Establishment method of blood group immunodetection platform based on fluorescence difference labeling

Abstract

The invention relates to a blood group antibody detection method established through differential fluorescence labeling reagent red blood cells, the method uses fluorescence and biotin-avidin for independent or mixed labeling, cells can be pre-stained, and the fluorescence uses single fluorescence to label different cells. The invention also provides a biotin working solution and biotinylated red blood cells. According to the invention, biotin is used for labeling, and half-life period and storage life tests (50d, 90d) prove that labeled cells can be effectively distinguished within the storage life (2-3 months) of reagent red blood cells in the actual use process. The device can be prefabricated, the detection process is greatly shortened, and precious time is saved. According to the present invention, the single fluorescence difference is adopted to stably mark the cell reagent, such that various types of cells can be mixed, and the marking only occupies one channel so as to synchronously perform multi-parameter detection on the sample, such that the obvious intensive improvement is provided.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for establishing a blood typing immunoassay platform based on fluorescent differential labeling. Background Technology

[0002] Blood typing requires the use of multiple reagent cells to test the sample (e.g., antibody screening red blood cell reagents typically consist of 3-4 red blood cell reagents with different blood types, while antibody identification cell products require 10-15). Flow cytometry, however, lacks a "fluorescence compensation" channel, making it difficult to use a complete set of reagents with different fluorescent labels (e.g., for antibody identification cells). Current domestic methods mainly rely on cell agglutination, and no patents for reagents or methods based on flow cytometry have been found. The existing international document (CA2977436) describes a method where reagent red blood cells are labeled with different fluorescent markers and then detected using flow cytometry.

[0003] The main drawbacks of agglutination methods are: 1. Cumbersome operation, high dependence on operator experience, and experimental results easily influenced by subjective judgment; 2. Requires the use of dozens of reagents, with only one reagent usable per experiment, resulting in low levels of automation and time consumption; 3. Essentially a qualitative reaction, making quantitative analysis extremely difficult.

[0004] The main problems with the existing international patent (International Document No.: CA2977436) are: 1. Different cells are labeled with different fluorescent monochromatic markers. When multiple cells are combined for experiments, the available channels for antibody detection are very limited. 2. Based on the objective fact of problem 1, it is impossible to detect multiple antibody types simultaneously in one experiment. 3. Due to the monochromatic labeling of cells, the number of detection channels required increases significantly, which places high demands on hardware and is not conducive to carrying out complex experiments on conventional instruments. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a blood group antibody detection system that uses differentially fluorescently labeled red blood cells to establish a system that can determine the presence, specificity, immunoglobulin type, and quantification of blood group antibodies in a single test. This provides a feasible labeling scheme for the automated detection of cell-based in vitro diagnostic reagents.

[0006] In a first aspect, the present invention provides a method for establishing a blood group immunoassay platform based on fluorescent differential labeling. The method uses fluorescence and biotin-avidin for single or mixed labeling, which can pre-stain cells. The fluorescence is a single fluorescence used to label different cells.

[0007] As a preferred example, the method includes the following steps:

[0008] S1: Titration of biotinylated erythrocyte labeling concentration:

[0009] S11: Preparation of red blood cell suspension: Aliquot the sample, centrifuge to remove the supernatant, and make up the volume with 1xPBS. Then centrifuge and wash three times to prepare the packed red blood cells, and dilute with the washing buffer to prepare red blood cell suspension for later use.

[0010] S12: Preparation of biotin working solution: Dissolve 10 mg sulfo-NHS-biotin in 10 ml of 0.9% physiological saline. Use the current working solution concentration of 1 mg / ml as the initial concentration N0. Dilute N0 with physiological saline to obtain N1 = 500 ug / ml, N2 = 333 ug / ml, N3 = 167 ug / ml, N4 = 83 ug / ml, and N5 = 54 ug / ml.

[0011] S13: Biotinylated erythrocytes: Prepare biotin working solution (NO and N...) 2-5 Mix with red blood cell suspension, incubate at room temperature, then centrifuge, remove supernatant, wash twice, and resuspend in preservation solution for storage.

[0012] S14: Flow cytometry: FITC-avidin was diluted with 1xPBS, mixed with 0.8-1.0% red blood cell suspension at a final concentration of 1:300, incubated in the dark, centrifuged to remove supernatant, washed twice, resuspended, and then subjected to flow cytometry.

