Detection method of urine exfoliated podocyte

Abstract

The invention discloses an absolute quantitative detection method for urine exfoliated podocyte, relates to the technical field of biomedical detection, and aims to solve the problem that the urine exfoliated podocyte cannot be accurately and absolutely quantified due to uncertain cell loss in a sample treatment process in the prior art. The method comprises the following steps: before carrying out any physical treatment on a urine sample, adding a known number of internal standard reference substances with physical characteristics similar to those of cells into the urine sample, carrying out co-enrichment treatment on a mixed sample containing target podocytes and the internal standard reference substances, and preparing a cell slide by adopting a standardized slide preparation technology; and carrying out podocyte specific immunofluorescence staining on the slide. Through the design of internal standard preposition and whole-course synchronous calibration, the cell loss error in the operation process is effectively overcome, the traditional semi-quantitative detection is improved into accurate and repeatable absolute quantification, and the reliability and clinical application value of a detection result are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to a method for detecting urinary podocytes. Background Technology

[0002] Podocytes are a key component of the glomerular filtration barrier in the kidneys, and their damage or shedding is a central factor in the development of various glomerular diseases, such as diabetic nephropathy, focal segmental glomerulosclerosis, and lupus nephritis. Under normal physiological conditions, podocytes are almost undetectable in urine. However, when the glomeruli are diseased, damaged podocytes detach from the glomerular basement membrane and are excreted in the urine, resulting in "podocyte urine." Therefore, detecting detached podocytes in urine has become a promising non-invasive indicator for the early diagnosis, disease monitoring, and efficacy evaluation of kidney diseases.

[0003] Currently, methods for detecting podocytes exfoliated in urine mainly rely on immunological techniques. This involves using antibodies that specifically recognize podocyte surface or intracellular markers (such as podocalyxin and podocin), combined with immunofluorescence or immunohistochemistry, to identify and count podocytes in urine sediment. However, existing detection technologies generally suffer from inherent limitations in achieving accurate quantification.

[0004] The standard procedure for existing methods typically involves centrifuging and enriching urine samples, preparing smears from the precipitate, fixing and staining, and finally counting under a microscope. Cell loss is unavoidable in this complex and multi-step sample processing. From centrifugation and discarding the supernatant to resuspension, transfer, and smearing, each step results in the loss of a variable number of cells. The magnitude of this loss is influenced by various unstable factors such as sample viscosity, operator technique, and equipment conditions, leading to a lack of reproducibility and predictability. Furthermore, traditional smear preparation methods easily result in uneven cell distribution on the slide, with some areas showing overlapping cell clusters while others are too sparse, making subsequent microscopic observation and counting extremely difficult and highly random. These factors combined mean that the number of cells observed on the slide ultimately fails to accurately reflect their actual concentration in the original urine sample. Therefore, existing technologies can usually only produce semi-quantitative or relatively quantitative results such as "number of cells per unit field of view" or "+, ++", which have poor accuracy and repeatability, severely restricting the ability of this detection indicator to be used for refined dynamic disease monitoring and standardized application in clinical practice. Summary of the Invention

[0005] The main objective of this invention is to provide a method for detecting podocytes shed in urine, aiming to solve the problems of inaccurate quantification and poor repeatability in existing technologies, so as to achieve accurate and repeatable absolute quantitative detection of the number of podocytes in urine samples.

[0006] To achieve the above objectives, the present invention provides a method for detecting urinary exfoliated podocytes, the method comprising the following steps: First, a urine sample is provided for testing, and a known amount of internal standard reference is added to it before any processing steps are performed. In some embodiments, the internal standard reference is a fluorescent microsphere. To ensure that the physical behavior of the internal standard reference and the podocytes is as consistent as possible during subsequent processing, the diameter of the fluorescent microsphere can be set to 10–20 μm. Simultaneously, to clearly distinguish the target cells from the reference in subsequent detection, the fluorescence color emitted by the fluorescent microsphere should have different spectral characteristics from the fluorescence color subsequently used to label the shed podocytes in the urine, to avoid signal overlap.

