Reagent composition, kit and method for predicting risk of senile deafness

Abstract

The invention is applicable to the technical field of biomedicine and immunological detection, and provides a reagent composition, a kit and a method for risk prediction of senile deafness, the reagent composition contains three types of fluorescence labeled antibody combinations: T cell surface marker antibodies CD3, CD4 and CD8; a T cell differentiation and activation marker antibody CCR7, CD45RA, HLA-DR and CD38; and natural killer cell functional marker antibodies CD56 and NKP30. The expression level of related lymphocyte subpopulations and functional markers thereof in peripheral blood is detected through flow cytometry, the'immune-auditory axis' association of immune aging and auditory degeneration is revealed, and a multi-parameter prediction model integrating age and immune indexes is constructed. The model can realize early recognition, risk stratification and accurate evaluation of senile deafness of middle-aged and elderly people. The method has the advantages of high prediction accuracy, early warning, non-invasiveness, simplicity and convenience in operation and the like, and has a wide application prospect.

Description

A reagent composition, kit, and method for predicting the risk of age-related hearing loss. Technical Field

[0001] This application belongs to the field of biomedical and immunological detection technology, and particularly relates to a reagent composition specifically used for detecting the phenotype and functional status of peripheral blood lymphocyte subsets, a kit containing the composition, and a method for constructing a risk prediction model for age-related hearing loss based on multi-parameter immune indicators. Background Technology

[0002] Presbycusis is the most common chronic sensory impairment among the elderly, and its prevalence increases significantly with age, making it a major global public health problem. It not only leads to difficulties in verbal communication but is also closely related to cognitive decline, depression, and social isolation, severely impacting the quality of life and mental and physical health of the elderly.

[0003] Currently, clinical diagnosis mainly relies on pure-tone audiometry. This method is a passive diagnosis after hearing loss has occurred, and can only make a judgment when there is already obvious damage to hearing function. It lacks effective early warning and risk prediction methods, and cannot achieve early detection and early intervention of the disease.

[0004] Studies have shown that the pathological process of age-related hearing loss is not simply a matter of "wear and tear" on the auditory organs, but is closely related to systemic "immunosenescence" and chronic low-grade inflammation ("inflammatory aging"). Dysregulation of systemic immune function may affect the cochlear microenvironment through multiple pathways, promoting local inflammatory responses and accelerating damage and death of auditory cells. Previous studies have found abnormal proportions of immune cells in the peripheral blood of patients with age-related hearing loss, such as increased infiltration of pro-inflammatory immune cells, suggesting a potential link between immune indicators and the occurrence and development of age-related hearing loss.

[0005] However, existing technologies have significant shortcomings: First, there is a lack of standardized testing protocols for systematic, multi-dimensional analysis of lymphocyte subsets (including proportion, differentiation status, and functional biomarkers), failing to comprehensively capture the immune characteristics associated with age-related hearing loss; second, key immune indicators have not yet been organically combined with core influencing factors such as age to construct a highly accurate risk prediction model that can be directly applied to clinical practice. Therefore, there is an urgent need to develop a non-invasive, highly accurate early risk assessment tool for age-related hearing loss based on peripheral blood immune characteristics to overcome the deficiencies of existing technologies. Summary of the Invention

[0006] The purpose of this application is to provide a reagent composition, kit, and detection method that can effectively predict the risk of age-related hearing loss. By detecting the expression levels of a key lymphocyte subset and its functional markers in peripheral blood, the "immune-auditory axis" relationship between immune aging and hearing degeneration is revealed. A multi-parameter prediction model integrating age and immune indicators is constructed to achieve early identification, risk stratification, and accurate assessment of age-related hearing loss in middle-aged and elderly populations, providing a scientific basis for early clinical intervention and filling the gap in the field of early warning of age-related hearing loss.

[0007] This application is implemented as follows: a reagent composition for predicting the risk of age-related hearing loss, characterized in that it comprises a combination of fluorescently labeled antibodies for specifically detecting the phenotype and functional status of peripheral blood lymphocyte subsets. The antibody combination consists of the following three types of antibodies: T cell surface marker antibodies CD3, CD4, and CD8; T cell differentiation and activation marker antibodies CCR7, CD45RA, HLA-DR, and CD38; and natural killer cell functional marker antibodies CD56 and NKP30.

[0008] Another objective of this application is a kit for predicting the risk of developing age-related hearing loss, comprising the above-described reagent composition, red blood cell lysis buffer, and phosphate buffer.

