A method for improving the accuracy of detection of allogeneic blood reinfusion

Abstract

This invention improves the detection rate and accuracy of allogeneic transfusion detection by screening and combining blood type antigens suitable for allogeneic transfusion detection, and by combining multiple conditions such as blood type antibody selection, fluorescent antibody screening, automated cell washing program control, washing solution selection, anti-erythrocyte aggregation, and allogeneic erythrocyte determination methods.

Description

Technical Field

[0001] This invention belongs to the field of doping detection, specifically relating to a method for improving the accuracy of allogeneic blood reinfusion detection. Background Technology

[0002] Allogeneic blood transfusion refers to the method of transfusing blood from another person with the same ABO blood type into the human body. Allogeneic blood transfusion can rapidly increase red blood cell count, enhance oxygen transport, and improve aerobic performance. Athletes typically receive allogeneic blood stored in blood bags intravenously one to two weeks before a competition to increase their own red blood cell count and thus improve athletic performance. Allogeneic blood transfusion is a method explicitly prohibited by the World Anti-Doping Agency (WADA) in sports.

[0003] Blood type is a genetic polymorphism of the blood system. Human blood group antigens are determined by blood group genes and controlled by regulatory or modifying genes, exhibiting individual and racial differences. In 2002, Australian scholar Nelson proposed using flow cytometry for blood typing serology to detect allogeneic blood transfusions in athletes. The principle is based on the differences in red blood cell antigen profiles between individuals, using immunoassay for fluorescent labeling, and then applying flow cytometry to detect the presence of small amounts of allogeneic red blood cells. Giraud, Donati, Marchand, and others have studied allogeneic blood transfusion detection methods, focusing on method validation, method improvement, and comparisons with other methods. Their research demonstrates the feasibility of using flow cytometry for blood typing serology to detect allogeneic blood transfusions in athletes, the applicability of improved methods, and the existing problems with this approach.

[0004] International doping control laboratories typically test only eight red blood cell antigens (C, c, E, Jka, Jkb, Fya, Fyb, S) to determine the presence of allogeneic transfusions. This increases the risk of false negatives in allogeneic transfusion doping tests. While theoretically expanding the target antigen set could improve the differentiation between donor and recipient blood, it's impossible to infinitely expand the types of antigens tested as a standard testing protocol; therefore, antigen selection is necessary. However, it is well known that human blood type polymorphism varies significantly across different ethnic groups. Among the 30 internationally recognized major blood types, over 300 different antigens have been identified, and the expression of different antigens varies in different populations. Whether these antigens are suitable for allogeneic transfusion testing is uncertain. Current commercially available antibodies cannot effectively detect all expression types, leading to uncertainty in results. Furthermore, current testing methods require different fluorescent antibodies for staining different types of blood group antigens, increasing the requirements for antibody types and staining protocols, significantly increasing the number of test tubes, testing time and cost, and compromising timeliness.

[0005] Meanwhile, due to the significant racial differences among athletes in international competitions, although blood samples are collected from athletes during doping tests, all personal information is de-identified before the samples are delivered to the testing laboratory due to the confidentiality requirements of the World Anti-Doping Code. Testing personnel have no way of knowing the racial situation of the athlete corresponding to the sample, which makes it difficult to select the appropriate allogeneic blood transfusion antigen. Furthermore, existing methods determine blood type antigens based on the presence or absence of a positive signal, which cannot distinguish between homozygous and heterozygous types. This can easily lead to misinterpreting weak signals from heterozygous types as false negatives / false positives, or failing to identify homozygous / heterozygous components in mixed samples. Therefore, it is necessary to provide an allogeneic blood transfusion testing method to improve testing accuracy and reduce false positives or false negatives when the testing population is unknown. Summary of the Invention

[0006] In view of the defects and problems existing in the current allogeneic blood transfusion detection, the present invention provides a method to improve the accuracy of allogeneic blood reinfusion detection.

[0007] A method for improving the accuracy of allogeneic blood reinfusion detection includes the following steps:

[0008] S1. Determine the detection antigen: Select C, c, E, Jka, Jkb, S, Fya, Fyb, Lea, Leb, M, and N erythrocyte surface antigens as allogeneic transfusion detection antigens;

[0009] S2. Antibody Optimization: Add antibody diluent to the antibody for serial dilution. Optimize the antibody using different concentrations of antibody in homozygous and heterozygous samples. Select the antibody concentration that can be detected in both homozygous and heterozygous samples as the optimal concentration of the antibody.

[0010] S3. Sample collection and processing: Collect venous blood from the upper limbs using EDTA anticoagulant tubes, dilute whole blood with red blood cell preservation solution at a ratio of 1:10, and store in a refrigerator for later use.

[0011] S4. Antibody incubation and washing: Mix the whole blood anticoagulated sample with the fluorescent recognition antibody and immediately vortex in the dark. After incubation in the dark, add washing solution and centrifuge to remove free antibodies. Add fluorescent tracer antibody, incubate in the dark and wash before use.

