A method for fluorescent staining of urinary exfoliated cells

Abstract

The application discloses a urine exfoliative cell fluorescent staining method, which can be applied to non-invasive screening of urothelial tumors. In the application, DAPI can be combined with cell nucleus DNA strongly and stably, and even in the case that acridine orange staining fails due to inflammation, degeneration and other factors, DAPI can clearly and completely outline the shape and profile of the cell nucleus, and completely solves the problems of uneven and non-staining of the cell nucleus in the prior art. Meanwhile, acridine orange makes the cytoplasm present green fluorescence, and DAPI makes the cell nucleus present blue fluorescence, the color distinction degree of the two is high, and they are not overlapped, so that an interpreter can easily distinguish the cell nucleus and the cytoplasm, accurately identify key tumor indexes such as nuclear atypia and nucleoplasm ratio, effectively avoid mistakes of considering hemoglobin and other impurities as tumor cells, significantly reduce the false positive and false negative rates, and improve the interpretation accuracy and consistency.

Description

Technical Field

[0001] This invention relates to the field of biological sample staining technology, specifically to a method for fluorescent staining of exfoliated urine cells. Background Technology

[0002] Urine exfoliative cytology is the preferred non-invasive screening method for urothelial tumors of the urinary system, such as bladder cancer, ureteral cancer, and renal pelvis cancer, with advantages such as ease of operation, high patient compliance, and repeatability. Traditional urine exfoliative cytology often uses hemoglobin (HE) staining, but because it cannot effectively distinguish between hemoglobin and tumor cells in urine sediment, the staining process can stain hemoglobin, leading to a high false-positive rate and affecting diagnostic accuracy.

[0003] To address these issues, fluorescent staining techniques were introduced into this field. Acridine orange is a fluorescent chrome yellow dye that binds to double-helical DNA molecules via insertion, forming an AO-DNA complex. The edematic acid in the dye breaks down RNA molecules into single strands and, through electrostatic adsorption, attaches to these single-stranded RNA molecules, gradually accumulating to form an AO-RNA complex. Because DNA and RNA adsorb acridine orange differently, they exhibit different fluorescence reactions. Bladder cancer cells show different fluorescence colors due to differences in nucleic acid content in the cytoplasm and nucleus. The cytoplasm and nucleolus, containing more RNA, display orange-red or flame-red fluorescence; while the chromosomes in the nucleus, containing a large amount of DNA, display yellow-green or yellow fluorescence. Normal cells exhibit homogeneous green or yellow-green fluorescence under a fluorescence microscope. However, in apoptotic cells, due to chromatin breakage and condensation, acridine orange accumulates, emitting yellow-green fluorescence. The binding of acridine orange to ethidium bromide can also distinguish dead cells. Compared to traditional hematoxylin and eosin (HE) staining for urine exfoliative cytology, AO-F staining is simpler, more efficient, and more sensitive, simplifying the work of pathologists and improving examination efficiency. Secondly, AO-F staining does not stain hemoglobin during the process, significantly reducing the false positive rate in urine exfoliative cytology. Therefore, based on the fluorescence changes observed in AO-F staining combined with cell morphological changes, it is easy to determine whether cells are benign or malignant, demonstrating high specificity.

[0004] However, in actual clinical applications, due to the complex conditions of patients with urothelial tumors, often accompanied by inflammation, drug treatment, or surgical effects, and the variable composition of urine, the staining effect of acridine orange is extremely unstable, frequently resulting in problems such as light staining of cell nuclei, uneven staining, or even complete lack of staining. Figure 1 , Figure 2As shown, this directly leads to difficulties in interpreting staining results, resulting in false negatives and false positives, severely limiting its clinical application value. Therefore, developing a method that can stably and clearly stain urinary exfoliated cells, especially cell nuclei, in a complex urinary environment is crucial for improving the accuracy of urinary cytology. Summary of the Invention

[0005] The purpose of this invention is to provide a method for fluorescent staining exfoliated cells in urine, so as to solve the technical problems in existing fluorescent staining techniques for exfoliated cells in urine, such as unstable staining of cell nuclei, light staining, and no staining due to the complex composition of urine.