[0007] Subsequently, the urine sample containing the internal standard reference is processed to obtain a test slide containing the urinary podocytes and the internal standard reference. This processing step preferably includes centrifuging the urine sample to obtain a precipitate containing both urinary podocytes and the internal standard reference. In one specific embodiment, the centrifugation speed can be set to 1800–2200 rpm, and the centrifugation time to 5–10 minutes. After obtaining the precipitate, it is further processed using liquid-based thin-layer cell preparation technology to prepare a test slide with a uniform distribution of cells and the internal standard reference. After the test slide is prepared and before subsequent immunological staining, the method may further include a step of fixing the test slide with ethanol to maintain the morphology and antigen sites of the cells.

[0008] Next, the slide to be tested is subjected to immunological staining to achieve specific labeling of urinary podocytes. In a preferred embodiment, this immunological staining utilizes an antibody that specifically recognizes the Podocalyxin protein on the surface of podocytes. The staining process can be specifically broken down as follows: first, the slide to be tested is permeabilized; then, a primary antibody recognizing the Podocalyxin protein is added and incubated; finally, a secondary antibody with a fluorescent group is added for incubation and color development, thereby causing the podocytes to exhibit a specific fluorescent signal. To facilitate observation and cell identification, this immunological staining step may further include a step of counterstaining the nuclei of all cells on the slide using a nuclear dye (e.g., DAPI).

[0009] Finally, under a microscope, the specifically labeled urinary podocytes and the internal standard reference on the test slide are counted. Based on the count values ​​of both and the known quantity of the initially added internal standard reference, the absolute number of urinary podocytes in the test urine sample is determined. Specifically, this counting step can be performed under a fluorescence microscope, by switching different fluorescence channels to clearly identify and independently count the urinary podocytes emitting specific fluorescence signals and the internal standard reference emitting its own fluorescence signals.

[0010] In summary, the present invention has at least one of the following beneficial technical effects: 1. This invention fundamentally solves the problem of inaccurate quantification caused by the uncertainty of cell loss during sample processing in existing technologies by introducing a known quantity of internal standard reference material at the initial stage of urine sample processing. This technical feature ensures that the internal standard reference material and the target podocytes undergo synchronous loss during all subsequent physical operations such as centrifugation and slide preparation. By calculating the final ratio of their quantities, systematic errors throughout the process can be accurately corrected, thus achieving absolute quantitative detection of shed podocytes in urine for the first time, greatly improving the accuracy of the detection results.

[0011] 2. This invention establishes a standardized testing procedure, significantly enhancing the repeatability of test results and the comparability between different laboratories. Its core lies in the fact that the internal standard calibration mechanism effectively eliminates differences in cell loss caused by uncontrollable factors such as operator technique and equipment condition. Regardless of the absolute amount of cell loss during the specific operation, this method can minimize its impact on the final result through the built-in calibration system, ensuring highly consistent quantitative results for the same sample at different times and by different personnel.

[0012] 3. This invention significantly improves the reliability of accurate identification and counting of target cells and internal standard references by synergistically integrating internal standard calibration technology with liquid-based thin-layer cell preparation technology. Liquid-based thin-layer preparation technology can distribute cells and internal standard references in the sample in a uniform and clear monolayer on the slide, effectively avoiding the cell overlap, aggregation, and background clutter problems common in traditional smears. This creates ideal observation conditions for subsequent accurate counting under a microscope without omissions or repetitions.

[0013] 4. The method system of this invention has a built-in quality control function, which greatly enhances the reliability of the entire detection process. Since the initial amount of internal standard reference is known, its recovery rate on the final slide can itself serve as an objective process monitoring indicator. By setting a reasonable recovery rate threshold, it is possible to immediately determine whether any major errors have occurred in the experimental operation (such as improper centrifugation, sample splashing, etc.), thereby effectively avoiding the risk of false negatives or distorted results due to undetected operational errors.