[0012] S5. Flow cytometry detection: After the flow cytometer is powered on and calibrated, the red blood cell gating strategy is determined, and the sample is loaded onto the instrument to collect and analyze the expression of red blood cell surface antigens in the sample blood.

[0013] S6. Result Analysis and Judgment:

[0014] (1) If two or more blood group antigens are detected with a few expression or a few non-expression in a double peak, it can be determined as a positive allogeneic transfusion.

[0015] (2) If only one blood type antigen shows a few expression or a few non-expression bimodal peaks, then it is necessary to further determine whether there is a significant antibody dose effect between homozygous and heterozygous expression of the allele antigen. If there is an antibody dose effect, it can be determined as positive for allogeneic blood transfusion.

[0016] Conversely, it is negative.

[0017] The aforementioned method for improving the accuracy of allogeneic blood reinfusion detection uses PE-labeled mixed antibodies, including anti-C (human IgM) diluted 50 times, anti-c (human IgM) diluted 40 times, anti-E (human IgM) diluted 40 times, anti-Jka (human IgM) diluted 10 times, anti-Jkb (human IgM) diluted 20 times, anti-S (human IgM) diluted 40 times, anti-Fya (human IgG) diluted 10 times, anti-Fyb (human IgG) diluted 5 times, anti-Lea (mouse IgM) diluted 3 times, anti-Leb (mouse IgM) diluted 3 times, anti-M (mouse IgG) diluted 20 times, and anti-N (mouse IgG) diluted 10 times.

[0018] In the above-mentioned method for improving the accuracy of allogeneic blood reinfusion detection, the antibody diluent is a 1×PBS + 0.1% BSA solution.

[0019] In the above-described method for improving the accuracy of allogeneic blood reinfusion detection, the washing solution in step S4 is a PBS solution.

[0020] The above-mentioned method for improving the accuracy of allogeneic blood reinfusion detection involves a red blood cell gating strategy: first, delineating a single red blood cell gate on the FSC-H / FSC-A scatter plot, and then delineating the main particles to determine the core population on the FS / SS scatter plot within that gate.

[0021] The method described above for improving the accuracy of allogeneic blood reinfusion detection, step S5 further includes using a pipette to agitate the sample before loading it onto the instrument to reduce red blood cell aggregation.

[0022] In the above-described method for improving the accuracy of allogeneic blood reinfusion detection, the fluorescent tracer antibody in step S5 is any one of the following: 400-fold diluted goat anti-human IgM-FITC, 400-fold diluted goat anti-human IgM-PE, 400-fold diluted goat anti-human IgM-AF488, 50-fold diluted goat anti-human IgG-FITC, 100-fold diluted goat anti-human IgG-PE, 100-fold diluted goat anti-human IgG-AF488, 400-fold diluted goat anti-mouse IgM-FITC, 400-fold diluted goat anti-mouse IgM-PE, 400-fold diluted goat anti-mouse IgM-AF488, 100-fold diluted goat anti-mouse IgG-FITC, 100-fold diluted goat anti-mouse IgG-PE, or 100-fold diluted goat anti-mouse IgG-AF488.

[0023] The above-mentioned method for improving the accuracy of allogeneic blood transfusion detection, in step S6, the fluorescence signal peak shape of the blood group antigen corresponding to the allele: if a first signal peak and a second signal peak are detected in the blood sample of the recipient, wherein the first signal peak is a few expression double peaks or a few non-expression double peaks of one blood group antigen corresponding to the allele, and the second signal peak is an expression-expression double peak of another blood group antigen corresponding to the allele, then the recipient is determined to be positive for allogeneic blood transfusion.

[0024] The method described above for improving the accuracy of allogeneic blood reinfusion detection, step S6 further includes determining whether the sample to be tested is an RzR2 haplotype by comparing the expression patterns of the spectrum cells and the blood group antigens D, C, c, E, and e of the blood group to be tested with those of the RzR2 type C antigen.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] 1. This invention innovatively adds four additional antigens to the eight internationally recognized antigens, thereby significantly reducing the probability of false positives or false negatives in allogeneic blood transfusion detection through the specific combination of 12 blood type antigens. At the same time, this invention optimizes multiple factors such as blood type antibody selection, fluorescent antibody comparison, automated cell washing program control, washing solution selection, anti-erythrocyte aggregation, and allogeneic erythrocyte determination methods, thereby more effectively improving the detection rate of positive allogeneic blood transfusion stimulant samples in my country.

[0027] 2. This invention adds the allele antibody dose effect as a criterion for judgment on the basis of the original positive judgment criteria, which effectively solves the problem that the existing judgment method cannot make a judgment on only one double peak, thus leading to false negatives, and improves the detection accuracy and precision.