[0013] S2: Fluorescent dye-labeled erythrocyte concentration titration:

[0014] Labeled red blood cells were prepared by mixing the dye stock solution diluted with 1xPBS with the red blood cell suspension. After staining and washing, the red blood cells were resuspended in red blood cell preservation solution.

[0015] As a preferred example, the method includes step S3:

[0016] S3: Verification of the effectiveness of differentially labeled mixed reagents for erythrocyte antibody identification

[0017] S31: Mixed Reagent Red Blood Cell Preparation: Mix biotin-labeled red blood cells prepared as described above: ①-high concentration / ②-low concentration / ③-unlabeled;

[0018] S32: Simulated antibody and reagent red blood cell reaction: Using monoclonal antibody reagent to simulate patient serum, take mixed BioRBCs, centrifuge to remove supernatant, add IgA-Leb, diluted IgM-e and IgG-E, incubate at room temperature, remove supernatant, wash and resuspend;

[0019] S33: Flow cytometry antibody screening experiment.

[0020] As a preferred example, S12: Biotin working solution is prepared with 0.9% physiological saline at pH=5 10-20 minutes before use.

[0021] As a preferred example, S13: Biotin working solution (N0 and N2-5) and 11% red blood cell suspension are mixed at a volume ratio of 1:10, incubated at room temperature, centrifuged, supernatant removed, washed twice with 6 ml of preservation solution, resuspended in 4k preservation solution to 5%, and stored.

[0022] As a preferred example, S2: The dye stock solution was diluted with 1xPBS to a final concentration of 0.5uM to 8uM as the application solution, and mixed with 2% red blood cell suspension at a ratio of 1:1 to prepare labeled red blood cells. After staining and washing, the cells were resuspended in 1% red blood cell preservation solution.

[0023] Secondly, the present invention provides a biotin working solution, which is prepared by the following method: sulfo-NHS-biotin is dissolved in physiological saline, and the initial concentration N0 is 1 mg / ml; N0 is diluted with physiological saline to obtain N1 = 500 ug / ml, N2 = 333 ug / ml, N3 = 167 ug / ml, N4 = 83 ug / ml, and N5 = 54 ug / ml. The biotin working solution is used in the method for establishing blood type antibody detection using fluorescently labeled red blood cells.

[0024] As a preferred example, the sulfo-NHS-biotin is 10 mg and the saline solution is 10 ml of 0.9% saline solution.

[0025] Thirdly, the present invention provides a biotinylated erythrocyte: the biotinylated erythrocyte is prepared by the following method: a biotin working solution (NO and N...) is prepared... 2-5 Mix 1% red blood cell suspension with 11% red blood cell suspension at a volume ratio of 1:10, mix well, incubate, centrifuge, remove supernatant, wash twice, and resuspend in preservation solution for storage.

[0026] The advantages of this invention are:

[0027] This invention utilizes biotin labeling. After half-life and shelf-life testing (50 days and 90 days), the labeling method designed in this invention has proven effective in distinguishing labeled cells within the actual red blood cell shelf-life (2-3 months) of practical use. Furthermore, fluorescent labeling methods suffer from fluorescence migration issues during long-term mixed storage, while biotin, due to its covalent bonding, exhibits good stability and does not have migration problems. This invention allows for pre-fabrication, significantly shortening the detection process and saving valuable time. Because this invention utilizes a single fluorescence difference for stable labeling of cell-based reagents, it can mix multiple cell types. Simultaneously, since labeling only occupies one channel, multiple parameters can be detected simultaneously, resulting in significant efficiency improvements. If this invention is applied, it will replace traditional cell agglutination experiments with immunofluorescence detection, establishing a fully automated red blood cell blood typing system based on flow cytometry, revolutionizing existing blood typing methods. It will provide a novel red blood cell blood typing antibody detection solution to over 5000 domestic laboratories. Attached Figure Description

[0028] Figure 1 Differentially labeled biotinylated erythrocytes by flow cytometry.

[0029] Figure 2 Differentially labeled red blood cells by flow cytometry.

[0030] Figure A shows the results of fluorescent dye labeling (unpublished). The red peak is the unstained negative control; the green peak is 1.5 μM; the purple peak is 3 μM; and the yellow peak is 4 μM. Figure B shows the results of biotin-avidin labeling.

[0031] Figure 3 Biotinylated reagent for flow cytometry antibody screening of red blood cells.