[0014] 5. This invention, by employing optimized sample processing conditions, maximizes the preservation of the biological integrity of urinary podocytes while ensuring effective enrichment, thereby improving detection sensitivity. The gentle processing procedure defined in this method effectively avoids physical damage or destruction of surface antigen sites in fragile podocytes caused by excessive mechanical or chemical treatment, ensuring that the vast majority of existing podocytes are presented in a state that can be recognized by specific antibodies, thus improving the detection capability for low-concentration samples. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the method flow of the present invention. Detailed Implementation

[0016] The technical solutions in 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. Example Example

[0017] This embodiment provides a method for detecting urinary exfoliated podocytes, the specific steps of which are as follows: Sample pretreatment and internal standard addition: Take 50 mL of freshly collected midstream urine sample.

[0018] Add 50 μL of a red fluorescent microsphere suspension (15 μm in diameter) with a concentration of 1.0 x 10^5 microspheres / mL to the urine sample, which means adding a total of 5000 internal standard references.

[0019] Cover the container tightly and gently invert to mix 12 times.

[0020] Co-enrichment of cells and internal standard and standardized slide preparation: The above samples were centrifuged at 2000 rpm for 7 minutes at room temperature.

[0021] Discard the supernatant, retain about 5 mL of precipitate at the bottom of the tube, and resuspend it in 5 mL of cell preservation medium (RPMI-1640 medium containing 25 mM HEPES, 3% BSA, and 0.08% sodium azide).

[0022] A liquid-based thin-layer cell slide preparation machine was used, with the speed set to 800 rpm and centrifugation for 4 minutes to prepare the slides.

[0023] The prepared glass slide was immersed in 95% ethanol for 12 minutes to fix it.

[0024] Podocyte-specific immunofluorescence staining: Remove the slide, rinse with PBS, add 0.3% Triton X-100 in PBS solution, and allow it to permeate at room temperature for 20 minutes.

[0025] After rinsing with PBS, add PBS blocking buffer containing 3% BSA and block at room temperature for 45 minutes.

[0026] Discard the blocking solution, add the mouse anti-human Podocalyxin monoclonal antibody working solution diluted at a ratio of 1:200, and incubate in a humidified chamber at 37°C for 45 minutes.

[0027] After rinsing with PBS, add Alexa Fluor 488-labeled goat anti-mouse IgG secondary antibody working solution diluted at a ratio of 1:500, and incubate at room temperature in the dark for 45 minutes.

[0028] After rinsing with PBS, add 5 µg / mL DAPI solution and counterstain at room temperature in the dark for 7 minutes.

[0029] Result interpretation and counting: After final rinsing with PBS, the slides are mounted using mounting medium containing an anti-fluorescence quencher.

[0030] Under a fluorescence microscope, by switching the FITC and TRITC channels, green fluorescently labeled podocytes and red fluorescent microspheres were counted in 20 random high-power fields of view. Example

[0031] This embodiment provides a method for detecting urinary exfoliated podocytes, the specific steps of which are as follows: Sample pretreatment and internal standard addition: Take 40 mL of freshly collected midstream urine sample.

[0032] Add 40 μL of orange fluorescent microsphere suspension (10 μm in diameter) with a concentration of 1.0 x 10^5 microspheres / mL to the urine sample, which means a total of 4000 internal standard references are added.

[0033] Seal the container tightly and gently invert it to mix 10 times.

[0034] Co-enrichment of cells and internal standard and standardized slide preparation: The above samples were centrifuged at 1800 rpm for 10 minutes at room temperature.

[0035] Discard the supernatant, retain about 4 mL of precipitate at the bottom of the tube, and resuspend it in 4 mL of cell preservation medium (RPMI-1640 medium containing 20 mM HEPES, 1% FBS, and 0.05% sodium azide).