[0028] 3. To address the issue of RzR2 type C antigen presenting as intertwined double-core scattering / joined double peaks, which makes it prone to being positive, this invention aims to eliminate the interference of false positives for RzR2 type C antigen by comparing the expression patterns of blood group antigens D, C, c, E, and e between spectrophotometer cells and the blood sample to be tested, thereby determining whether the sample to be tested is RzR2 haplotype and avoiding false positives.

[0029] 4. After verification, the method of this invention can detect single blood sample + 95% / 5% mixed blood sample after 28 days of refrigerated storage of whole blood sample, and partially mixed blood sample after 35 days; samples diluted with stabilizing solution can still be accurately detected after 35 days of refrigerated storage, and the blood test has good stability. Attached Figure Description

[0030] Figure 1a -b indicates a positive allogeneic blood transfusion pattern; Figure 1a These are a minority of heterologous erythrocytes; Figure 1b These are a minority of red blood cells that do not express heteroplasm;

[0031] Figure 2 To express heterogeneous erythrocyte atlases;

[0032] Figure 3 Configure and gate settings for the DxFLEX flow cytometer protocol;

[0033] Figure 4 ABO blood type expression results for 100 subjects;

[0034] Figure 5 This invention yields consistent results using 8 antigen combinations and 12 antigen combinations.

[0035] Figure 6 For R z R2C flow cytometry analysis diagram. Detailed Implementation

[0036] To address the problems and shortcomings of current allogeneic blood transfusion testing, this invention provides a method to improve the accuracy of allogeneic blood transfusion testing by screening suitable blood type antigens for allogeneic blood transfusion testing and by combining multiple conditions such as blood type antibody selection, fluorescent antibody comparison, automated cell washing program control, washing solution selection, anti-erythrocyte aggregation, and allogeneic erythrocyte determination methods. This effectively increases the detection rate of positive allogeneic blood transfusion stimulant samples.

[0037] This method mainly includes the following:

[0038] S1. Identification of Detection Antigens: Through population screening and analysis, C, c, E, Jka, Jkb, S, Fya, Fyb, Lea, Leb, M, and N erythrocyte surface antigens were selected as allogeneic transfusion detection antigens.

[0039] S2. Antibody Optimization: 1×PBS + 0.1% BSA solution was added to the antibody as a serial dilution buffer for serial dilution. Compared with the traditional dilution buffer, the BSA concentration was reduced, thus reducing red blood cell aggregation. Different concentrations of antibody were used to optimize the antibody concentration using homozygous and heterozygous samples. The antibody concentration that could be detected in both homozygous and heterozygous samples was selected as the optimal concentration of the antibody.

[0040] A comparison of four fluorescent labeling methods—FITC, AF488, FITC / AF488, and PE—revealed that using the PE fluorescently labeled antibody, which exhibits stronger fluorescence intensity, makes it easier to separate the two groups of red blood cells. By using the same fluorescent mixed antibody, the number of blank control test tubes is reduced, and fluorescence compensation is not required, ensuring accurate results while saving detection time and costs. The PE-labeled mixed antibody includes anti-C (human IgM) diluted 50-fold, anti-c (human IgM) diluted 40-fold, anti-E (human IgM) diluted 40-fold, anti-Jka (human IgM) diluted 10-fold, anti-Jkb (human IgM) diluted 20-fold, anti-S (human IgM) diluted 40-fold, anti-Fya (human IgG) diluted 10-fold, anti-Fyb (human IgG) diluted 5-fold, anti-Lea (mouse IgM) diluted 3-fold, anti-Leb (mouse IgM) diluted 3-fold, and anti-M (mouse IgM) diluted 20-fold. After adding fluorescent antibody to each tube (IgG) and anti-N (mouse IgG) diluted 10 times, immediately vortex to ensure uniform staining. This also makes the vortexed tubes easier to distinguish from those without antibody, avoiding the risk of missing or incorrect antibody addition.

[0041] S3. Sample collection and processing: Collect venous blood from the upper limbs using EDTA anticoagulant tubes, dilute whole blood with red blood cell preservation solution at a ratio of 1:10, and store in a refrigerator for later use.

[0042] S4. Antibody Incubation and Washing: After mixing the whole blood anticoagulated sample with the fluorescent recognition antibody, immediately vortex in the dark to avoid red blood cell aggregation affecting antigen-antibody binding. After incubation in the dark, add PBS solution as a washing buffer and use it in conjunction with an automated washing centrifuge to remove free antibodies. Add fluorescent tracer antibody, incubate in the dark, and wash before use. By using a 24-position automated cell washing centrifuge, the programmed steps of adding washing buffer, forward centrifugation, reverse centrifugation to discard the washing buffer, and then forward centrifugation to collect cells are automatically completed. This effectively removes free antibodies, saves detection time, standardizes multi-tube washing conditions, and reduces manual cross-contamination, achieving the goal of automated cell washing. At the same time, using PBS solution as a washing agent reduces the use of commercial washing buffer, lowering costs.

