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CN119023640A - A non-invasive urinary system urine tumor fluorescent colorimetric reagent and its use method - Google Patents

A non-invasive urinary system urine tumor fluorescent colorimetric reagent and its use method Download PDF

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Publication number
CN119023640A
CN119023640A CN202411501608.2A CN202411501608A CN119023640A CN 119023640 A CN119023640 A CN 119023640A CN 202411501608 A CN202411501608 A CN 202411501608A CN 119023640 A CN119023640 A CN 119023640A
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urinary system
phosphate
buffer
chromogenic reagent
fluorescent
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CN202411501608.2A
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Chinese (zh)
Inventor
任锋
李运生
李鹏飞
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Hangzhou Huidekang Medical Technology Co ltd
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Hangzhou Huidekang Medical Technology Co ltd
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Abstract

本发明公开一种无创泌尿系统尿液肿瘤荧光显色试剂及其使用方法，所述的荧光显色试剂组份由荧光素、稳定剂、抗漂白剂、抗凝剂和缓冲液组成，荧光显色试剂组份配比如下：氯化钠0.1%‑2%、丙三醇0.1%‑5%、乙二胺四乙酸或其钠盐或钾盐0.01%‑1%、荧光素含量0.001%‑0.01%、抗漂白剂浓度为1‑10 mmol/L，缓冲液浓度为0.01‑1 mol/L。在荧光素染色后，胞核呈黄绿色或黄色荧光，胞质中因存在较多RNA而呈橘红色或火焰色荧光，采用细胞形态学和化学荧光相结合特点，可同时观察细胞形态学和化学荧光变化，在敏感性和特异性弥补传统巴氏细胞学染色不足。

The present invention discloses a non-invasive urinary system urine tumor fluorescence colorimetric reagent and a method for using the same. The fluorescence colorimetric reagent component is composed of fluorescein, a stabilizer, an anti-bleaching agent, an anticoagulant and a buffer solution. The fluorescence colorimetric reagent component ratio is as follows: sodium chloride 0.1%-2%, glycerol 0.1%-5%, ethylenediaminetetraacetic acid or its sodium or potassium salt 0.01%-1%, fluorescein content 0.001%-0.01%, anti-bleaching agent concentration of 1-10 mmol/L, and buffer concentration of 0.01-1 mol/L. After fluorescein staining, the nucleus shows yellow-green or yellow fluorescence, and the cytoplasm shows orange-red or flame-colored fluorescence due to the presence of more RNA. The characteristics of combining cell morphology and chemical fluorescence are adopted, and cell morphology and chemical fluorescence changes can be observed simultaneously, which makes up for the insufficiency of traditional Papanicolaou cytology staining in terms of sensitivity and specificity.