[0009] Another objective of this application is to provide a method for predicting the risk of developing age-related hearing loss, comprising the following steps:

[0010] (1) Obtain peripheral venous blood samples from the subjects and use the above-mentioned reagent composition or the above-mentioned kit to detect the following indicators by flow cytometry: CD4+ T cell ratio, CD8+ T cell ratio, CD4+ / CD8+ T cell ratio, CD8+HLA-DR+ T cell ratio, CD8+HLA-DR+CD38+ T cell ratio, terminally differentiated CD8+ T cell ratio, and NKP30+ NK cell ratio;

[0011] (2) Combine the results of the above detection indicators with the subject's age information and substitute them into a multivariate binary logistic regression model to calculate the probability P of the risk of age-related hearing loss. The model formula is: ln (P / (1−P)) = −8.913 −0.078×CD8+HLA-DR+CD38+T cell proportion− 0.021×NKP30+NK cell proportion+ 0.165× age;

[0012] (3) Determine the risk of presbyopia based on the risk probability P: if P>0.620, it is considered high risk; if P≤0.620, it is considered low risk.

[0013] Another objective of this application is to provide a computer-readable storage medium storing computer program instructions that, when executed by a processor, implement the aforementioned method for predicting the risk of age-related hearing loss.

[0014] The beneficial effects of the embodiments in this application compared with the prior art are:

[0015] High predictive accuracy: The immune markers selected in this application (CD8+HLA-DR+CD38+T cells, NKP30+NK cells, etc.) are highly correlated with the risk of age-related hearing loss. The constructed multi-parameter model integrates immune indicators and age factors, achieving an AUC of 0.828, significantly outperforming single-indicator prediction. After leave-one-out cross-validation (LOOCV), the model's AUC was 0.782, demonstrating good robustness.

[0016] Achieving early warning: This application can capture early immune changes associated with age-related hearing loss through peripheral blood testing, identifying high-risk individuals before significant hearing loss occurs. This breaks through the limitation of traditional diagnosis, which can only passively detect existing hearing loss, and buys time for early intervention.

[0017] Non-invasive and easy to operate: Only routine peripheral venous blood collection from the subject is required, without any invasive procedures; the testing process is standardized and easy to promote and apply.

[0018] Scientific Mechanism Support: Based on the "immune-auditory axis" theory, this application provides new scientific evidence for the study of the pathogenesis of age-related hearing loss by revealing the association between changes in immune cell subsets and age-related hearing loss.

[0019] The application prospects are broad: This kit can be used for large-scale screening of age-related hearing loss in middle-aged and elderly populations, long-term monitoring of high-risk groups, providing technical support for public health prevention and control, and providing potential targets for the development of intervention drugs and targeted therapy programs. Attached Figure Description

[0020] Figure 1 shows the distribution of lymphocyte subsets provided in the embodiments of this application in people with different hearing levels under the age of 75 (including 75 years old);

[0021] Figure 2 is a ROC curve diagram of the multi-factor regression model construction provided in the embodiment of this application;

[0022] Figure 3 is the ROC curve of the leave-one-out method used to verify the model according to the embodiments of this application. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0024] This application provides a reagent composition for predicting the risk of age-related hearing loss, comprising a combination of fluorescently labeled antibodies for specifically detecting the phenotype and functional status of peripheral blood lymphocyte subsets. The antibody combination consists of the following three classes of antibodies:

[0025] T cell surface marker antibodies: CD3, CD4, CD8;

[0026] T cell differentiation and activation marker antibodies: CCR7, CD45RA, HLA-DR, CD38;

[0027] Natural killer (NK) cell functional marker antibodies: CD56, CD337 (NKP30);

[0028] All antibodies are monoclonal antibodies conjugated with fluorescent dyes, preferably stored in lyophilized or liquid form, to meet the needs of multicolor flow cytometry detection. By accurately capturing the expression characteristics of the above-mentioned markers, a systematic analysis of the proportion and functional status of peripheral blood lymphocyte subsets can be achieved, thereby providing core detection basis for risk assessment of age-related hearing loss.

[0029] The fluorescent dye is selected from at least one of APC-AF700, APC-H7, PE, BB515, PE-Cy7, BV421, FITC, BB700, and Pacific Blue AF467.