[0032] Figure A shows the IgG-E detection results, Figure B shows the IgM-e detection results, and Figure C shows the IgA-Leb detection results. Table D shows the MFI values ​​of each component of erythrocytes using biotinylated reagents, where C represents the control group results and T represents the experimental group results.

[0033] Figure 4 Differences in fluorescence intensity among different components of the mixed reagent cells during the storage period. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0035] Example 1

[0036] The following description of erythrocyte biotinylation and detection procedures is based on existing in vivo methods for the detection of labeled erythrocytes. 1-4 Since this system is mainly designed for in vitro transfusion immunoassay, there are some differences in the operation procedures, biotin-labeled reagents, and washing solutions.

[0037] 1. Experimental Materials

[0038] The main reagents used in system construction and validation are as follows: sulfosuccinyl biotin (Sulfo-NHS-Biotin, Sangon Biotech, C100213-0050), FITC-conjugated avidin (FITC-conjugated avidin, Sangon Biotech). Biotech, D111086-0100), IgM-e (Shanghai Blood Biotechnology, 20243301), IgG-E (gifted from Osaka Blood Research Institute), IgA-Leb (Sanquin, 8000461365), PE-IgM (Jackson, 709-116-073, 1:200 used), 405-IgG (Jackson, 209-475-097, 1:50 used), APC-IgA (Jackson, 109-135-011, 1:50 used), 20xPBS buffer, ddH2O, 0.9% physiological saline.

[0039] The red blood cell samples used in the system construction and validation were all taken from the research blood used within the station (n=8).

[0040] 2 methods

[0041] 2.1 Titration of biotinylated erythrocyte labeling concentration

[0042] 1) Preparation of red blood cell suspension: After thorough mixing, aliquot 15 ml of sample from the blood bag, centrifuge at 2400 g for 3 min to remove the supernatant, and then make up the volume with 1xPBS (pH 7.4). Subsequently, centrifuge at 2400 g for 3 min and wash three times to prepare the hematocrit red blood cells, which are then diluted with washing buffer to an 11% red blood cell suspension for later use. Three samples were screened in this experiment, with RhCE typing as follows: ①Ccee; ②ccEe; ③ccEE.

[0043] 2) Preparation of Biotin Working Solution: Dissolve 10 mg of sulfo-NHS-biotin in 10 ml of 0.9% physiological saline. Use this initial working solution concentration of 1 mg / ml as the starting concentration N0. Dilute N0 with physiological saline to obtain N1 = 500 ug / ml, N2 = 333 ug / ml, N3 = 167 ug / ml, N4 = 83 ug / ml, and N5 = 54 ug / ml. Note that to avoid inactivation of the sulfo-NHS-biotin reagent, prepare the biotin working solution with 0.9% physiological saline at pH = 5 10-20 minutes before use. Do not prepare a biotin stock solution; prepare and use immediately, discarding all unused reagent solutions.

[0044] 3) Biotinylated erythrocytes: Prepare biotin working solution (NO and N...) 2-5 The labeled Bio-RBCs were mixed with 11% red blood cell suspension at a 1:10 volume ratio, inverted 10 times, and incubated at room temperature for 30 min. Then, the mixture was centrifuged at 800g for 3 min to completely remove the supernatant. The cells were washed twice with approximately 6 ml of preservation buffer (4K, patent application number 202010613045.1, invention title: A Reagent Red Blood Cell Preservation System and its Preparation Method) to a final concentration of 5%, and stored at 4°C. Labeled Bio-RBCs should not be stored at room temperature for more than 4 hours, but can be stored at 4°C for more than 35 days.

[0045] 4) Flow cytometry: FITC-avidin was diluted to two gradients: 1:100 and 1:10000. After mixing with the red blood cell suspension, the mixture was incubated in the dark for 30 min, centrifuged at 200g for 3 min, the supernatant was removed, and the mixture was washed twice and resuspended.

[0046] Perform flow cytometry detection.

[0047] 2.2 Fluorescent dye-labeled erythrocyte concentration titration

[0048] The dye stock solution (1 mM, DMSO) was diluted with 1xPBS to a final concentration of 0.5 μM to 8 μM as the application solution. It was then mixed with 2% red blood cell suspension (1×PBS) at a 1:1 ratio to prepare labeled red blood cells. After staining and washing, the cells were resuspended in 1% red blood cell preservation solution (twice the volume of the original suspension).

[0049] 2.3 Verification of the effectiveness of differentially labeled mixed reagents for red blood cell antibody identification

[0050] 1) Preparation of mixed reagent red blood cells: Mix biotin-labeled red blood cells prepared as described above in a 1:1:1 volume ratio: ①-high concentration / ②-low concentration / ③-unlabeled.