Description

Noninvasive urinary system urine tumor fluorescent chromogenic reagent and application method thereof
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a noninvasive urinary system urine tumor fluorescent chromogenic reagent applied to bladder cancer and a use method thereof.
Background
The current clinical diagnosis of bladder cancer includes imaging, cystoscopy and biopsy methods, fluorescence in situ hybridization (Fluorescence in situ hybridization, FISH), urine abscission cytology and detection of bladder tumor markers (NMP 22, BTA) in urine, urine DNA methylation detection, etc. The advantages and disadvantages of these methods are briefly described below.
With the popularization of fluorescence microscopy, a fluorescent examination method (Acridine orange fluorescence, AO-F) for acridine orange staining of urine abscission cells is increasingly paid attention to, the method combines cell morphology with fluorescence chemistry, the defects of Papanicolaou staining are overcome in sensitivity and accuracy, and the displayed cell images are rapid and vivid and are easy to judge. In terms of detection principle, acridine orange can bind to DNA and RNA in cells simultaneously but shows fluorescence of different colors, with excitation peaks of about 492 nm and fluorescence emission peaks of 530 nm (DNA) and 640 nm (RNA). The binding mode of the fluorescent dye and the double-stranded DNA is to intercalate between double strands, and the fluorescent dye and the double-stranded DNA and RNA are accumulated on phosphate groups of the fluorescent dye by electrostatic attraction, and the different binding methods cause the difference of fluorescent colors. Under visible light excitation from 488-502 nm, the nuclei fluoresce green (about 530 nm), and the nucleoli and cytoplasmic RNAs fluoresce orange (about 640 nm). Malignant tumor cells grow rapidly, nucleic acid metabolism, especially DNA content is higher than that of normal cells, and abnormal increase of DNA content in the nucleus can be used as an important marker for identifying tumor cells. From morphology, the tumor cells have little cytoplasm, fluorescent orange or flame red, large nuclei and obvious deformity, mitotic phases, nuclear-to-plasma ratio imbalance and yellow-green or yellow fluorescence. Therefore, the acridine orange staining method combines the morphological characteristics of cells with the fluorescent chemistry characteristics of cells, overcomes the defect that Papanicolaou staining is simply dependent on the morphology of cells, and can be used as an auxiliary diagnosis method of bladder cancer. The identification of tumor cells by using acridine orange as a coloring agent is reported in the literature, but the methods are complicated to operate, have low detection rate of about 70%, and also fail to solve the problem that acridine orange is easy to photobleach. Therefore, the detection reagent for assisting and improving the bladder tumor by seeking a noninvasive, convenient and accurate diagnosis method has important scientific significance and clinical value for timely and accurately diagnosing early and recurrent bladder cancer.
Disclosure of Invention
Based on the analysis, the invention discloses a noninvasive urinary system urine tumor fluorescent chromogenic reagent and a use method thereof.
The technical scheme adopted by the invention is as follows: the fluorescent chromogenic reagent for the urine tumor of the noninvasive urinary system consists of fluorescein, a stabilizer, an anti-bleaching agent, an anticoagulant and a buffer solution, wherein the pH value of the fluorescent chromogenic reagent is 4.0-8.0;
The fluorescent chromogenic reagent comprises the following components in proportion: sodium chloride 0.1-2%, glycerol 0.1-5%, ethylenediamine tetraacetic acid or its sodium salt or potassium salt 0.01-1%, fluorescein 0.001-0.01%, bleaching inhibitor concentration 1-10 mmol/L, and buffer concentration 0.01-1 mol/L.
Preferably, the fluorescein is acridine orange.
Preferably, the buffer is one or more of a tris buffer, a citric acid-phosphate buffer, an acetic acid-acetate buffer or a phosphate buffer.
The phosphate in the citric acid-phosphate buffer solution is any one or a mixture of more than one of disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium phosphate and potassium phosphate.
Acetate in the acetic acid-acetate buffer solution is any one or two of sodium acetate and ammonium acetate.
The phosphate buffer solution is any one or two of sodium dihydrogen phosphate-disodium hydrogen phosphate or potassium dihydrogen phosphate-dipotassium hydrogen phosphate.
Preferably, the stabilizer is a glycerol-sodium chloride solution.
Preferably, the bleaching inhibitor is one or more of vitamin C, tri (2-carboxyethyl) phosphine, 6-hydroxy-2, 5,7, 8-tetramethyl-chromane-2-carboxylic acid, cyclooctatetraene or glutathione.
Preferably, the anticoagulant is ethylenediamine tetraacetic acid or a sodium salt or potassium salt thereof.
In another aspect, the invention provides a method for using a fluorescent chromogenic reagent for a urine tumor of a noninvasive urinary system, which comprises the steps of taking urine of a detected person for centrifugation at 50-200 mL, taking 10-15 mu L of cell residues at the bottom, taking 15-18 mu L of fluorescent chromogenic reagent for a urine tumor of a noninvasive urinary system, dripping the reagent on the glass for staining, adding the reagent under a fluorescent microscope for observation, and judging according to a detection result, wherein the detection result is as follows: observing the stained cytoplasm under a microscope to display orange or flame fluorescence; the nuclei are yellow-green or yellow fluorescent, and are large and irregular, namely, the bladder cancer tumor cells are judged, otherwise, the cells are normal.
