uniformly mix the sample on the swab with the purified water to prepare a sample solution, and covering and sealing the sample test tube for later use.
3. Detailed description of the preferred embodiments
(1) And opening the outer package of the prepared reagent slide, and taking out one reagent slide.
(2) The cap of the collected specimen test tube was opened, and 50. Mu.l of the specimen liquid was aspirated by a pipette.
(3) And (3) flatly holding the reagent glass slide, dripping the sample liquid on the middle right side of the reagent film position, and uniformly coating the sample by using a pipette tip.
(4) The test results were evaluated within 1 minute.
4. And (4) judging the standard:
(1) Visual inspection (chemochromic method): direct observation of the reactant color produced by the above embodiments can present 4 possible color changes of dark blue, light blue, and colorless.
(2) Microscopic slide reading (pathological staining method): using a high power lens to observe the staining of cells in the reaction generated on the reagent slide, 2 possible staining changes of blue and colorless can be presented.
(3) And (4) judging a result: the results of the test are determined according to the following table, based on the indications presented by the reactants:
determination standard of pathological staining method by chemical color development method
Dark blue, light blue-positive
Positive by colorless blue
Colorless and colorless negative
As can be seen from the above table, if the chemochromic method is positive, the pathological staining interpretation step can be omitted; if the color is not developed by the chemical color development method, the color is further judged by a microscope. Only if the pathological staining method is positive, the test can be determined as positive reaction. Positive reaction shows the sample containing FH.
The above expressions show that the single-detection double-judgment FH determination method has the beneficial effects of convenience, rapidness, economy, accuracy and bedside diagnosis.
Those who do not specify specific conditions in the above description proceed according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
To illustrate the scientificity and innovation of the present invention, the mechanism of producing Free Heme (FH) in nasopharyngeal carcinoma tissue cells and the principle of detecting FH with the reagent of the present invention are demonstrated as follows:
FH is an important member of the heme family, is one of the forms of heme present in humans, and is the active group of heme-binding proteins. It is made up by combining protoporphyrin IX with ferrous ion. Based on different molecular structures and functions, the tumor is divided into more than ten subtypes such as heme a, b, c, o and the like, is released in the process of epithelial cell tumorigenesis, and belongs to a medicine-related biomarker. Carcinogens cause epithelial sugar metabolism to be abnormal, tumor cells consume ten times as much sugar as normal cells, and the pentose phosphate pathway is metabolically active, known as the "warburg effect". In the process of transferring energy metabolism electrons of tumor cells, hydroxyl free radicals, peroxy radicals and the like generated by Fenton's reaction cause oxidative damage to mitochondria, change the polarity magnitude of cell protein, release FH (hemoglobin binding protein) in a hydrophobic nucleus (also called a hemoglobin pocket) of hemopexin in a mitochondrial basement membrane, increase the FH content in exudate, and the release amount of FH is positively correlated with the degree of epithelial cell canceration.
FH has the effect of peroxidase, catalyzing the oxidation-reduction reaction with benzidine as the main participant. A reagent stock solution A preset in the reagent slide manufactured by the technology contains hydrogen donor dianiline, and a reagent stock solution B contains oxygen donor cumyl hydroperoxide. Benzidine, which contains two readily oxidizable amino groups, can be oxidized to aniline blue by both one-electron and two-electron pathways. The 371nm and 652nm bands are charge transfer complexes with benzidine as donor and a diimine ion (TNB 2) acceptor. The benzidine deoxidizes the peroxide by the electron transfer principle of quantum mechanics, and oxidizes the hydrogen donor into the substance benzidine blue containing the color-developing group. The benzidine blue is extremely unstable and continues to be oxidized to generate the benzidine hydrazone. Benzidine blue appears blue in the reactant solution, which binds to iron ions in the tissue cells to make the cells blue. Whether the reactant solution is blue or not or/and whether the reactant cells are stained blue or not can be used for displaying whether FH exists in the specimen liquid or not, but the observation time is not suitable to be too long so as to prevent the benzidine blue from being converted into the benzidine hydrazone to lighten the blue color.
To demonstrate the effectiveness of the present invention, verification of the sensitivity, specificity and jordan index of the method of single-test double-judge mode reagent slides is provided herein, as well as the EB virus antibody (VCA/IgA) test (commercially available finished kit) as a control, with the following data:
sensitivity (sensitivity): the sensitivity is also called as the proportion that the screening method can correctly judge the actually sick people as patients; specificity (specificity): this is a ratio in which a screening method can accurately judge a person who is actually free of a disease as a non-patient.
1. Sensitivity verification
1.1 Specimen source: 100 parts of nasopharyngeal liquid seepage specimens of nasopharyngeal cancer patients are contained in a clinical laboratory of a certain medical unit. Nasopharyngeal carcinoma is diagnosed by pathology.