[0036] A liquid-based thin-layer cell slide preparation machine was used, with the speed set to 700 rpm and centrifuged for 5 minutes to prepare the slides.

[0037] The prepared glass slide was immersed in 90% ethanol for 15 minutes to fix it.

[0038] Podocyte-specific immunofluorescence staining: Remove the slide, rinse with PBS, add 0.1% Triton X-100 in PBS solution, and allow it to permeate at room temperature for 25 minutes.

[0039] After rinsing with PBS, add PBS blocking buffer containing 1% BSA and block at room temperature for 60 minutes.

[0040] Discard the blocking solution, add mouse anti-human Podocalyxin monoclonal antibody working solution diluted at a ratio of 1:500, and incubate in a humidified chamber at 4°C for 18 hours (overnight).

[0041] After rinsing with PBS, add FITC-labeled goat anti-mouse IgG secondary antibody working solution diluted at a ratio of 1:1000 and incubate at room temperature in the dark for 60 minutes.

[0042] After rinsing with PBS, add 1 µg / mL DAPI solution and counterstain at room temperature in the dark for 10 minutes.

[0043] Result interpretation and counting: After final rinsing with PBS, the slides are mounted using mounting medium containing an anti-fluorescence quencher.

[0044] Under a fluorescence microscope, by switching the FITC channel and the Cy3 channel, green fluorescent labeled podocytes and orange fluorescent microspheres were counted in 10 random high-power fields of view. Example

[0045] II. Experimental Data Table 2: Effects of different slide preparation methods on cell and internal standard counts

[0046] III. Experiment Summary The experimental data in Table 2 show that the slides prepared using the standardized liquid-based thin-layer preparation technique in Example 1 exhibited good consistency and stability in the counts of podocytes and internal standard microspheres in three repeated experiments. In stark contrast, the traditional smear technique used in Comparative Example 2, even with identical starting samples, showed huge and irregular fluctuations in the counts in three repeated experiments. Not only did the podocyte count jump significantly between 52 and 93, but the internal standard microsphere count also showed similar patterns, indicating extremely poor repeatability.

[0047] This result profoundly reveals the mechanism of synergistic action of the various technical modules in the method of this invention. The basis of absolute quantification in this invention lies in the precise calculation of the ratio of target cells to internal standard reference on the final slide. Traditional smear methods, due to their inherent defects, are prone to uneven distribution of cells and microspheres on the slide, resulting in sparse areas and overlapping areas. This physical inhomogeneity directly leads to the unrepresentative nature of random field-of-view counting under the microscope, and cell overlap can cause missed detections, making accurate counting of podocytes and internal standard reference extremely difficult, thus undermining the basis for accurate ratio calculation.

[0048] Therefore, this invention integrates standardized liquid-based thin-layer cell preparation technology into the detection process. This is not a simple addition of technologies, but an indispensable step in achieving the ultimate goal. Through its working principle, this technology ensures that cells and internal standards are clearly distributed in a uniform monolayer on the slide, providing the necessary technical prerequisite for accurate and repeatable counting. This fully demonstrates that this invention, through the synergistic integration of multiple technical modules such as internal standard calibration, standardized slide preparation, and specific staining, constructs a complete and interconnected technical solution. The absence or improper substitution of any key link will prevent the achievement of the precise and reliable absolute quantitative effect desired by this invention.

[0049] Process parameter influence verification experiment I. Experimental Procedure Sample preparation: To eliminate differences between samples, a large volume (≥300mL) of positive urine sample was taken, thoroughly mixed, and divided into 6 equal portions, each 50mL. These 6 samples were randomly divided into two groups: Example 1 group (3 replicates) and Comparative Example 3 group (3 replicates).