The fluorescent chromogenic reagent has the following characteristics or advantages:
1. accurately distinguish: by adopting the combination characteristic of cell morphology and chemical fluorescence, the change of cell morphology and chemical fluorescence can be observed at the same time, and the defect of traditional Papanicolaou cytology staining is overcome in sensitivity and specificity;
2. high detection: the sensitivity and the specificity are high;
3. Interference elimination: the main clinical symptom of the urinary system tumor is hematuria, and the hemoglobin has fluorescence inhibition, so the method is not interfered by the hematuria;
4. Is not easy to bleach: acridine orange is easy to photobleaching, is unfavorable for observing cell states for a long time, is clinically limited, and overcomes the defect;
5. the determination is rapid: the Papanicolaou staining method or other staining procedures using acridine orange in the literature are complex, are not beneficial to large-scale screening, and the preparation method is simple, quick and easy to operate, and is accurate and quick when being used for fluorescent color development of the urine tumor of the noninvasive urinary system;
6. the application is wide: is suitable for various tissues of human body to shed cells.
Drawings
FIG. 1 is a schematic view of the present invention for detecting fluorescence imaging of a patient with bladder cancer.
Detailed Description
In order that the objects and advantages of the invention will become more apparent, the invention will be further described with reference to the following examples; it should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are merely for explaining the technical principles of the present invention, and are not intended to limit the scope of the present invention.
Example 1 phosphate-citrate buffer, ph6.0, was prepared: 8 g sodium chloride, 8 mL glycerol, 0.3 g ethylenediamine tetraacetic acid and 0.25 g Trolox are dissolved in 992 mL pure water, and the liquid is respectively prepared: 0.18 The pH of the mixture of the two solutions was adjusted to 6.0 with sodium hydroxide after mixing the two solutions of disodium hydrogen phosphate and citric acid of 0.08 mol/L. Preparing acridine orange stock solution: 1g of acridine orange is taken and dissolved in 1L pure water, namely 1 mg/mL of acridine orange stock solution. Preparing fluorescent staining application liquid with pH of 6.0: diluting 1 mg/mL of acridine orange stock solution 40 mL in 920 mL of phosphate-citrate buffer with pH of 6.0 to obtain fluorescent staining application solution; then adding a stabilizer glycerol-sodium chloride solution with the concentration of 1 mg/mL of 40 mL; 20mL of tris (2-carboxyethyl) phosphine at a concentration of 2 mmol/L was added; then adding 0.2g of ethylenediamine tetraacetic acid; the fluorescent chromogenic reagent for the urine tumor of the noninvasive urinary system is obtained.
The reagent prepared from the above example was designated sample 1.
Example 2 phosphate buffer pH7.0 was prepared, 10 g sodium chloride, 8 mL glycerol, 0.3 g ethylenediamine tetraacetic acid sodium salt and 1g Trolox were dissolved in 992 mL pure water, 0.1 mol/L disodium hydrogen phosphate solution and 0.1 mol/L monosodium phosphate were prepared from the solutions, respectively, and the pH was adjusted to 7.0 with potassium hydroxide after mixing the two. Preparing acridine orange stock solution: 0.8 g acridine orange is taken and dissolved in 1L pure water, namely 0.8 mg/mL acridine orange stock solution. Preparing pH7.0 fluorescent staining application liquid: taking 0.8 mg/mL of acridine orange stock solution 40 mL, diluting with 920 mL of phosphate buffer solution with pH7.0 to obtain fluorescent staining application liquid, and then adding a stabilizer glycerol-sodium chloride solution with the concentration of 1 mg/mL into 40 mL; adding 20mL of vitamin C with the concentration of 2 mmol/L as an anti-bleaching agent; then adding 0.1g of ethylenediamine tetraacetic acid; the fluorescent chromogenic reagent for the urine tumor of the noninvasive urinary system is obtained.
The reagent prepared from the above example was designated sample 2.
Example 3A pH5.5 acetic acid-sodium acetate buffer was prepared, 8 g sodium chloride, 10mL glycerol, 0.3. 0.3 g sodium ethylenediamine tetraacetate and 1.76. 1.76 g vitamin C were dissolved in 950mL pure water, 0.2 mol/L sodium acetate solution was prepared from this solution, and the pH was adjusted to 5.5 with glacial acetic acid. Preparing an acridine orange stock solution, and dissolving 1g of acridine orange in 1L of pure water to obtain 1 mg/mL of acridine orange stock solution. Preparing fluorescent dyeing application liquid with pH of 5.5, taking 1 mg/mL acridine orange stock solution 20mL, diluting with 990 mL of acetic acid-sodium acetate buffer solution with pH of 5.5 to obtain fluorescent dyeing application liquid, and then adding stabilizer glycerol-sodium chloride solution with concentration of 40 mL of 1 mg/mL; 20mL of 6-hydroxy-2, 5,7, 8-tetramethylchromane-2-carboxylic acid at a concentration of 2 mmol/L was added; then adding 0.2g of sodium ethylenediamine tetraacetate; the fluorescent chromogenic reagent for the urine tumor of the noninvasive urinary system is obtained.
The reagent prepared from the above example was designated sample 3.