[0051] 2) Simulated antibody and reagent red blood cell reaction: Using monoclonal antibody reagent to simulate patient serum, take 50ul of mixed BioRBC (5%), centrifuge to remove supernatant, add 50ul of IgA-Leb, 50ul of 1:10 diluted IgM-e and 50ul of IgG-E, incubate at room temperature for 30min, remove supernatant and wash three times, resuspend to 1ml, at which time the cell concentration is about 2.5‰.

[0052] 3) Flow cytometry antibody screening experiment: After adding the samples as shown in Table 1, incubate at room temperature in the dark for 30 min, discard the supernatant, wash twice (200 μL) and then perform the analysis. The recommended antibody concentrations are as follows: PE-IgM 1:200, 405-IgG 1:50, APC-IgA 1:50, FITC-Avidin 1:100. Use 1xPBS for both washing and dilution.

[0053] Table 1

[0054]

[0055] 3 Results

[0056] 3.1 Flow cytometry detection of the discriminative power of different concentrations of biotin labeling

[0057] Flow cytometry analysis of FITC-avidin diluted 1:10000 showed no positive signal, suggesting that the dilution was too low to label biotinylated erythrocytes. The results of flow cytometry analysis of FITC-avidin diluted 1:100 are as follows (…). Figure 1 Among them, the detection signal peaks of the three treatment groups, Bio-RBC-B, Bio-RBC-L, and Bio-RBC-H, did not overlap, providing practical evidence for the feasibility of preparing differentially labeled erythrocyte anti-screening reagents using different concentrations of biotin labeling. Figure 2 Furthermore, differential labeling of erythrocyte membranes using different concentrations of fluorescent dyes can also achieve this, but issues such as label migration and loss occur, and this system still requires further optimization. Therefore, to obtain stable experimental results, subsequent experiments will still be conducted in the biotin-labeled system.

[0058] 3.2 Interpretation of Antibody Identification Results

[0059] Flow cytometry antibody screening experiments verified that the results of the simulated samples all matched the predicted results, confirming that the differentially labeled biotinylated erythrocytes can initially achieve single-well detection of mixed blood group antibodies. Further large-scale repeated experiments are needed to verify the stability and sensitivity of the above detection system. For flow cytometry antibody screening of biotinylated erythrocytes, see [link to details]. Figure 3 .

[0060] 3.3 Changes in resolution and stability of labeled red blood cells during preservation

[0061] This experiment tested mixed reagent red blood cells stored on day 0, day 50, and day 90 to evaluate their resolution and stability. Resolution was expressed as the MFI ratio (…). Figure 3 The signal-to-noise ratio (SNR = ΔMean / rSD) is used to represent stability; stability refers to the ability to distinguish different components of mixed reagent red blood cells (BIO-RBC-B / L / H), mainly based on differences in fluorescence intensity and coefficient of variation. Figure 4 ).

[0062] The signal-to-noise ratio is calculated as follows:

[0063] SNR = |Mean group1 -Mean group2 | / √(rSD group1 2 +rSD group2 2 SNR (>3) indicates good resolution.

[0064] • BIO-RBC-Lvs. BIO-RBC-B:

[0065] oD0 = 4.59

[0066] oD50 = 3.87

[0067] oD90 = 4.57

[0068] • BIO-RBC-H vs. BIO-RBC-B:

[0069] oD0 = 7.63

[0070] oD50 = 6.69

[0071] oD90=7.33

[0072] The decrease in the intergroup fluorescence intensity ratio during the storage period was mainly due to the increased background. The intergroup signal-to-noise ratio remained greater than 3, indicating good resolution. There was a moderate shift in the three detection results, but the reproducible frequency response (rCV) improved or stabilized in most populations. This suggests that the detection system has a certain degree of reproducibility, but sample or operational factors (such as storage time and instrument calibration) may introduce bias.

[0073] On day 0 of the storage period, the SNR was >7, indicating good resolution. On day 50, the resolution decreased slightly, mainly due to increased background. Both BIO-RBC-L and BIO-RBC-H maintained good resolution throughout. There was a moderate shift in the results between the two tests, but the precision (rCV) improved or stabilized in most populations. This suggests that the detection system has some reproducibility, but sample or operational factors (such as storage time and instrument calibration) may introduce bias.