[0050] Experimental grouping and operation: Example 1 group (n=3): The operation was carried out exactly as described in Example 1, with the key centrifugation step using a rotation speed of 2000 rpm.

[0051] Comparative Example 3 (n=3): Except for the centrifugation step, all other operations (including internal standard addition, slide preparation, fixation, and staining) were exactly the same as in Example 1. The centrifugation step was as follows: the samples were centrifuged at 10,000 rpm at room temperature.

[0052] Data acquisition and recording: For the slides prepared in both groups of experiments, under the same fluorescence microscope conditions, the number of green fluorescent labeled podocytes (N_podocytes) and the number of red fluorescent microspheres (N_microspheres) were accurately counted and recorded in 20 random high-power fields (HPF).

[0053] II. Experimental Data Table 3: Effect of different centrifugation speeds on podocyte detection

[0054] III. Experiment Summary The experimental results in Table 3 clearly demonstrate that the centrifugation speed, a key process parameter, has a decisive impact on the detection results. Using the mild centrifugation conditions of 2000 rpm as described in Example 1, a relatively high number of positive podocytes could be consistently detected. However, in Comparative Example 3, simply increasing the centrifugation speed to 10000 rpm resulted in a precipitous decrease in the final countable number of positive podocytes, reducing it by approximately 75%–85%. It is noteworthy that the recovery count of the internal standard, which is physically stable, did not differ significantly between the two conditions, ruling out the possibility that excessive centrifugal force prevented the effective precipitation of microspheres.

[0055] This phenomenon profoundly reveals the intrinsic mechanism by which the method of this invention protects the integrity of biological samples. As biological cells, exfoliated podocytes in urine are relatively fragile. Excessively high centrifugation speeds (e.g., 10,000 rpm) generate enormous mechanical shear forces, sufficient to disrupt cell membrane integrity, leading to cell breakage, or causing the shedding or conformational alteration of surface antigen proteins such as podocalyxin, which serve as recognition targets. Once the cell structure is damaged or key antigenic sites are lost, subsequent specific antibodies cannot bind effectively, resulting in these actually present but damaged cells appearing negative in immunofluorescence detection, causing serious missed detections.

[0056] Therefore, the range of process parameters defined in this invention is not arbitrarily chosen, but rather a crucial component of the overall technical solution to ensure accuracy. By employing optimized and mild enrichment conditions, this method can effectively concentrate the sample while maximally protecting the biological integrity of the target cells, which is the biological basis for subsequent specific identification and accurate quantification. This fully demonstrates that the innovation of this invention is not only reflected in the calibration approach of introducing an internal standard, but also in the systematic optimization of the entire preparation process to ensure that every step serves the core objective of final accurate quantification.

[0057] Key Step (Fixed) Function Verification Experiment I. Experimental Procedure Sample preparation: To eliminate differences between samples, a large volume (≥300mL) of positive urine sample was taken, thoroughly mixed, and divided into 6 equal portions, each 50mL. These 6 samples were randomly divided into two groups: Example 1 group (3 replicates) and Comparative Example 4 group (3 replicates).

[0058] Experimental grouping and operation: Example 1 group (n=3): The procedure was performed exactly as described in Example 1. The key difference was that after the liquid-based thin-film preparation was completed, the glass slides were immersed in 95% ethanol for fixation.

[0059] Comparative Example 4 (n=3): Except for the fixation step, all other operations (including internal standard addition, centrifugation, slide preparation and staining) were exactly the same as in Example 1. The specific operation was as follows: after the liquid-based thin-layer slide preparation was completed, the slides were briefly air-dried, and then directly proceeded to the subsequent immunofluorescence staining step, omitting ethanol fixation throughout the process.

[0060] Data acquisition and recording: For the slides prepared in both groups of experiments, under the same fluorescence microscope conditions, the number of green fluorescent labeled podocytes (N_podocytes) and the number of red fluorescent microspheres (N_microspheres) were accurately counted and recorded in 20 random high-power fields (HPF).