Example 4, preparing phosphate-citrate buffer with pH of 8.0, dissolving 8g sodium chloride, 8 mL glycerol, 0.4 g ethylenediamine tetraacetic acid sodium salt and 2.86 g tris (2-carboxyethyl) phosphine in 952 mL pure water, preparing 0.25 mol/L disodium hydrogen phosphate solution and 0.15 mol/L citric acid buffer respectively from the above solutions, mixing the two solutions, and adjusting pH to 8.0 with potassium hydroxide; preparing an acridine orange stock solution, and dissolving 1g of acridine orange in 1L of pure water to obtain 1 mg/mL of acridine orange stock solution. Preparing fluorescent staining application liquid with pH of 8.0, taking 1 mg/mL acridine orange stock solution 30 mL, diluting with 970 mL phosphate-citrate buffer with pH of 8.0 to obtain fluorescent staining application liquid, and then adding stabilizer glycerol-sodium chloride solution with concentration of 40 mL of 1 mg/mL; 20mL of cyclooctatetraene or glutathione at a concentration of 2 mmol/L is added; then adding 0.5g of potassium ethylenediamine tetraacetate to obtain the noninvasive urinary system urine tumor fluorescent chromogenic reagent.
The reagent prepared from the above example was designated sample 4.
Example 5A pH5.0 citric acid buffer was prepared, 8g sodium chloride, 20mL glycerol, 0.3g sodium ethylenediamine tetraacetate and 8. 8mg cyclooctatetraene were dissolved in 940 mL pure water, and a 0.1 mol/L citric acid solution was prepared from this solution, and then the pH was adjusted to 5.0 with potassium hydroxide. Preparing an acridine orange stock solution, and dissolving 1g of acridine orange in 1L of pure water to obtain 1 mg/mL of acridine orange stock solution; preparing fluorescent dyeing application liquid with pH of 5.0, taking 1 mg/mL acridine orange stock solution 60 mL, diluting with 940 mL citric acid buffer solution with pH of 6.0 to obtain fluorescent dyeing application liquid, and then adding stabilizer glycerol-sodium chloride solution with concentration of 40 mL of 1 mg/mL; 20mL of tris (2-carboxyethyl) phosphine at a concentration of 2 mmol/L was added; then adding 0.6g of sodium ethylenediamine tetraacetate to obtain the noninvasive urinary system urine tumor fluorescent chromogenic reagent.
The reagent prepared from the above example was designated sample 5.
Example 6 phosphate-citrate buffer ph7.0 was prepared: 15g sodium chloride, 15 mL glycerol, 0.5g of sodium ethylenediamine tetraacetate and 3.07g of glutathione are dissolved in 945 and mL pure water, 0.18 mol/L disodium hydrogen phosphate solution and 0.1 mol/L citric acid solution are respectively prepared from the solutions, and the pH value of the two solutions is adjusted to 7.0 by potassium hydroxide after mixing. Preparing an acridine orange stock solution, and dissolving 0.8g acridine orange in 1L of pure water to obtain 0.8 mg/mL acridine orange stock solution. Preparing pH7.0 fluorescent staining application liquid, taking 0.8 mg/mL acridine orange stock solution 60 mL, diluting with 940mL pH7.0 phosphate-citrate buffer to obtain fluorescent staining application liquid, and then adding 20mL stabilizer glycerol-sodium chloride solution with the concentration of 1 mg/mL; 20mL of tris (2-carboxyethyl) phosphine at a concentration of 2mmol/L was added; then adding 0.8g of potassium ethylenediamine tetraacetate to obtain the noninvasive urinary system urine tumor fluorescent chromogenic reagent.
The reagent prepared from the above example was sample 6.
In the embodiment 7, urine 50 mL of a detected person is taken for centrifugation, supernatant is discarded, the supernatant is resuspended in 100 mu L PBS, 10-15 mu L of cell residues at the bottom are taken for smearing, 15-18 mu L of noninvasive urinary system urine tumor fluorescent chromogenic reagent is taken for dripping on the glass for staining and adding the cover glass, and the glass is placed under a fluorescent microscope for observation and is judged according to the detection result. Observing the stained cytoplasm under a microscope to display orange or flame fluorescence; the nuclei are yellow-green or yellow fluorescent, and are large and irregular, namely, the bladder cancer tumor cells are judged, otherwise, the cells are normal.
Samples 1-6 prepared from examples 1-6 stained for tumor markers as follows in Table 1:
TABLE 1
As can be seen from Table 1 and FIG. 1, the fluorescence chromogenic reagent for treating tumor in urine of the noninvasive urinary system prepared by the invention has higher occupation ratio and accurate effect on tumor cell staining marks. Wherein, the medium+ of the cell staining intensity indicates weak cell staining and incomplete cell staining; ++ means strong and complete cell staining intensity; ++ represents cells the dyeing degree is extremely high, the color is extremely high, the cell staining was complete and clear.
Example 1 was applied to the results of a hospital test in which test samples 1-10 were normal and test samples 11-20 were tumor patients, and the results were as shown in Table 2 below:
TABLE 2
From table 2, it is observed that 10 normal persons all detect no tumor cells, 10 patients detect tumor cells except 17 cases of the detected samples, and the detection rate of the present sample is 90%. Note that table 2 is used for testing in a hospital, and is affected by sample acquisition time and other factors, and the actual testing effect is slightly lower than the laboratory testing effect of table 1, but its 90% accuracy meets the existing testing needs. Wherein, Indicating that no tumor cells were detected, + indicating that tumor cells were detected.
Thus far, the technical solution of the present invention has been described in connection with the preferred embodiments shown in the drawings, but it is easily understood by those skilled in the art that the scope of protection of the present invention is not limited to these specific embodiments. Equivalent modifications and substitutions for related technical features may be made by those skilled in the art without departing from the principles of the present invention, and such modifications and substitutions will be within the scope of the present invention.