[0030] Specifically, the specific information of the antibody reagents used in the specific embodiments of this application is shown in Table 1 below:

[0031] Table 1 Antibody Reagent Information

[0032]

[0033] This application also provides a kit for predicting age-related hearing loss based on peripheral blood lymphocyte subsets. The kit comprises the antibody reagent combination described in the above technical solution, and further includes the following auxiliary components:

[0034] Red blood cell lysis buffer: used to lyse red blood cells in peripheral blood and remove interference from red blood cells in lymphocyte detection;

[0035] Phosphate-buffered saline (PBS): Used for sample washing, antibody dilution, and cell resuspending to maintain normal cell physiological state;

[0036] Flow cytometer-specific sample loading tubes: adapted for flow cytometer detection, ensuring standardized sample testing;

[0037] Detailed experimental operation instructions: clearly define the standardized procedures for antibody working solution preparation, sample processing, staining, and instrument detection, and guide users to operate in a standardized manner.

[0038] All reagents in the kit have been optimized in proportion and can be stored in lyophilized or liquid form as needed to ensure the stability and repeatability of the testing process and meet the needs of different laboratories and clinical scenarios.

[0039] In addition, the kit may include a negative control antibody or blank control setup to calibrate the fluorescence signal of the flow cytometer, eliminate interference from nonspecific binding, and improve the accuracy of the test results.

[0040] This application also provides a method for predicting the risk of developing age-related hearing loss, the method comprising the following steps:

[0041] Sample Collection and Processing: Peripheral venous blood was collected from the subjects and anticoagulated using sodium heparin. The anticoagulated blood samples were stored at room temperature (25°C) and staining, erythrocyte lysis, and washing were performed within 72 hours to avoid changes in cell state due to prolonged sample storage, which could affect the accuracy of the test results.

[0042] Indicator Detection: Using the reagent composition or kit described in this application, the following key immune indicators were detected by flow cytometry: CD4+ T cell percentage, CD8+ T cell percentage, CD4+ T cell to CD8+ T cell ratio, CD8+HLA-DR+ T cell percentage, NKP30+ NK cell percentage, terminally differentiated CD8+ T cell percentage, and CD8+HLA-DR+CD38+ T cell percentage. Terminally differentiated CD8+ T cells were defined using a combination of CD8+CCR7-CD45RA+ markers; the percentages of each T cell subset were all percentages of the total lymphocyte population, and the NK cell percentage was the percentage of CD3-CD56+ cells in the total lymphocyte population.

[0043] Multi-parameter risk prediction model calculation: The detected proportions of CD8+HLA-DR+CD38+T cells and NKP30+NK cells are combined with the subject's age information and substituted into a multivariate binary logistic regression model to calculate the risk probability P of the subject developing age-related hearing loss. The model formula is: ln (P / (1−P)) = −8.913 − 0.078×CD8+HLA-DR+CD38+T cell proportion − 0.021×NKP30+NK cell proportion + 0.165×age.

[0044] Risk assessment: The risk probability P is assessed based on the optimal cutoff value calculated using the Youden index: if P > 0.620, the risk of developing age-related hearing loss is considered high; if P ≤ 0.620, the risk of developing age-related hearing loss is considered low.

[0045] Model Validation: The model was validated using Leave-One-out Cross-Validation (LOOCV). The validation approach is to leave one sample from the dataset as the test set in each iteration, train the model using all the remaining samples, and then test it on the left-out sample. This process is repeated so that each sample is used as the test set exactly once. Finally, the average of all test results is used as an estimate of the model performance.

[0046] The above methods can be used for early screening, risk stratification, or monitoring of intervention effects for age-related hearing loss in middle-aged and elderly people aged 50 and above.

[0047] This application also provides a computer-readable storage medium storing computer program instructions. When these instructions are executed by a processor, the method for predicting the risk of age-related hearing loss described above is implemented. Specifically, the computer program instructions are used to: receive peripheral blood lymphocyte subset data and subject age information obtained by flow cytometry, substitute them into the aforementioned multivariate binary logistic regression model to calculate the risk probability P, and automatically output the risk assessment result for age-related hearing loss (high risk or low risk) based on the P value. This enables rapid analysis of detection data and automated output of risk results, improving detection efficiency and the standardization of result assessment.

[0048] The following detailed description of the reagent composition, kit, and method for predicting the risk of age-related hearing loss provided in this application is provided through specific embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; and the materials and reagents used, unless otherwise specified, are conventional products that can be obtained commercially.