[0006] This invention is implemented by the following technical solution: A method for fluorescent staining of exfoliated urine cells includes the following steps: (1) Preparation of dye solution: Acridine orange dye solution and DAPI dye solution were prepared separately, and the prepared dye solutions were aliquoted in the dark and stored in an environment of -20℃. (2) Sample pretreatment: Take a urine sample, centrifuge at 3000 rpm for 5-7 minutes, collect the cell pellet, and resuspend and fix it with 200 μL of 95% ethanol to obtain a cell suspension; (3) Slide preparation: Take 20 μL of the cell suspension obtained in the specimen pretreatment in step (2), spread it evenly on a glass slide, and dry it naturally or by heating to make a cell smear; (4) Two-color fluorescent staining: Add 1 drop of acridine orange staining solution prepared in step (1) to the cell smear obtained in step (3) and stain at room temperature in the dark for 60 seconds. Then add 1 drop of DAPI staining solution prepared in step (1) and stain at room temperature in the dark for 60 seconds. Pour off the excess staining solution, add a coverslip, and obtain the stained sample. (5) Microscopic examination and interpretation: The stained sample obtained in step (4) dual-color fluorescence staining is placed under a fluorescence microscope and observed and interpreted through dual channels. The DAPI channel shows that the cell nucleus is blue fluorescent and the acridine orange channel shows that the cytoplasm is green fluorescent. The results are interpreted according to the Paris Reporting System for Urine Cytology 2022.

[0007] Furthermore, in step (1) of the preparation of the dye solution, the method for preparing the acridine orange dye solution is as follows: weigh acridine orange powder and dissolve it in sterile deionized water to prepare a 1 mg / mL solution, vortex for 2-3 minutes until completely dissolved, and then filter it through a disposable needle filter to remove bacteria. The method for preparing DAPI staining solution is as follows: Weigh DAPI powder and dissolve it in 1×PBS buffer with a pH of 7.2-7.4 to prepare a 1 mg / mL solution. Vortex for 1-2 minutes until completely dissolved, and then filter through a disposable needle filter to remove bacteria.

[0008] Furthermore, the acridine orange powder is of molecular biology grade with a purity of ≥95%.

[0009] Furthermore, the acridine orange staining solution and DAPI staining solution should be dispensed in 100-200 μL / tube, stored at -20℃ protected from light, and have a shelf life of 12 months.

[0010] Furthermore, in step (2) of specimen pretreatment, the urine sample is the first half of the second morning urine or the first half of the urine collected more than 2 hours after the last urination. The sample volume is 30-50 mL, and it should be stored at room temperature for no more than 2 hours or at 2-8°C for no more than 24 hours. If the cell count is insufficient, the cell suspension resuspended in ethanol should be transferred to a 1.5 mL centrifuge tube, centrifuged at 600 g for 5 minutes, the supernatant discarded, and then resuspended in 200 μL of 95% ethanol.

[0011] Furthermore, in step (4) of the two-color fluorescent staining, the volume of each drop of dye solution is 10 μL.

[0012] Furthermore, in step (5) microscopic examination, the excitation wavelength of the DAPI channel is 358 nm and the emission wavelength is 461 nm; the excitation wavelength of the acridine orange channel is 488 nm and the emission wavelength is 520 nm.

[0013] Advantages of this invention: In this invention, because DAPI can bind strongly and stably to nuclear DNA, even when acridine orange staining fails in urine samples due to inflammation, degeneration, or other factors, DAPI can still clearly and completely delineate the morphology and outline of the cell nucleus, completely solving the problems of uneven or no staining of the cell nucleus in existing technologies. Simultaneously, since acridine orange fluoresces green in the cytoplasm and DAPI fluoresces blue in the nucleus, the two colors are highly distinguishable and do not overlap, allowing interpreters to easily differentiate between the nucleus and cytoplasm, accurately identify key tumor indicators such as nuclear atypia and nuclear-cytoplasmic ratio, effectively avoiding misidentification of hemoglobin and other impurities as tumor cells, significantly reducing false positive and false negative rates, and improving interpretation accuracy and consistency. Furthermore, DAPI staining is fast and can be added directly after acridine orange staining without additional rinsing or blocking steps. The entire staining process is essentially the same as the original acridine orange staining procedure, requiring no new instruments or complex operating procedures, making it easy to promote and popularize in clinical pathology departments. Furthermore, DAPI has a high affinity for DNA and is not easily affected by impurities in the sample, cellular degeneration, or pH changes. The dual staining system of this invention exhibits higher stability and durability against complex urine backgrounds, ensuring the reliability of staining results. Attached Figure Description

[0014] Figure 1To assess the effectiveness of using single acridine orange fluorescent staining in staining urine exfoliated cells using existing techniques. Figure 1 ; Figure 2 To assess the effectiveness of using single acridine orange fluorescent staining in staining urine exfoliated cells using existing techniques. Figure 2 ; Figure 3 The image shows the effect of staining urine exfoliated cells using the staining method of Example 1. Figure 4 This is a diagram showing the effect of staining urine exfoliated cells using the staining method of Example 2. Detailed Implementation

[0015] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1 A method for fluorescent staining of exfoliated urine cells, specifically including the following steps: (1) Preparation of dye solution: Preparation of acridine orange staining solution: In a fume hood, weigh 10 mg of acridine orange powder with a purity ≥95%, add it to 10 mL of sterile deionized water, vortex for 2 minutes until completely dissolved, filter sterilize using a 0.22 μm filter membrane, dispense into light-protected centrifuge tubes, 100 μL per tube, label with concentration and date, and store at -20℃ protected from light.