Claims (10)

1. The fluorescent chromogenic reagent for the urine tumor of the noninvasive urinary system is characterized by comprising fluorescein, a stabilizer, an anti-bleaching agent, an anticoagulant and a buffer solution, wherein the pH value of the fluorescent chromogenic reagent is 4.0-8.0;
The fluorescent chromogenic reagent comprises the following components in proportion: sodium chloride 0.1-2%, glycerol 0.1-5%, ethylenediamine tetraacetic acid or its sodium salt or potassium salt 0.01-1%, fluorescein 0.001-0.01%, bleaching inhibitor concentration 1-10 mmol/L, and buffer concentration 0.01-1 mol/L.
2. The non-invasive urinary system urine tumor fluorescent chromogenic reagent as claimed in claim 1, wherein said fluorescein is acridine orange.
3. The non-invasive urinary system urine tumor fluorescent chromogenic reagent as claimed in claim 1, wherein said buffer is one or more of a tris buffer, a citric-phosphate buffer, an acetic-acetate buffer or a phosphate buffer.
4. A non-invasive urinary system urine tumor fluorescent chromogenic reagent as claimed in claim 3, wherein the phosphate in said citric acid-phosphate buffer is any one or more of disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium phosphate or potassium phosphate.
5. A non-invasive urinary system urine tumor fluorescent chromogenic reagent as claimed in claim 3, wherein acetate in said acetate-acetate buffer is any one or two of sodium acetate and ammonium acetate.
6. A non-invasive urinary system urine tumor fluorescent chromogenic reagent according to claim 3, wherein the phosphate buffer solution is any one or two of sodium dihydrogen phosphate-disodium hydrogen phosphate or potassium dihydrogen phosphate-dipotassium hydrogen phosphate.
7. The non-invasive urinary system urine tumor fluorescent chromogenic reagent as claimed in claim 1, wherein said stabilizer is a glycerol-sodium chloride solution.
8. The non-invasive urinary system urine tumor fluorescent chromogenic reagent according to claim 1, wherein the bleach inhibitor is one or more of vitamin C, tris (2-carboxyethyl) phosphine, 6-hydroxy-2, 5,7, 8-tetramethylchromane-2-carboxylic acid, cyclooctatetraene or glutathione.
9. A non-invasive urinary system urine tumor fluorescent chromogenic reagent according to claim 1, wherein said anticoagulant is ethylenediamine tetraacetic acid or sodium or potassium salts thereof.
10. A method for using the noninvasive urinary system urine tumor fluorescent-developing reagent according to any one of claims 1-9, wherein the urine of a detected person is centrifuged at 50-200 mL, 10-15 [ mu ] L of bottom cell residues are smeared, 15-18 [ mu ] L of the noninvasive urinary system urine tumor fluorescent-developing reagent is dripped on the glass for staining and cover glass addition, and the glass is placed under a fluorescence microscope for observation.
CN202411501608.2A 2024-10-25 2024-10-25 A non-invasive urinary system urine tumor fluorescent colorimetric reagent and its use method Pending CN119023640A (en)