[0049] 1. Grouping of experimental samples

[0050] The study population was divided into five groups based on hearing level, with the following specific grouping criteria: normal hearing (0-25 dBHL), mild hearing impairment (26-40 dBHL), moderate hearing impairment (41-60 dBHL), severe hearing impairment (61-80 dBHL), and complete hearing impairment (>80 dBHL). The mild hearing impairment group and the moderate hearing impairment group were combined into the mild to moderate hearing impairment group (MMHL), and the severe hearing impairment group and the complete hearing impairment group were combined into the severe to complete hearing impairment group (SPHL) for subsequent analysis of differences in immune indicators. Subject information is shown in Table 2 below.

[0051] Table 2 Subject Information

[0052]

[0053] 2. Experimental Procedure (to be conducted in complete darkness)

[0054] (1) Preparation of mixed antibody working solution: After removing the antibody from the 4℃ freezer, briefly centrifuge at 3000 rpm. Referring to the instructions of the corresponding antibody in Table 1 above, prepare the antibody working solution in a 1.5 ml EP tube at a ratio of 1:10 (the final antibody concentration ratio in the total system is 1:100), gently pipette to mix, and then place on ice for later use. A blank control tube should be set up simultaneously for each experiment to eliminate non-specific interference.

[0055] (2) Sample incubation: Take 100 μl of peripheral venous blood from the subject and add it to a labeled flow cytometer tube. Then add 10 μl of mixed antibody working solution (or add 10 μl of mixed antibody first and then add 100 μl of whole blood). Gently shake to mix the sample and antibody thoroughly and incubate at 4°C in the dark for 30 min.

[0056] (3) Red blood cell lysis: After incubation, add 2 ml of red blood cell lysis buffer to the flow cytometry tube, shake to mix immediately, and lyse at room temperature (20~30℃) for 10 min. Observe the clarity of the liquid. If it is clear, centrifuge at 1000 rpm for 5 min. If the lysis is incomplete, add 200 μL of lysis buffer, mix well, continue lysis at room temperature for 3 min, and then centrifuge at 1000 rpm for 5 min.

[0057] (4) Sample washing and resuspension: After centrifugation, remove the flow cytometry tube, discard the liquid in the tube, and gently invert it onto absorbent paper to blot away any remaining liquid at the tube opening. Add 2 ml of 1×PBS buffer to wash, centrifuge at 1500 rpm for 5 min, discard the supernatant, and then add 200 μl of 1×PBS buffer to resuspend the cells. Mix well and wait for testing. All samples must be tested within 3 hours using a BD FACSlyric flow cytometer.

[0058] 3. Statistical analysis and difference analysis of immune indicators

[0059] As shown in Figure 1, seven core immune indicators were detected and statistically analyzed by flow cytometry, including the proportion of CD4+ T cells, the proportion of CD8+ T cells, the CD4+ / CD8+ T cell ratio, the proportion of CD8+HLA-DR+ T cells, the proportion of NKP30+ NK cells, the proportion of terminally differentiated CD8+ T cells, and the proportion of CD8+HLA-DR+CD38+ T cells.

[0060] Statistical analysis employed targeted methods: analysis of variance was used for continuously distributed data that followed a normal distribution, while non-parametric tests were used for non-normally distributed data. Specific differences are as follows:

[0061] In individuals aged ≤75 years with mild to moderate hearing impairment (26-60 dB HL), compared to the group with normal hearing, the proportion of peripheral blood CD8+HLA-DR+CD38+ T cells was significantly increased, while the proportion of NKP30+NK cells was significantly decreased.

[0062] Compared with the normal hearing group, individuals with severe to complete hearing impairment (≥61 dB HL) had a decreased proportion of CD4+ T cells, an increased proportion of CD8+ T cells, a decreased CD4+ / CD8+ T cell ratio, and a decreased proportion of CD8+ HLA-DR+ T cells.

[0063] Compared with the mild to moderate hearing impairment group, individuals with severe to complete hearing impairment showed a further decrease in the proportion of CD4+ T cells, a continued increase in the proportion of CD8+ T cells, a decrease in the CD4+ / CD8+ T cell ratio, and a significant increase in the proportion of terminally differentiated CD8+ T cells.

[0064] 4. Prediction Model Construction and Validation

[0065] (1) Model building

[0066] Univariate logistic regression analysis was performed on the above key immune indicators, and receiver operating characteristic (ROC) curves were plotted to screen out effective predictive indicators. A multivariate logistic regression prediction model was constructed by incorporating age factors, and ROC curves were plotted as well (the results are shown in Figure 2).