[0074] It should be noted that this invention differs from International Document No.: CA2977436 in that;

[0075] 1. Different basic design principles: This invention utilizes a single fluorescent species to stably label different cells based on differences in intensity; the comparative document uses different fluorescent markers to distinguish different cells. Blood typing requires multiple reagent cells to test the sample (e.g., antibody screening red blood cell reagents typically consist of 3-4 red blood cell reagents with different blood types, while antibody identification cell products require 10-15). Flow cytometry, however, lacks a limited "fluorescence compensation" channel, making it difficult to use different fluorescent markers for the entire reagent set (e.g., antibody identification cells).

[0076] 2. The labeling methods for pre-staining differ: This invention uses fluorescence and biotin-avidin for single or mixed labeling, allowing for pre-staining of cells; the reference document must be used immediately after staining. Routine blood typing often occurs in scenarios where patients require blood transfusions and testing time is critical. This patent allows for pre-staining, significantly shortening the testing process and saving valuable time.

[0077] 3. Difference in fluorescence stability: This invention uses a biotin-avidin labeling method, ensuring labeling stability; the prior art cannot avoid fluorescence decay during storage. Flow cytometry is highly sensitive, and fluorescence decay can cause a decrease in the resolution of labeled cell recognition. This patent utilizes biotin labeling, and after half-life and storage period tests (50 days and 90 days), the labeling method designed in this patent proves that it can effectively distinguish labeled cells within the actual use of reagent red blood cells during their storage period (2-3 months). Furthermore, fluorescent labeling methods suffer from fluorescence migration issues during long-term mixed storage, while biotin, due to its covalent bond labeling, has good stability and does not have migration problems.

[0078] 4. Difference in the degree of integration: This invention utilizes a single fluorescence difference to stably label cell-based reagents, allowing for the simultaneous detection of multiple cell types after mixing. Furthermore, since the labeling occupies only one fluorescence channel, it can be used with different types of antibody reagents for simultaneous multi-parameter detection of samples. In contrast, the comparative paper uses different fluorescence labels, requiring splitting the experiment when detecting 10-15 cells. Additionally, due to the labeling space, simultaneous multi-parameter detection of samples is usually not possible. This patent, by utilizing a single fluorescence difference to stably label cell-based reagents, allows for the mixing of multiple cell types. Moreover, since the labeling occupies only one channel, simultaneous multi-parameter detection of samples is possible, resulting in a significant improvement in integration compared to the comparative paper.

[0079] 5. Different Future Automated Prospects: This invention, due to differential fluorescent labeling, has a wide range of applicable labeling differences. For experiments detecting multiple cell types, there is no issue of reusing fluorescence, thus allowing automated software to identify the corresponding cells. In contrast, the comparative method uses fluorescent labeling, which leads to the problem of reusing fluorescence when different cell types are labeled in experiments detecting multiple cell types, hindering automation. Automation is an effective method to improve detection efficiency. The comparative method inevitably involves the reuse of fluorescence in multi-cell detection, which is detrimental to automated result interpretation.

[0080] Main references:

[0081] 1.Mock DM,Nalbant D,Kyosseva SV,et al.Development,validation,andpotential applications of biotinylated red blood cells for posttransfusionkinetics and other physiological studies:evidenced-based analysis and recommendations.Transfusion.2018;58(8):2068-2081.doi:10.1111 / trf.14647

[0082] 2. Mock DM, Matthews NI, Zhu S, et al. Red blood cell (RBC) survival determined in humans using RBCs labeled at multiple biotindensities. Transfusion. 2011; 51(5): 1047-1057. doi: 10.1111 / j.1537-2995.2010.02926.x

[0083] 3. Donnenberg AD, Kim-Shapiro DB, Kanias T, et al. Optimizing interpretation of survival studies of fresh and aged transfused biotin-labeled RBCs. Transfusion. 2023; 63(1): 35-46. doi:10.1111 / trf.17192

[0084] 4.Vimonpatranon S, Chotivanich K, Sukapirom K, Lertjuthaporn S, Khowawisetsut L, Pattanapanyasat K. Enumeration of the Invasion Efficiency ofPlasmodium falciparum In Vitro in Four Different Red Blood Cell PopulationsUsing a Three-Color Flow Cytometry-Based Method. A.2019;95(7):737-745.doi:10.1002 / cyto.a.23750

[0085] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A method for establishing a blood typing immunoassay platform based on fluorescent differential labeling, characterized in that, The method described uses fluorescence and biotin-avidin for labeling, either alone or in combination, to pre-stain cells. The fluorescence is achieved by using a single fluorescence to label different cells.