[0043] S5. Flow Cytometry Detection: After the flow cytometer is powered on and calibrated, a red blood cell gate fixation scheme is adopted, which involves "first delineating individual red blood cell gates on the FSC-H / FSC-A scatter plot, and then delineating the main particles to determine the core population on the FS / SS scatter plot within the gate". This scheme can effectively reduce background noise while maximizing the proportion of minority red blood cell populations. Subsequently, the sample is loaded onto the instrument to collect and analyze data on the expression of red blood cell surface antigens in the sample blood. In order to reduce red blood cell aggregation in this step, the sample is pipetted before loading the sample to disperse the red blood cells and avoid aggregation.

[0044] S6. Result Analysis and Judgment:

[0045] Traditional testing methods define a positive allogeneic transfusion result as the detection of two or more blood group antigens with a few expressed or few not expressed in a biphasic pattern, which is considered a positive allogeneic transfusion stimulant test. Figure 1a and Figure 1b As shown. However, if only one antigen shows the aforementioned double peak, a positive result cannot be determined, leading to false negatives. To solve this problem, this invention introduces the antibody dose effect of allele antigens based on existing positive determination methods. When only one blood type antigen shows a double peak of minority expression or minority non-expression, it further determines whether there is a significant antibody dose effect between homozygous and heterozygous expression of that allele antigen. Based on this characteristic, if the donor and recipient have homozygous and heterozygous expression of an antigen determined by a certain allele, respectively, the recipient will show a double peak of minority expression or minority non-expression of one antigen, and a double peak of expression of the other antigen, i.e., a double peak appears in the antigen expression region. Therefore, if an antibody dose effect exists, a positive result can be determined. Figure 2 As shown.

[0046] Conversely, it is negative.

[0047] The specific technology of the present invention will be further described below with reference to specific embodiments.

[0048] Example 1: Instrument parameter setting and red blood cell gating strategy optimization

[0049] Using Makorpanel 16 cells manufactured by Sanquin as quality control samples, routine antigen detection was performed. Instrument testing protocols were created and optimized, and test protocol templates were established and standardized.

[0050] (1) A detection scheme for FITC, AF488, and PE fluorescence was established on a DxFLEX flow cytometer. The DxFLEX is a new type of flow cytometer produced in China. Compared with the FC500, the optical path abandons the plug-in integrated circuit method, resulting in more stable performance; the liquid path adopts a peristaltic pump injection method, making the instrument compact; the software analysis section adds FSC-H and FSC-A parameters, making the single-cell gate setting more precise, while reducing the interference of cell debris in the expression area, resulting in a cleaner detection background in the expression area; in addition, in terms of acquisition speed, the DxFLEX can complete the acquisition of more samples in the same amount of time, improving analysis efficiency. The instrument scheme settings are as follows.

[0051] Table 1. DxFLEX Instrument Parameter Settings

[0052]

[0053] (2) Experimental subjects

[0054] Positive Quality Control (QCP): Standard profile of mixed cell samples (95% expressing cells / 5% non-expressing cells, or vice versa);

[0055] Quality control negative (QCN): Standard profile cells (single blood sample with or without expression type);

[0056] Reagent blank control: blood sample + mixed secondary antibody (without specific primary antibody);

[0057] Sample type (blood group antigen expression type):

[0058] Single blood sample expression type, single blood sample non-expression type, mixed blood sample majority expression type (95% expression), mixed blood sample majority non-expression type (95% non-expression).

[0059] (3) Setting and evaluation of erythrocyte phylogenetics: The following scheme is used to accurately delineate individual erythrocyte populations and refine the analysis area based on this.

[0060] Option A: On the FS / SS scatter plot, directly delineate the gates that contain most of the particles at locations the size of a single red blood cell.

[0061] Option B: On the FS / SS scatter plot, directly delineate the phylum containing the main granules (core population) at the location of a single red blood cell.

[0062] Option C: On the FS / SS scatter plot, directly delineate the strict gate that contains only the core particle at the location of a single red blood cell.

[0063] Option D: First, delineate individual erythrocyte phyla (excluding adhesions) on the FSC-H / FSC-A scatter plot, and then delineate most of the particles on the FS / SS scatter plot within that phylum.

[0064] Option E: First, delineate individual erythrocyte phyla on the FSC-H / FSC-A scatter plot, and then delineate the major granules (core population) on the FS / SS scatter plot within that phylum.

[0065] Option F: First, delineate a single erythrocyte phylum on the FSC-H / FSC-A scatter plot, and then delineate the core particle on the FS / SS scatter plot within that phylum.

[0066] The advantages and disadvantages of the different gate configuration schemes (Scheme AF) mentioned above are evaluated based on key indicators such as detection capability, single-sample background interference, and non-specific binding level:

[0067] Minority population detection capability: On the same mixed sample (e.g., QCP), compare the proportion of particles detected by minority red blood cell populations (e.g., 5% non-expressing or expressing) in different gate settings. The higher the proportion, the stronger the ability of the protocol to detect low-frequency minority cell populations.

