CN115997754A - Preparation method of pretreatment liquid for urine exfoliated cells - Google Patents
Preparation method of pretreatment liquid for urine exfoliated cells Download PDFInfo
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- CN115997754A CN115997754A CN202211543565.5A CN202211543565A CN115997754A CN 115997754 A CN115997754 A CN 115997754A CN 202211543565 A CN202211543565 A CN 202211543565A CN 115997754 A CN115997754 A CN 115997754A
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- urine
- cells
- pretreatment liquid
- phosphate buffer
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a preparation method of a pretreatment liquid for urine exfoliated cells, which comprises the following steps: s1, adding 273.6mmol/L NaCl,5.2mmol/L KCl,16.2mmol/L LNa2HPO4 and 3.5mmol/L KH2PO4 into ultrapure water respectively to prepare 0.02mol/L phosphate buffer solution, and using DEPC water with water of 2 per mill; s2, mixing the prepared 0.02mol/L phosphate buffer solution with ethanol/methanol 1:1, mutually dissolving; s3, adding 3% of polyethylene glycol 1500,2% of mercaptoethanol, 0.1% of dithiothreitol and 0.1% of tween-20; s4, adding 0.83% of ammonium chloride, 0.1% of potassium bicarbonate and 0.04% of disodium ethylenediamine tetraacetate. The invention can keep the urine cells for a longer time before examination, can keep the cells in the urine, can keep the cells at room temperature for 3-5 days, and can directly carry out subsequent cytological detection, nucleic acid detection and the like.
Description
The application is a divisional application of a patent application named as 'preparation method of urine desquamation cell pretreatment liquid', the application date of the original application is 2022, 03 and 07, and the application number is 202210217490. X.
Technical Field
The invention relates to the technical field of cell treatment fluid, in particular to a preparation method of urine shed cell pretreatment fluid.
Background
Urine is produced by the kidneys, stored through the ureters to the bladder, and then expelled from the body through the urethra. Due to the physiological nature of urine, urine samples are common in clinical testing and are a very excellent source of liquid biopsy samples for urinary system tumors, where urine cytology is a pathological examination method used to discover urinary system malignancies.
Because urine shed cells are less in content and the proportion of abnormal cells is less, a morning urine sample is usually needed for ensuring enough shed cells, urine is more in quantity and the cell content is high, but because morning urine is more concentrated, organic components such as urea are high in concentration, ph value is lower, osmotic pressure is unfavorable for cell preservation, so that the normal temperature of urine cannot exceed 2 hours for the examination of abnormal cells of urine and the like, and the abnormal cells of urine can be preserved for 4 hours at 4 ℃, which greatly limits the application of the examination of abnormal cells of urine. However, most products stored in cells are only capable of maintaining the morphology, DNA and the like of the cells, and products stored in RNA are less.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a preparation method of pretreatment liquid for urine exfoliated cells, which can ensure that urine cells can be kept for a longer time before examination, and is convenient for subsequent examination of cell morphology and nucleic acid abnormality.
In order to achieve the above object, the present invention provides the following solutions:
a preparation method of a pretreatment liquid of urine exfoliated cells comprises the following steps:
s1, adding 273.6mmol/LNaCl,5.2mmol/LKCl,16.2mmol/LNa2HPO4 and 3.5mmol/LKH2PO4 into ultrapure water respectively to prepare 0.02mol/L phosphate buffer, and adding DEPC water with water of 2%o;
s2, mixing the prepared 0.02mol/L phosphate buffer solution with ethanol/methanol 1:1, mutually dissolving;
s3, adding 3% of polyethylene glycol 1500,2% of mercaptoethanol, 0.1% of dithiothreitol and 0.1% of tween-20;
s4, adding 0.83% of ammonium chloride, 0.1% of potassium bicarbonate and 0.04% of disodium ethylenediamine tetraacetate;
the phosphate buffer is used for an osmotic pressure maintaining agent; the ethanol/methanol/EDTA-Na is used as a fixing agent for inhibiting bacteria; the polyethylene glycol 1500 is used for maintaining the integrity of a cytoskeleton and preventing cell swelling; the dithiothreitol is used as a mucolytic agent; the ethylenediamine tetraacetic acid is used for an anti-aggregation reagent, so that cell accumulation is avoided; the CT value of the cell pretreatment liquid prepared by the preparation method of the urine exfoliated cell pretreatment liquid by using the ammonium chloride and the potassium bicarbonate for lysing erythrocytes is less than 20.
According to the specific embodiment provided by the invention, the invention discloses the following technical effects:
the invention can keep the urine cells for a longer time before examination, can keep the cells in the urine, can keep the cells at room temperature for 3-5 days, and can directly carry out subsequent cytological detection, nucleic acid detection and the like.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Reference herein to "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment may be included in at least one embodiment of the present application. The appearances of such phrases in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments. Those of skill in the art will explicitly and implicitly appreciate that the embodiments described herein may be combined with other embodiments.