1.2 The detection method comprises the following steps: the single-check double-judgment mode reagent slide prepared by the invention is used for carrying out FH detection on the 100 samples and judging according to the prepared FH detection. Meanwhile, a control study was performed with a commercially available EB virus antibody (VCA/IgA) detection reagent. The results of the measurements are shown in the following table.
| Detection method | Positive for | Negative of | Suspected positivity | Sensitivity (%) |
| Single-detection double-judgment mode reagent slide | 88 | 11 | 1 | 88 |
| EB virus antibody (VCA/IgA) detection | 46 | 50 | 4 | 50 |
As can be seen from the above table, the sensitivity difference of the two methods used as the marker detection samples is high. Statistically, the single-test double-judgment mode reagent slide test has a very significant difference (P < 0.01) compared with the detection sensitivity of EB virus antibody (VCA/IgA).
2. Specificity verification
2.1 specimen origin: 100 parts of nasopharyngeal liquid specimen of a patient with non-nasopharyngeal carcinoma, which is contained in the same clinical laboratory of the medical unit in the step 1. Patients with non-nasopharyngeal carcinoma need pathological examination for other reasons, and all patients with non-nasopharyngeal carcinoma are excluded.
2.2 detection method: in the same way as in step 1.2, the results are shown in the following table.
| Detection method | Positive for | Negative of | Suspected positivity | Degree of specificity (%) |
| Single-detection double-judgment mode reagent slide | 1 | 93 | 6 | 93 |
| EB virus antibody (VCA/IgA)) Detection of | 7 | 57 | 36 | 57 |
As can be seen from the above table, the specificity difference between the two methods used as the marker for detecting the sample is high. Through statistical treatment, the single-detection double-judgment mode reagent slide test has a very significant difference (P < 0.01) compared with the detection specificity of EB virus antibody (VCA/IgA).
3. Joden index
The joden index refers to: the sum of the sensitivity and the specificity minus 1 represents the total ability of the screening method to find real patients and non-patients, and the larger the index is, the better the screening test is and the greater the authenticity is.
3.1 The johnson index of single-test double-judgment mode reagent slide detection is 0.81;
3.2 The john index of EB virus antibody (VCA/IgA) detection is 0.07;
3.3 Statistically, the single-test double-judgment mode reagent slide test also has very significant difference (P < 0.01) compared with EB virus antibody (VCA/IgA) detection Youyang index.
In conclusion, the FH detection contained in the nasopharyngeal effusion of the single-detection double-judgment mode reagent slide is used for detecting the canceration condition of the nasopharyngeal epithelial cells for the first time, the surprising result is that the sensitivity reaches 88%, the specificity reaches 93%, the Youyang index reaches 0.81, each index has obvious statistical difference with the detection and detection of EB virus antibody (VCA/IgA), the missed diagnosis rate and the misdiagnosis rate are both very low, the requirements of a screening reagent are met, the method is suitable for screening the nasopharyngeal carcinoma of a large-data population, and the method is convenient and quick and is a biologically suitable technology for screening the nasopharyngeal carcinoma.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A single-detection double-judgment method for free heme is characterized in that a glass slide with a preset color reagent is used as a sample liquid carrying tool, the sample liquid is taken and dripped at a reagent position of the glass slide to complete single-detection operation, if the free heme exists, the color of the free heme reacts with the color reagent and then is changed, the free heme is combined with substances in cells to dye the cells, the color change condition of the sample liquid is visually observed, whether the cells are dyed is observed under a microscope, and whether the free heme is contained in the sample liquid is judged by a chemical color development method and a pathological dyeing method.
2. The method of claim 1, wherein the chromogenic reagent comprises a benzidine reagent stock solution A containing a hydrogen donor and a cumene hydroperoxide reagent stock solution B containing an oxygen donor, and the chromogenic reagent is dried on a glass slide to form a film.
3. The method for single-test and double-test of free heme according to claim 1, wherein the sample solution is any one of nasopharyngeal fluid, rectal fluid, sputum, cervical fluid, prostatic fluid, uterine fluid, and vaginal fluid.
4. The method for detecting free heme individually and determining free heme according to claim 1, wherein the amount of the sample solution is 50 microliters.
5. The method for single-test and double-test of free heme according to claim 1, wherein the determination result is determined within 1 minute after the sample solution is added.
6. The method for detecting free heme by single test and double judgment according to claim 1, wherein the chemochromic method is: the color of the reactant generated by the specimen liquid can be directly observed by naked eyes, and the color can be changed into dark blue, light blue and colorless.
7. The method for single-examination and double-judgment of free heme according to claim 1, wherein the pathological staining method is that the staining condition of cells is observed by reading under a microscope, and 2 staining changes of blue and colorless can be presented.
8. The single-detection double-judgment method for free heme according to claim 1, wherein the double judgment means that the positive of a chemochromic method is observed by naked eyes when a specimen solution is dripped; the pathological staining method is positive when the cell staining in the specimen liquid is blue under the microscope, and the test is judged to be positive when one of the chemical coloration method and the pathological staining method is positive.
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