[0068] Single-sample background interference: On the same single sample (such as QCN or a single non-expressive type), compare the proportion of background particles (i.e. non-specific signals) in different gate settings. The lower the proportion, the less background interference the scheme has.

[0069] Non-specific binding level: On the reagent blank control of all samples, the proportion of background particles in different gate settings was compared. The lower the proportion, the less non-specific binding signal the scheme has.

[0070] Taking into account the detection capability of the indicators, background interference in single samples, and non-specific binding levels, the gate setting scheme is optimized to minimize background interference and non-specific binding (reduce the risk of false positives) while maintaining or increasing the detection rate of a small number of red blood cell populations (avoiding false negatives due to overly strict gate settings).

[0071] Based on the above assessment, the results are as follows: Figure 3 As shown, scheme E significantly removes the clutter peaks of aggregated cell debris, minimizing background interference and non-specific binding while increasing the detection rate of a small number of red blood cell populations.

[0072] Example 2: Antibody Optimization

[0073] Using Sanquin's Makorpanel 16 cells as quality control samples, a series of antibody concentrations were used to detect the expression type of the antigen corresponding to the antibody, determine the optimal antibody concentration, and ensure that when the quality control samples were tested at 50%, 100%, and 200% of this concentration, at least two concentrations met the criteria for clearly distinguishing between antigen-expressing and non-expressing samples, or clearly determining between samples with majority or minority expression.

[0074] (1) Optimization of fluorescence quality control antibodies

[0075] Cell samples were used as control samples with different concentrations of antibodies. At least two of the three concentrations must meet the following criteria: Glycophorin A (CD235a) must be expressed in the expression region, and its isotype control, Mouse IgG1, must be expressed in the non-expression region. The antibody concentrations that meet these criteria are used as the optimal antibody concentrations. The optimal concentrations of the fluorescent control antibodies are shown in Table 2 below.

[0076] Table 2 Optimal Concentration of Fluorescent Quality Control Antibody

[0077]

[0078]

[0079] (2) Antibody recognition optimization

[0080] Antibody optimization: The antibody was serially diluted with antibody diluent, and different concentrations of antibody were used to optimize the antibody concentration for homozygous and heterozygous samples. Samples with homozygous or heterozygous alleles corresponding to the antigen were selected and mixed with samples where the antigen was not expressed to prepare blood samples. The differences in antigen expression levels between homozygous and heterozygous alleles for each antigen were compared. The antibody concentration that could be detected in both homozygous and heterozygous samples was selected as the optimal antibody concentration, ensuring the detection of samples with different antigen expression levels and guaranteeing the detection of both high and low expression levels of the antigen. In this example, 1×PBS + 0.1% BSA solution was used as the antibody diluent, which reduced the BSA concentration compared to traditional diluents, effectively reducing erythrocyte aggregation.

[0081] By comparing four fluorescent labeling methods—FITC, AF488, FITC / AF488, and PE—it was found that using the PE fluorescently labeled antibody, which has a stronger fluorescence intensity, makes it easier to separate the two groups of red blood cells.

[0082] This embodiment uses the same fluorescent (PE-labeled) mixed antibody, which not only reduces the number of blank control test tubes but also eliminates the need for fluorescence compensation, ensuring test results while saving test time and costs; the optimal concentration of the identification antibody is shown in Table 3 below.

[0083] Table 3 Optimal concentration of recognition antibody

[0084]

[0085]

[0086] (3) Optimization of fluorescent tracer antibodies

[0087] The optimal antibody concentration for each fluorescent antibody was determined by optimizing it with corresponding type-identifying antibodies using different fluorescent antibodies. The optimal concentrations of the fluorescent tracer antibodies are shown in Table 4 below.

[0088] Table 4 Optimal Concentration of Fluorescent Tracing Antibody

[0089]

[0090] Example 3: Antigen Screening

[0091] One hundred student volunteers were recruited from Beijing Sport University. Four milliliters of venous blood were collected from the upper limbs using EDTA anticoagulant tubes. Whole blood was diluted with red blood cell preservation solution at a ratio of 1:10 and stored in a refrigerated refrigerator until testing. The tests included ABO blood typing and the detection of the aforementioned 17 optimized blood group antigens.

[0092] (1) ABO blood type

[0093] ABO blood typing was performed using an ABO reverse typing reagent. The ABO blood type expression results of 100 subjects are as follows: Figure 4 As shown.

[0094] (2) Expression status of 17 antigens

[0095] The expression of 17 blood type antigens in 100 subjects is shown in Table 5 below.