The terms "first," "second," "third," and "fourth" and the like in the description and in the claims, are used for distinguishing between different objects and not for describing a particular sequential order. Furthermore, the terms "comprise" and "have," as well as any variations thereof, are intended to cover a non-exclusive inclusion. For example, inclusion of a list of steps, processes, methods, etc. is not limited to the listed steps but may alternatively include steps not listed or may alternatively include other steps inherent to such processes, methods, products, or apparatus.
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof.
Example 1
A preparation method of a pretreatment liquid of urine exfoliated cells comprises the following steps:
s1, adding 273.6mmol/L NaCl,5.2mmol/L KCl,16.2mmol/L LNa2HPO4 and 3.5mmol/L KH2PO4 into ultrapure water respectively to prepare 0.02mol/L phosphate buffer solution, and using DEPC water with water of 2 per mill;
s2, mixing the prepared 0.02mol/L phosphate buffer solution with ethanol/methanol 1:1, mutually dissolving;
s3, adding 3% of polyethylene glycol 1500,2% of mercaptoethanol, 0.1% of dithiothreitol and 0.1% of tween-20;
s4, adding 0.83% of ammonium chloride, 0.1% of potassium bicarbonate and 0.04% of disodium ethylenediamine tetraacetate.
In the above embodiment, the technical principle of each component is as follows:
phosphate buffer: an osmotic pressure maintenance agent;
ethanol/methanol/EDTA-Na: fixative to inhibit bacteria;
polyethylene glycol 1500: maintaining cytoskeletal integrity, preventing cell swelling;
dithiothreitol: a mucolytic agent;
ethylenediamine tetraacetic acid: an anti-aggregation agent that prevents cell accumulation;
ammonium chloride, potassium bicarbonate: red blood cells were lysed.
Example 2
As shown in Table 1, the pretreatment liquid for cells prepared by the method for preparing the pretreatment liquid for urinary shed cells of example 1 has good RNA preservation effect.
TABLE 1 RNA preservation Effect after treatment with cell preservation solution
The preparation method of the pretreatment liquid for the urine shed cells can enable the urine cells to be kept for a longer time before examination, can keep the cells in the urine, can be stored for 3-5 days at room temperature, and can be used for directly carrying out subsequent cytological detection, nucleic acid detection and the like.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The principles and embodiments of the present invention have been described herein with reference to specific examples, the description of which is intended only to assist in understanding the methods of the present invention and the core ideas thereof; also, it is within the scope of the present invention to be modified by those of ordinary skill in the art in light of the present teachings. In view of the foregoing, this description should not be construed as limiting the invention.
Claims (1)
1. The preparation method of the pretreatment liquid for the urine exfoliated cells is characterized by comprising the following steps:
s1, adding 273.6mmol/L NaCl,5.2mmol/L KCl,16.2mmol/L LNa2HPO4 and 3.5mmol/L KH2PO4 into ultrapure water respectively to prepare 0.02mol/L phosphate buffer solution, and using DEPC water with water of 2 per mill;
s2, mixing the prepared 0.02mol/L phosphate buffer solution with ethanol/methanol 1:1, mutually dissolving;
s3, adding 3% of polyethylene glycol 1500,2% of mercaptoethanol, 0.1% of dithiothreitol and 0.1% of tween-20;
s4, adding 0.83% of ammonium chloride, 0.1% of potassium bicarbonate and 0.04% of disodium ethylenediamine tetraacetate;
the phosphate buffer is used for an osmotic pressure maintaining agent; the ethanol/methanol/EDTA-Na is used as a fixing agent for inhibiting bacteria; the polyethylene glycol 1500 is used for maintaining the integrity of a cytoskeleton and preventing cell swelling; the dithiothreitol is used as a mucolytic agent; the ethylenediamine tetraacetic acid is used for an anti-aggregation reagent, so that cell accumulation is avoided; the ammonium chloride and the potassium bicarbonate are used to lyse red blood cells; the CT value of the cell pretreatment liquid prepared by the preparation method of the urine shed cell pretreatment liquid is less than 20.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202211543565.5A CN115997754A (en) | 2022-03-07 | 2022-03-07 | Preparation method of pretreatment liquid for urine exfoliated cells |
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| CN202211543565.5A CN115997754A (en) | 2022-03-07 | 2022-03-07 | Preparation method of pretreatment liquid for urine exfoliated cells |
| CN202210217290.XA CN114514923A (en) | 2022-03-07 | 2022-03-07 | Preparation method of pretreatment solution for urine exfoliated cells |
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| JPWO2010134246A1 (en) * | 2009-05-20 | 2012-11-08 | オリンパス株式会社 | Method for preparing nucleic acid-containing sample |
| CN108770836A (en) * | 2018-08-21 | 2018-11-09 | 生工生物工程（上海）股份有限公司 | Liquid based cell preservative fluid and its preparation method and application |
| CN110760567A (en) * | 2019-11-12 | 2020-02-07 | 杭州昱鼎生物科技有限公司 | Urine sample RNA stabilizing solution and preparation method thereof |
| CN112760318B (en) * | 2020-12-30 | 2023-11-17 | 苏州白垩纪生物科技有限公司 | Reagent composition for stabilizing nucleic acid molecules and application thereof |
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