[0017] DAPI staining solution preparation: In a fume hood, weigh 10 mg of DAPI powder and add it to 10 mL of 1×PBS buffer at pH 7.4. Vortex for 1 minute until completely dissolved. Filter through a disposable needle filter to remove bacteria. Aliquot into light-protected centrifuge tubes, 100 μL per tube, label with concentration and date, and store at -20°C in the dark.

[0018] (2) Specimen pretreatment: Collect 40 mL of the first and midstream urine from the patient’s second morning urine and leave the sample at room temperature for no more than 1 hour. Take 30 mL of urine and centrifuge at 3000 rpm for 5 minutes; Discard the supernatant, add 200 μL of 95% ethanol to the cell pellet, gently pipette to mix and fix the cells, and obtain a cell suspension.

[0019] (3) Production: Take 20 μL of the above cell suspension, spread it evenly on a glass slide, and let it air dry for 30 minutes to prepare a cell smear.

[0020] (4) Two-color fluorescent staining: Add 1 drop of acridine orange staining solution to the prepared cell smear and stain at room temperature in the dark for 60 seconds; Add another drop of DAPI staining solution and stain at room temperature in the dark for 60 seconds. Gently tilt the slide to remove excess staining solution, then cover with a coverslip to obtain the stained sample.

[0021] (5) Microscopic interpretation: The stained samples were placed under a fluorescence microscope and observed using both the DAPI and acridine orange channels.

[0022] According to the 2022 Paris Reporting System for Urine Cytology: normal cells show blue fluorescence in the nucleus and green fluorescence in the cytoplasm, such as... Figure 3 As shown.

[0023] Example 2 A method for fluorescent staining of exfoliated urine cells, specifically including the following steps: (1) Preparation of dye solution: Same as in Example 1.

[0024] (2) Specimen pretreatment: Collect 30 mL of the first and midstream urine from the patient’s second morning urine and store the sample at 4°C for 20 hours. Take 30 mL of urine and centrifuge at 3000 rpm for 7 minutes.

[0025] Discard the supernatant, add 200 μL of 95% ethanol to the cell pellet to resuspend, and mix by pipetting.

[0026] Concerned that not enough cells were collected, the cell suspension resuspended in ethanol was transferred to a 1.5 mL centrifuge tube, centrifuged at 600 g for 5 minutes, and then the supernatant was removed and the cells were resuspended again with 200 μL of 95% ethanol to obtain the cell suspension.

[0027] (3) Preparation: Same as in Example 1.

[0028] (4) Two-color fluorescent staining: Same as in Example 1.

[0029] (5) Microscopic examination and interpretation: Same as in Example 1.

[0030] Images observed under a fluorescence microscope, such as Figure 4 As shown, even in areas where acridine orange staining is poor, DAPI can still clearly display the complete morphology and outline of the cell nucleus.

[0031] Comparative Example 1 This comparative example used the exact same urine exfoliated cell samples and pretreatment methods as Example 1, but in the staining step, only 1 drop of acridine orange staining solution was added, and the staining was performed at room temperature in the dark for 120 seconds. After discarding the excess staining solution, the slide was directly mounted for observation, without DAPI staining.

[0032] Comparative Example 2 This comparative example used the exact same urine exfoliated cell samples and pretreatment methods as Example 1. However, in the staining step, acridine orange staining solution and DAPI staining solution were premixed at a 1:1 volume ratio. Then, 1 drop of the mixed staining solution was added to the cell smear, stained at room temperature in the dark for 120 seconds, and the excess staining solution was poured off before mounting and observation.

[0033] Experimental Example Urine samples from 60 clinically diagnosed bladder cancer patients were stained using the methods described in Example 1, Comparative Example 1, and Comparative Example 2 of this invention, respectively. Two independent pathologists interpreted the samples without the patients' knowledge, comparing the staining quality and diagnostic efficacy. The results are shown in Table 1. Table 1 shows the interpretation results after staining using the methods of Example 1, Comparative Example 1, and Comparative Example 2 of the present invention, respectively.