[0061] II. Experimental Data Table 4: Effect of cell fixation steps on detection results

[0062] III. Experiment Summary The experimental results in Table 4 clearly reveal the crucial role of the cell fixation step in this method. Under the conditions of Example 1, a large number of positive podocytes could be consistently detected using the standard fixation procedure. However, in Comparative Example 4, simply omitting the ethanol fixation step resulted in a sharp decrease in the number of identifiable and countable podocytes to single digits, almost equivalent to detection failure. Meanwhile, the count values ​​of internal standard microspheres remained relatively stable in both groups of experiments, indicating that the slide preparation process itself was effective in capturing the microspheres; the problem mainly lay in the handling of the biological cells.

[0063] This result profoundly reflects the inherent scientific mechanism of the method of this invention. Cell fixation is the core bridge connecting physical cell preparation and biochemical staining. First, fixatives (such as ethanol) can firmly adhere cells to the slide surface by denaturing proteins. Without this step, cells loosely attached to the slide are easily detached during subsequent rinsing, incubation, and other liquid treatments, resulting in a large loss of cells. This is the direct reason for the extremely low podocyte count in Comparative Example 4. Second, fixation can instantly stop the cell's life activities and autolysis process, effectively preserving the cell's fine morphological structure and the spatial conformation of antigen sites, providing a material basis for the subsequent specific recognition and binding of antibodies.

[0064] Therefore, the overall solution of this invention is not a simple accumulation of isolated technologies, but a precisely designed organic whole with internal logical connections. The fixed steps are essential to ensure that cells captured at the physical level can smoothly enter and successfully complete the immunological recognition process. This demonstrates that the innovation of this method lies in the systematic construction of the entire detection process. By integrating a series of key and optimized steps, it ensures that every step from sample pretreatment to final data acquisition is stable and reliable, thus collectively serving the ultimate goal of achieving accurate, reproducible, and absolute quantification.

[0065] The timing of internal standard addition affects the verification experiment. I. Experimental Procedure Sample preparation: To eliminate differences between samples, a large volume (≥300mL) of positive urine sample was taken, thoroughly mixed, and divided into 6 equal portions, each 50mL. These 6 samples were randomly divided into two groups: Example 1 group (3 replicates) and Comparative Example 5 group (3 replicates).

[0066] Experimental grouping and operation: Example 1 group (n=3): The procedure was performed exactly as described in Example 1. The key difference was that 5000 red fluorescent microspheres were added to the 50 mL urine sample before any further processing.

[0067] Comparative Example 5 (n=3): The timing of internal standard addition was varied. First, 50 mL of urine sample was centrifuged and enriched according to the conditions of Example 1. After discarding the supernatant, cell preservation solution was added to the precipitate at the bottom of the tube for resuspending. Then, 5000 red fluorescent microspheres were added to this cell suspension. The subsequent liquid-based thin-layer preparation, fixation, and staining steps were exactly the same as in Example 1.

[0068] Data acquisition and recording: For the slides prepared in both groups of experiments, under the same fluorescence microscope conditions, the number of green fluorescent labeled podocytes (N_podocytes) and the number of red fluorescent microspheres (N_microspheres) were accurately counted and recorded in 20 random high-power fields (HPF).

[0069] II. Experimental Data Table 5: The impact of the timing of internal standard addition on test results

[0070] III. Experiment Summary The experimental results in Table 5 clearly demonstrate the decisive significance of the timing of internal standard addition for the method of this invention. Using the method of adding the internal standard beforehand in Example 1, the final calculated absolute number of podocytes was stable and within a reasonable range. However, in Comparative Example 5, simply moving the internal standard addition step to after centrifugation resulted in a final calculated "absolute quantification" result that was more than an order of magnitude lower than in Example 1, and the result itself was completely distorted, due to the extremely high count of the internal standard microspheres.