[0067] The optimal cutoff values ​​for the univariate model are: age > 70 years, peripheral blood CD8+HLA-DR+ T cell ratio < 12.3%, and NKP30+NK cell ratio < 54.4%. Individuals meeting these criteria have a higher probability of developing hearing impairment.

[0068] The multi-factor joint prediction model formula is: ln(P / (1−P)) = −8.913 − 0.078×CD8+HLA-DR+CD38+T cell proportion − 0.021×NKP30+NK cell proportion + 0.165× age. Based on the Youden index, the optimal risk cutoff value P=0.620 was determined. When P>0.620, it was considered a high risk of age-related hearing loss; when P≤0.620, it was considered a low risk. Furthermore, the multi-factor joint prediction efficacy was significantly better than the single-factor model.

[0069] (2) Model validation

[0070] Leave-one-out cross-validation (LOOCV) is used to verify the robustness of the model. The core idea is as follows: in each iteration, one sample is reserved as the test set, and all remaining samples are used to train the model. The model performance is then validated on the test set. This process is repeated until all samples have been validated as the test set once. Finally, the model performance is evaluated by the average of all test results.

[0071] The area under the ROC curve (AUC) plotted based on the validation results reached 0.782, indicating that the model has good robustness and clinical application value (as shown in Figure 3).

[0072] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of this patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

[0073] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A reagent composition for predicting the risk of age-related hearing loss, characterized in that, It includes a fluorescently labeled antibody ensemble for the specific detection of peripheral blood lymphocyte subset phenotypes and functional status, the antibody ensemble consisting of the following three classes of antibodies: T cell surface marker antibodies CD3, CD4, and CD8; T cell differentiation and activation marker antibodies CCR7, CD45RA, HLA-DR, and CD38; and natural killer cell functional marker antibodies CD56 and NKP30.

2. The reagent composition according to claim 1, characterized in that, The fluorescently labeled antibody is a monoclonal antibody conjugated with a fluorescent dye.

3. The reagent composition according to claim 2, characterized in that, The fluorescent dye is selected from at least one of APC-AF700, APC-H7, PE, BB515, PE-Cy7, BV421, FITC, BB700, and Pacific Blue AF467.

4. The reagent composition according to claim 1, characterized in that, The antibodies are stored in lyophilized or liquid form and are suitable for multicolor flow cytometry analysis.

5. The reagent composition according to claim 1, characterized in that, The antibody is used to detect the proportion and functional status of peripheral blood lymphocyte subsets by flow cytometry, thereby assessing the risk of age-related hearing loss in the subject.

6. A kit for predicting the risk of developing age-related hearing loss, characterized in that, It comprises the reagent composition according to any one of claims 1-5, red blood cell lysis buffer, and phosphate buffer.

7. The reagent kit according to claim 6, characterized in that, It also includes negative control antibody or blank control setups for calibrating the flow cytometer and eliminating non-specific signal interference.

8. A method for predicting the risk of developing age-related hearing loss, characterized in that, Includes the following steps: (1) Obtain peripheral venous blood samples from the subjects and use the reagent composition described in any one of claims 1-5 or the kit described in any one of claims 6-7 to detect the following indicators by flow cytometry: CD4+ T cell ratio, CD8+ T cell ratio, CD4+ / CD8+ T cell ratio, CD8+HLA-DR+ T cell ratio, CD8+HLA-DR+CD38+ T cell ratio, terminally differentiated CD8+ T cell ratio, and NKP30+NK cell ratio; (2) Combine the results of the above detection indicators with the subject's age information and substitute them into a multivariate binary logistic regression model to calculate the probability P of the risk of age-related hearing loss. The model formula is: ln (P / (1−P)) = −8.913 − 0.078×CD8+HLA-DR+CD38+ T cell ratio − 0.021×NKP30+NK cell ratio + 0.165× Age; (3) Determine the risk of presbyopia based on the risk probability P: if P>0.620, it is considered high risk; if P≤0.620, it is considered low risk.

9. The method according to claim 8, characterized in that, The peripheral venous blood samples were anticoagulated with heparin sodium, stored at room temperature, and the subsequent pre-processing for testing was completed within 72 hours.

10. A computer-readable storage medium, characterized in that, The device stores computer program instructions that, when executed by a processor, implement the method for predicting the risk of age-related hearing loss as described in any of claims 8-9.

Images