[0096] Table 5. Expression of 17 blood group antigens in 100 subjects

[0097]

[0098] As shown in Table 3, both D and S antigens were expressed in all 100 individuals, while K antigens were not expressed. These three antigens lacked discriminatory power in the population and therefore did not meet the criteria for selection as antigens for allogeneic transfusion testing. The expression frequencies of the remaining 14 blood group antigens in the 100 volunteers ranged from 0.08 to 0.98, meeting the criteria for antigen selection in allogeneic transfusion testing. However, the study found that e and P1 antibodies exhibited poor recognition, specifically manifested as broad expression peaks or left-shifted expression peaks. This is attributed to the large number of e antigen variants in the population, resulting in significant variations in the amount of e antigen recognized by anti-e antibodies. Furthermore, the P1 antigen showed poor stability, leading to low binding efficiency of anti-P1 antibodies. Comparison of different antibodies did not improve the detection results. While these two antigens can distinguish the expression of single blood samples, they may cause inability to differentiate between double peaks or result in missed detections when testing mixed blood samples. Therefore, they are not recommended as routine antigens for allogeneic transfusion testing.

[0099] (3) Comparison of three fluorescent tracer antibodies

[0100] Eight blood samples were randomly selected from 100 subjects. Twelve fluorescent tracer antibodies labeled with PE, FITC, and AF488 were used to bind to 17 blood group antigens. The results showed that PE fluorescence had high binding efficiency for IgM antibodies, strong fluorescence intensity, and a more obvious distinction between expressed and non-expressed peaks compared to the other two fluorescence types. The binding of IgG antibodies to the three fluorescence types varied among individuals. All three fluorescent secondary antibodies could distinguish between expressed and non-expressed antigens. This invention uses PE-labeled fluorescent antibodies as the initial screening fluorescent secondary antibody.

[0101] (4) Method confirmation:

[0102] a. Selectivity: Each antigen corresponds to 10 single blood samples, of which 5 blood samples express the antigen and 5 blood samples do not express the antigen. Selectivity detection was performed on 14 antigens, and the results are shown in Table 6 below.

[0103] Table 6 Results of 14 antigen tests in a single blood sample

[0104]

[0105]

[0106] Note: In the table, "+" indicates antigen expression, and "-" indicates antigen non-expression.

[0107] As shown in Table 6, all 10 blood samples tested negative, and the expression was consistent with the prepared expression profile.

[0108] b. Reliability: For each of the 12 antigens, 10 95% / 5% mixed blood samples were used, with 5 samples showing majority expression and 5 samples showing majority non-expression. The results are shown in Table 7 below.

[0109] Table 7 Results of Detection of 12 Antigens in Mixed Blood Samples

[0110]

[0111]

[0112] Note: In the table, "DPE" indicates majority bimodal expression, and "DPN" indicates majority non-bimodal expression.

[0113] As shown in Table 7, all samples showed bimodal or suspected bimodal peaks, and their expression patterns were consistent with the recorded expression profiles, indicating that the 12 antigens could detect mixed blood samples.

[0114] Based on the above considerations, 12 antigens, namely C, c, E, Jka, Jkb, S, Fya, Fyb, Lea, Leb, M, and N, were selected as antigens for allogeneic blood transfusion detection.

[0115] c. Detection limit

[0116] Two mixed blood samples were selected, and mixed blood samples with homozygous and heterozygous expression were prepared with non-expressing blood samples to prepare mixed blood samples with majority expression and majority non-expression, respectively. The mixing ratios were 95% / 5%, 97% / 3%, 98.5% / 1.5%, 99.5% / 0.5%, and 100% / 0%, respectively. The detection limits of 12 antigens with majority and minority expression were verified. The results are shown in Table 8 below.

[0117] Table 8. Detection limits for 12 antigens with majority and minority expression.

[0118]

[0119]

[0120] As can be seen from Table 8, the detection limits for the antigens that are mostly expressed and mostly not expressed are as low as 0.5% and as high as 5%, respectively, which can meet the requirement that the number of mixed red blood cells is no more than 5%.

[0121] d. Carrying pollution

[0122] A tube of deionized water was placed after the sample, and the contamination rate was calculated (the number of particles in the erythrocyte phylum over 30 seconds was divided by 50,000, and then multiplied by 100%). The average contamination rate calculated from 100 trials was much less than 0.1%.

[0123] Example 4: Balance of expression and non-expression ratios of 12 antigens in the population

[0124] Because the expression ratio of certain antigens in the population is uneven, especially since Fya is expressed in the vast majority of subjects, in order to further verify the balance of the expression ratio of 12 antigens in the population, 150 subjects were recruited to study the expression of the 12 blood group antigens. The results are shown in Table 8 below.

[0125] Table 8. Expression of 12 blood group antigens in 150 subjects

[0126]

[0127]

[0128] As can be seen from Table 8, the expression and non-expression ratios of the four additional antigens added in this invention based on the eight internationally used antigens (C, c, E, Jka, Jkb, Fya, Fyb, S) are relatively balanced, which can effectively balance the relationship between antigen quantity and detection efficiency.

[0129] Simultaneously, a comparative analysis was conducted on 150 subjects using ABO blood type and 8 blood group antigens, as well as combinations of ABO blood type and 12 blood group antigens. The results are as follows: Figure 5 As shown.