2. The method according to claim 1, characterized in that, The method includes the following steps: S1: Titration of biotinylated erythrocyte labeling concentration: S11: Preparation of red blood cell suspension: Aliquot the sample, centrifuge to remove the supernatant, and make up the volume with 1xPBS. Then centrifuge and wash three times to prepare the packed red blood cells, and dilute with the washing buffer to prepare red blood cell suspension for later use. S12: Preparation of biotin working solution: Dissolve 10 mg sulfo-NHS-biotin in 10 ml of 0.9% physiological saline. Use the current working solution concentration of 1 mg / ml as the initial concentration N0. Dilute N0 with physiological saline to obtain N1 = 500 ug / ml, N2 = 333 ug / ml, N3 = 167 ug / ml, N4 = 83 ug / ml, and N5 = 54 ug / ml. S13: Biotinylated erythrocytes: Prepare biotin working solution (NO and N...) 2-5 Mix with red blood cell suspension, incubate at room temperature, then centrifuge, remove supernatant, wash twice, and resuspend in preservation solution for storage. S14: Flow cytometry: FITC-avidin was diluted with 1xPBS, mixed with 0.8-1.0% red blood cell suspension at a final concentration of 1:300, incubated in the dark, centrifuged to remove supernatant, washed twice, resuspended, and then subjected to flow cytometry. S2: Fluorescent dye-labeled erythrocyte concentration titration: Labeled red blood cells were prepared by mixing the dye stock solution diluted with 1xPBS with the red blood cell suspension. After staining and washing, the red blood cells were resuspended in red blood cell preservation solution.

3. The method according to claim 2, characterized in that, It also includes step S3: S3: Verification of the effectiveness of differentially labeled mixed reagents for erythrocyte antibody identification S31: Mixed Reagent Red Blood Cell Preparation: Mix biotin-labeled red blood cells prepared as described above: ①-high concentration / ②-low concentration / ③-unlabeled; S32: Simulated antibody and reagent red blood cell reaction: Using monoclonal antibody reagent to simulate patient serum, take mixed BioRBCs, centrifuge to remove supernatant, add IgA-Leb, diluted IgM-e and IgG-E, incubate at room temperature, remove supernatant, wash and resuspend; S33: Flow cytometry antibody screening experiment.

4. The method according to claim 1 or 2, characterized in that, S12: Biotin working solution preparation: Prepare biotin working solution with 0.9% physiological saline at pH=5 10-20 minutes before use.

5. The method according to claim 1 or 2, characterized in that, S13: Prepare the biotin working solution (NO and N) 2-5 Mix 1% red blood cell suspension with 11% red blood cell suspension at a volume ratio of 1:10, incubate at room temperature, then centrifuge, remove supernatant, wash twice with 6 ml of preservative solution, resuspend in 4K solution to 5%, and store.

6. The method according to claim 1 or 2, characterized in that, S2: Dilute the dye stock solution with 1xPBS to a final concentration of 0.5uM to 8uM as the application solution, mix it with 2% red blood cell suspension at a 1:1 ratio to prepare labeled red blood cells, and after staining and washing steps, resuspend them in 1% red blood cell preservation solution.

7. A biotin working solution, characterized in that, The biotin working solution is prepared by the following method: sulfo-NHS-biotin is dissolved in physiological saline, and the initial concentration N0 is 1 mg / ml; N0 is diluted with physiological saline to obtain N1 = 500 ug / ml, N2 = 333 ug / ml, N3 = 167 ug / ml, N4 = 83 ug / ml, and N5 = 54 ug / ml. The biotin working solution (concentration range 4.5 ug-1 mg / ml) is used in the method for establishing blood type antibody detection using fluorescently labeled erythrocytes.

8. The biotin working solution according to claim 7, characterized in that, The sulfo-NHS-biotin is 10 mg, and the physiological saline is 10 ml of 0.9% physiological saline.

9. A biotinylated erythrocyte: characterized in that, The biotinylated erythrocytes are prepared by the following method: using the biotin working solution (NO and N) as described in claim 7 or 8. 2-5 Mix 1% red blood cell suspension with 11% red blood cell suspension at a volume ratio of 1:10, mix well, incubate, centrifuge, remove supernatant, wash twice, and resuspend in preservation solution for storage.

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