[0034] Combine the data from Table 1 Figure 1 , Figure 2 As shown in the attached figures, after staining using the method of Comparative Example 1, the cell nuclei in most fields of view were lightly and unevenly stained, with some cell nuclei completely unstained, showing only a blurry cell outline; the red fluorescence of the cytoplasm was difficult to distinguish from background impurities, making interpretation difficult. After staining using the method of Comparative Example 2, the fluorescence intensity of both the cell nuclei and cytoplasm was significantly reduced, the blue fluorescence of DAPI and the orange-green fluorescence spectrum of acridine overlapped severely, resulting in poor image contrast, blurred cell nucleus boundaries, and an inability to clearly distinguish between nucleus and cytoplasm. However, after staining using the method of Example 1, combined with the data in Table 1 and... Figure 3 , Figure 4As can be seen from the images, cell nuclei exhibit bright, uniform blue fluorescence in all fields of view, with clear and complete outlines; the cytoplasm displays vivid green fluorescence, resulting in high contrast. Even in inflammatory backgrounds or samples with cell degeneration, DAPI can stably display cell nucleus morphology. Therefore, compared with single acridine orange staining and mixed staining, the sequential dual-color fluorescence staining method used in this invention, through the specific order of acridine orange followed by DAPI, fully utilizes the high affinity and stability of DAPI for nuclear DNA, increasing the cell nucleus staining integrity rate from approximately 66% to over 97%, and improving the interpretation consistency rate and diagnostic accuracy from 73% and 78%, respectively, to 95%. Simultaneously, it significantly reduces the proportion of interpretation difficulties caused by interference from impurity fluorescence. This invention effectively solves the technical problems of unstable, lightly stained, or unstained urinary exfoliated cell nuclei in existing technologies, and has significant clinical application value.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for fluorescent staining of exfoliated urine cells, characterized in that, Includes the following steps: (1) Preparation of dye solution: Acridine orange dye solution and DAPI dye solution were prepared separately, and the prepared dye solutions were aliquoted in the dark and stored in an environment of -20℃. (2) Sample pretreatment: Take a urine sample, centrifuge at 3000 rpm for 5-7 minutes, collect the cell pellet, and resuspend and fix it with 200 μL of ethanol to obtain a cell suspension; (3) Slide preparation: Take 20 μL of the cell suspension obtained in the specimen pretreatment in step (2), spread it evenly on a glass slide, and dry it naturally or by heating to make a cell smear; (4) Two-color fluorescent staining: Add 1 drop of acridine orange staining solution prepared in step (1) to the cell smear obtained in step (3) and stain at room temperature in the dark for 60 seconds. Then add 1 drop of DAPI staining solution prepared in step (1) and stain at room temperature in the dark for 60 seconds. Pour off the excess staining solution, add a coverslip, and obtain the stained sample. (5) Microscopic examination and interpretation: The stained sample obtained in step (4) of dual-color fluorescence staining is placed under a fluorescence microscope and observed through dual channels and the results are interpreted.

2. The method for fluorescent staining urine exfoliated cells according to claim 1, characterized in that, In step (1) of the preparation of the dye solution, the method for preparing acridine orange dye solution is as follows: weigh acridine orange powder and dissolve it in sterile deionized water to prepare a solution of 1 mg / mL. Vortex for 2-3 minutes until completely dissolved, and then filter to remove bacteria. The method for preparing DAPI staining solution is as follows: Weigh DAPI powder and dissolve it in 1×PBS buffer with a pH of 7.2-7.4 to prepare a 1 mg / mL solution. Vortex for 1-2 minutes until completely dissolved, and then filter to remove bacteria.

3. The method for fluorescent staining exfoliated urine cells according to claim 2, characterized in that, The acridine orange powder is of molecular biology grade with a purity of ≥95%.

4. The method for fluorescent staining urine exfoliated cells according to claim 1, characterized in that, Acridine orange and DAPI staining solutions are dispensed in 100-200 μL / tube and stored at -20°C in the dark for 12 months.

5. The method for fluorescent staining exfoliated cells in urine according to claim 1, characterized in that, In step (2) specimen pretreatment, the urine sample is the first and middle part of the second morning urine or the first and middle part of the urine at an interval of more than 2 hours from the last urination. The sample volume is 30-50 mL. It is stored at room temperature for no more than 2 hours or at 2-8℃ for no more than 24 hours.

6. The method for fluorescent staining urine exfoliated cells according to claim 1, characterized in that, In step (4) of the two-color fluorescent staining, the volume of each drop of dye solution is 10 μL.

7. The method for fluorescent staining urine exfoliated cells according to claim 1, characterized in that, In step (5) microscopic examination, the excitation wavelength of the DAPI channel is 358nm and the emission wavelength is 461nm; the excitation wavelength of the acridine orange channel is 488nm and the emission wavelength is 520nm.

8. The application of the urine exfoliated cell fluorescence staining method according to any one of claims 1-7 in non-invasive screening for urothelial tumors.

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