[0130] The results showed that: 1) The number of blood type antigen expression combinations was 115 in the former and 60 in the latter, with the latter being nearly double the former; 2) Among 150 people, 119 in the former were able to find at least one person with the same blood type as themselves, with a maximum of 11 people having completely identical results, while only 58 in the latter were able to find at least one person with the same blood type as themselves, with a maximum of 4 people having completely identical results; 3) Only 31 people in the former had blood types different from other subjects, while 92 people in the latter had blood types different from other subjects, which was nearly three times that of the former.

[0131] Therefore, the 12 antigen combinations used in this invention greatly reduce the proportion of people with the same blood type. That is, if these 150 subjects undergo allogeneic blood transfusions, the false negative rate of not being able to detect differences will also be greatly reduced.

[0132] Example 5: Blood Sample Stability

[0133] Fifty-two volunteers were randomly recruited, and 4 mL of venous blood was collected from their upper limbs using EDTA anticoagulant tubes to test their ABO blood type. Eight whole blood samples were selected from these samples, and two samples with the same ABO blood type were mixed in vitro to obtain four mixed blood samples as positive samples, with a mixing ratio of 95% / 5%. In addition, four single blood samples were randomly selected from the 52 blood samples as negative samples. All samples were stored under refrigerators until testing.

[0134] (1) Robustness

[0135] Four positive blood samples and one negative blood sample were prepared and divided into three equal parts. These parts were then tested by different personnel on different days using the method of this invention to test the robustness of the method in both human and daytime testing. The results of the three tests were consistent, indicating that the method of this invention has good robustness.

[0136] (2) Stability of whole blood and stability of blood samples diluted with stabilizing solution

[0137] Four mixed blood samples and four single blood samples were aliquoted into two 100 μL portions. One portion of whole blood was left untreated, while the other portion was diluted with 1000 μL of cell stabilizing solution on days 28 and 35 for preservation. The RBC values ​​of the stabilized samples were measured using Sysmex. The method of this invention was used to detect 12 blood group antigens.

[0138] Testing revealed that whole blood samples stored for 28 days could detect both single and 95% / 5% mixed blood samples; whole blood samples stored for 35 days could detect some mixed blood samples, while the results for single blood samples remained unchanged. Diluted blood samples stored in stabilizing solution, tested on day 35 of stabilization, could detect both single and 95% / 5% mixed blood samples.

[0139] (3) Blood sample testing

[0140] Fifty student volunteers were recruited from Beijing Sport University. Four mL of venous blood was collected from their upper limbs using EDTA anticoagulant tubes to test their ABO blood type. Fifty whole blood samples were used as negative samples. Twenty-four whole blood samples were selected, and two samples with the same ABO blood type were mixed in vitro to obtain 12 mixed blood samples, which were used as positive samples (mixing ratio 95% / 5%). All samples were refrigerated until testing. Results showed that all 50 individual samples tested negative, and all 12 mixed samples tested positive.

[0141] Example 6: Detection of RzR2 type C antigen

[0142] In the detection of spectrophotometers, this invention found that the RzR2 type C antigen, in the expression region of the flow cytometry analysis, presents as intertwined double-core scatter plots and connected double peaks, such as... Figure 6 As shown, its difference from a positive double peak is that, no matter how the antibody concentration is optimized, a separate double peak cannot be obtained. The possible reason for this phenomenon is that the expression level of RzR2 type C antigen is lower than that of other types of C antigen. When using a standard concentration of antibody to bind to the antigen, the antibody concentration is relatively too high, resulting in the illusion of two cell populations.

[0143] To eliminate the interference of false positives for RzR2 type C antigen, the following experiments were conducted in this embodiment:

[0144] 1) Compare the antibody detection results of different clones;

[0145] 2) Detection was performed using antibodies labeled with different fluorescent dyes such as FITC and PE;

[0146] 3) As the antibody concentration decreases, the two peaks gradually merge into a single peak;

[0147] 4) Detection was performed using the gel column antibody binding method.

[0148] The results showed that changing the antibody could not eliminate interference; using different fluorescently labeled antibodies for detection still could not eliminate interference; and reducing the antibody concentration gradually merged the biphasic peaks into a single peak, but the single peak appeared close to the non-expression region, making it impossible to determine whether the result was due to insufficient antibody; and although the gel column antibody binding method detected a small number of free cells, it was far less than the proportion observed on flow cytometry, indicating that the currently used methods cannot effectively eliminate the interference of false positives of RzR2 type C antigen.

[0149] Since the expression phenotypes of RzR2 type D, C, c, E, and e antigens are defined, this embodiment addresses this issue by comparing the expression phenotypes of blood group antigens D, C, c, E, and e between spectrophotometer cells and the blood sample being tested to determine whether the sample is an RzR2 haplotype. Simultaneously, by performing additional testing on a routine sample for the C antigen UDP status, and by detecting the D, C, c, E, and e antigen phenotypes and comparing them with the corresponding RzR2 type antigens on spectrophotometer cells, the sample was deemed to have consistent with the RzR2 haplotype detection results on spectrophotometer cells and was thus determined to be negative, effectively resolving the problem of false positive interference from RzR2 type C antigen.