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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5693484A (en) * 1991-05-14 1997-12-02 Toa Medical Electronics Co., Ltd. Method of classifying and counting cells in urine
WO2001032916A2 (en) * 1999-11-04 2001-05-10 Abbott Laboratories Automated lysophospholipid assay and methods of detecting cancer
CA2563189A1 (en) * 1999-11-16 2001-05-25 Betzdearborn Inc. Method of stabilizing dye solutions and stabilized dye compositions
KR20030094901A (en) * 2002-06-10 2003-12-18 김종기 Early detection of lung cancer using acridine staining and fluorescence sputum cytology
KR20050005250A (en) * 2003-07-01 2005-01-13 한국수력원자력 주식회사 Rapid determination of apoptotic cells by acridine orange dye staining method
CN1649968A (en) * 2002-03-28 2005-08-03 科学和工业研究委员会 A kind of natural non-toxic multicolor fluorescent protein dye obtained from marine invertebrates and composition containing said dye and application thereof
CN107607509A (en) * 2017-09-11 2018-01-19 武汉科技大学 A kind of fluorescent staining liquid is used for the method that Activity of Hemocytes of Oncomelania Hupensis is detected and classified
CN113670696A (en) * 2021-07-28 2021-11-19 上海睿钰生物科技有限公司 Staining solution for cell staining and preparation method thereof
CN116008045A (en) * 2022-12-12 2023-04-25 上海兰桥生物科技有限公司 Reagent for quantitatively analyzing reticulocyte ligand in human whole blood and application thereof

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5693484A (en) * 1991-05-14 1997-12-02 Toa Medical Electronics Co., Ltd. Method of classifying and counting cells in urine
WO2001032916A2 (en) * 1999-11-04 2001-05-10 Abbott Laboratories Automated lysophospholipid assay and methods of detecting cancer
CA2563189A1 (en) * 1999-11-16 2001-05-25 Betzdearborn Inc. Method of stabilizing dye solutions and stabilized dye compositions
CN1649968A (en) * 2002-03-28 2005-08-03 科学和工业研究委员会 A kind of natural non-toxic multicolor fluorescent protein dye obtained from marine invertebrates and composition containing said dye and application thereof
KR20030094901A (en) * 2002-06-10 2003-12-18 김종기 Early detection of lung cancer using acridine staining and fluorescence sputum cytology
KR20050005250A (en) * 2003-07-01 2005-01-13 한국수력원자력 주식회사 Rapid determination of apoptotic cells by acridine orange dye staining method
CN107607509A (en) * 2017-09-11 2018-01-19 武汉科技大学 A kind of fluorescent staining liquid is used for the method that Activity of Hemocytes of Oncomelania Hupensis is detected and classified
CN113670696A (en) * 2021-07-28 2021-11-19 上海睿钰生物科技有限公司 Staining solution for cell staining and preparation method thereof
CN116008045A (en) * 2022-12-12 2023-04-25 上海兰桥生物科技有限公司 Reagent for quantitatively analyzing reticulocyte ligand in human whole blood and application thereof

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