[0071] This significant difference profoundly confirms the core technical mechanism of this invention—pre-internal standard placement and synchronized calibration throughout the entire process. In the urine cell preparation process, centrifugation is the most significant and variable step in causing cell loss. This invention, by adding an internal standard before centrifugation, ensures that the internal standard reference and the target podocytes undergo this crucial loss process together. Therefore, the amount of internal standard remaining on the slide accurately reflects the total loss rate of the entire process, including centrifugation. Based on this proportional relationship of synchronized loss, the true concentration of podocytes in the original sample can be accurately deduced.

[0072] In contrast, in Comparative Example 5, the internal standard completely bypassed centrifugation, the most significant loss step. It only recorded the minor losses in subsequent steps such as resuspension and slide preparation after centrifugation. In this case, the internal standard and podocytes did not undergo a shared, proportional loss process, and their calibration basis was completely disrupted. Therefore, using a high-count internal standard with almost no loss to calibrate a low-count podocyte that has experienced significant loss will inevitably lead to a severely underestimated and erroneous conclusion. This fully demonstrates that the innovation of this invention lies not only in the use of an internal standard, but more importantly, in its creative "pre-positioning" of the calibration step at the very beginning of all processing steps. This design concept is the fundamental reason for achieving the leap from semi-quantitative to absolute quantification.

[0073] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for detecting urinary exfoliated podocytes, characterized in that, Includes the following steps: A. Add a known amount of internal standard reference to the urine sample to be tested; B. Process the urine sample containing the internal standard reference to obtain a test slide containing the urinary exfoliated podocytes and the internal standard reference; C. Perform immunological staining on the test slide to specifically label the exfoliated podocytes in the urine; D. Under a microscope, the urine exfoliated podocytes and the internal standard reference on the test slide that are specifically labeled are counted, and the number of urine exfoliated podocytes in the test urine sample is determined based on the count values ​​of the two.

2. The method for detecting urinary exfoliated podocytes according to claim 1, characterized in that, The internal standard reference is fluorescent microspheres.

3. The method for detecting urinary exfoliated podocytes according to claim 2, characterized in that, The fluorescent microspheres have a diameter of 10–20 μm, and their fluorescence color is different from the fluorescence color used to label urinary exfoliated podocytes in the immunological staining.

4. The method for detecting urinary exfoliated podocytes according to claim 1, characterized in that, Step B specifically includes: The urine sample containing the added internal standard was centrifuged and enriched to obtain a precipitate containing the shed podocytes and the internal standard. The precipitate was processed using liquid-based thin-layer cell preparation technology to prepare the glass slide to be tested.

5. The method for detecting urinary exfoliated podocytes according to claim 4, characterized in that, The centrifugation enrichment was carried out at a speed of 1800–2200 rpm for 5–10 minutes.

6. The method for detecting urinary exfoliated podocytes according to claim 1, characterized in that, The immunological staining was performed by labeling the urinary exfoliated foot cells with an antibody that specifically recognizes the Podocalyxin protein.

7. The method for detecting urinary exfoliated podocytes according to claim 6, characterized in that, The immunological staining specifically includes: The glass slide to be tested is subjected to a transparency treatment; Incubate with a primary antibody that recognizes the Podocalyxin protein; Incubation and color development were performed using a secondary antibody containing a fluorescent group.

8. The method for detecting urinary exfoliated podocytes according to claim 7, characterized in that, The immunological staining also includes the step of counterstaining the cell nucleus with nuclear dyes.

9. The method for detecting urinary exfoliated podocytes according to claim 1, characterized in that, Between steps B and C, there is also a step of fixing the glass slide to be tested with ethanol.

10. The method for detecting urinary exfoliated podocytes according to claim 1, characterized in that, Step D specifically includes: under a fluorescence microscope, identifying and counting the specifically labeled urine exfoliated podocytes and the internal standard reference through different fluorescence channels.

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