[0150] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions and improvements 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 of improving the accuracy of detection of allogeneic blood transfusion, characterized by: Includes the following steps: S1. Determine the detection antigen: Select red blood cell surface antigens as the detection antigens for allogeneic blood transfusion; the red blood cell surface antigens include C, c, E, Jka, Jkb, S, Fya, Fyb, Lea, Leb, M, and N; S2. Antibody Optimization: Add antibody diluent to the antibody for serial gradient dilution. Optimize the antibody using different concentrations of antibody in homozygous and heterozygous samples. Select the antibody concentration that can be detected in both homozygous and heterozygous samples as the optimal concentration of the antibody. S3. Sample collection and processing: Collect venous blood from the upper limbs using EDTA anticoagulant tubes, dilute whole blood with red blood cell preservation solution at a ratio of 1:10, and store in a refrigerator for later use. S4. Antibody incubation and washing: Mix the whole blood anticoagulated sample with the fluorescent recognition antibody and immediately vortex in the dark. After incubation in the dark, add washing solution and centrifuge to remove free antibodies. Add fluorescent tracer antibody, incubate in the dark and wash before use. S5. Flow cytometry detection: After the flow cytometer is powered on and calibrated, the red blood cell gating strategy is determined, and the sample is loaded onto the instrument to collect and analyze the expression of red blood cell surface antigens in the sample blood. S6. Result Analysis and Judgment: (1) If two or more blood group antigens are detected with a few expression or a few non-expression in a bimodal pattern, it can be determined as a positive allogeneic transfusion. (2) If only one blood type antigen shows a few expression or a few non-expression double peaks, then further determine whether there is a significant antibody dose effect between homozygous and heterozygous expression of the allele corresponding to the blood type antigen; if only the C antigen shows intertwined double core scattering and shoulder double peaks, and after S2 optimization of antibody concentration, it is still impossible to obtain mutually separated double peaks, then further compare whether the expression types of D, C, c, E, and e of the test sample and the RzR2 haplotype cells are consistent. If the test results of the sample and the RzR2 haplotype cells are consistent, then it is negative. If an antibody dose effect exists and is inconsistent with the results of the RzR2 haplotype detection in spectrocells, it can be determined as a positive allogeneic transfusion. (3) If it is different from the above (1) or (2), then it is negative.

2. The method of improving accuracy of detection of allogeneic blood reinfusion according to claim 1, wherein: The recognition antibody is a PE-labeled mixed antibody, including human IgM anti-C diluted 50 times, human IgM anti-c diluted 40 times, human IgM anti-E diluted 40 times, human IgM anti-Jka diluted 10 times, human IgM anti-Jkb diluted 20 times, human IgM anti-S diluted 40 times, human IgG anti-Fya diluted 10 times, human IgG anti-Fyb diluted 5 times, mouse IgM anti-Lea diluted 3 times, mouse IgM anti-Leb diluted 3 times, mouse IgG anti-M diluted 20 times, and mouse IgG anti-N diluted 10 times.

3. The method of improving accuracy of detection of allogeneic blood reinfusion according to claim 1, wherein: The antibody diluent was a 1×PBS + 0.1% BSA solution.

4. The method of improving accuracy of detection of allogeneic blood reinfusion according to claim 1, wherein: The cleaning solution mentioned in step S4 is a PBS solution.

5. The method of improving accuracy of detection of allogeneic blood reinfusion according to claim 1, wherein: The erythrocyte gating strategy is as follows: first, delineate a single erythrocyte gate on the FSC-H / FSC-A scatter plot, and then delineate the main particles to determine the core population on the FS / SS scatter plot within that gate.

6. The method of improving accuracy of detection of allogeneic blood reinfusion of claim 1, wherein: Step S5 also includes pipetting the sample before loading it onto the instrument to reduce red blood cell aggregation.

7. The method of improving accuracy of detection of allogeneic blood reinfusion of claim 1, wherein: The fluorescent tracer antibody mentioned in step S5 is any one of the following: 400-fold diluted goat anti-human IgM-FITC, 400-fold diluted goat anti-human IgM-PE, 400-fold diluted goat anti-human IgM-AF488, 50-fold diluted goat anti-human IgG-FITC, 100-fold diluted goat anti-human IgG-PE, 100-fold diluted goat anti-human IgG-AF488, 400-fold diluted goat anti-mouse IgM-FITC, 400-fold diluted goat anti-mouse IgM-PE, 400-fold diluted goat anti-mouse IgM-AF488, 100-fold diluted goat anti-mouse IgG-FITC, 100-fold diluted goat anti-mouse IgG-PE, or 100-fold diluted goat anti-mouse IgG-AF488.

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