A diagnostic kit for evaluating the state of rejection of a transplanted kidney

Abstract

This invention relates to a diagnostic kit for assessing transplant rejection status, belonging to the field of biomedical technology. The invention obtains data on the relative proportions and absolute counts of various immune cell populations in peripheral blood, and measures the ultrasound hemodynamic parameters of the transplanted kidney, including the interlobar artery resistance index and the renal aortic resistance index. It then uses the formula y1 = -1.228a + 0.008b – 0.009c to determine whether acute rejection exists. In the formula, the donor kidney type is defined as variable a, with living donor kidney = 0 and deceased donor kidney = 1; the absolute value of peripheral blood CD4 is defined as a continuous variable b; the absolute value of peripheral blood CD8 is defined as a continuous variable c; and when y1 > 0.5, acute rejection is determined. This invention makes the diagnosis of the immune status of transplanted kidneys non-invasive, optimizing the kidney transplant diagnosis and treatment process.

Description

Technical Field

[0001] This invention relates to a diagnostic kit for assessing transplanted kidney rejection, belonging to the field of biomedical technology. Background Technology

[0002] Currently, kidney transplantation is the best renal replacement therapy for end-stage renal disease. However, immune rejection caused by allogeneic transplantation remains the most important and unavoidable risk factor threatening the transplanted kidney. Various transplant complications, including rejection, can present with similar clinical manifestations; therefore, accurate assessment of the transplant rejection status is a prerequisite for making appropriate treatment decisions. The only current method for definitively determining transplant rejection status is renal biopsy. However: 1. This method has many limitations. Absolute contraindications for transplant biopsy include: significant bleeding tendency, severe hypertension, mental illness, or non-cooperation with the procedure. Relative contraindications include active pyelonephritis, renal tuberculosis, hydronephrosis or pyonephrosis, renal abscess, renal artery aneurysm, renal tumor, and obesity. Heart failure, severe anemia, hypovolemia, pregnancy, or advanced age are also relative contraindications. 2. Although transplant biopsy has been performed for many years and the technique is mature, due to the invasive nature of this technique, there is still a risk of various complications. Potential complications of transplant kidney biopsy may include hematuria, perirenal abscess, infection, arteriovenous fistula, etc.

[0003] The invasive procedure of kidney transplant biopsy is painful for patients, causes some damage to the transplanted kidney, and carries certain risks. Therefore, patient compliance with kidney transplant biopsy is relatively low, and it is often difficult to perform in clinical practice. Furthermore, kidney biopsy is expensive and has a long testing cycle. These factors limit the clinical application of kidney transplant biopsy and pose challenges to determining the immune status of the transplanted kidney. Therefore, there is an urgent need in this technical field for a non-invasive detection method that can replace kidney transplant biopsy. In current clinical practice, there are several non-invasive indicators related to kidney transplant rejection. Among them, the routine serum creatinine test after kidney transplantation is an important indicator for monitoring the status of the transplanted kidney. Serum creatinine is a reliable marker of kidney function impairment, but it often begins to rise some time after an acute rejection reaction occurs, thus lacking the ability to diagnose in a timely manner. While creatinine levels exceeding the upper limit or continuously rising suggest possible kidney transplant rejection, drug toxicity, viral infections, and other factors can also cause creatinine increases, so serum creatinine cannot be used alone as an indicator of rejection. It has been reported that when using serum creatinine, the AUC for diagnosing kidney transplant rejection is only 0.549. Donor-derived antibodies (DSA) are also used clinically to detect graft rejection. However, DSA is only related to antibody-mediated rejection, and it is difficult to determine whether other types of rejection have occurred. Ultrasound of the transplanted kidney is a commonly used diagnostic tool in clinical practice. It has been reported that the renal resistance index (RI) is often elevated in kidneys experiencing rejection, but this index cannot distinguish between graft rejection and acute tubular necrosis, and therefore is not entirely satisfactory. Summary of the Invention

[0004] The purpose of this invention is to solve the technical problem of how to obtain a non-invasive detection method and diagnostic kit for detecting transplanted kidney rejection status that can replace transplanted kidney biopsy.

[0005] To achieve the objectives of this invention, a method for assessing transplant rejection status is provided, comprising the following steps:

[0006] Step 1: For patients clinically suspected of kidney transplant rejection, draw 5ml of peripheral blood;

[0007] Step 2: Obtain the relative proportions and absolute counts of various immune cell populations in peripheral blood;

[0008] Step 3: Transplant kidney ultrasound. Clinical ultrasound was used to measure the ultrasound hemodynamic parameters of the transplant kidney on the same day as peripheral blood was drawn, including the renal interlobar artery resistance index and the renal aortic resistance index.

[0009] Step 4: Determine if there is acute rejection / if there is no acute rejection:

[0010] a) Define the type of donor kidney as variable a, where: living donor kidney = 0, deceased donor kidney = 1;

[0011] b) Define the absolute value of peripheral blood CD4 as a continuous variable b, with the unit being: cells;

[0012] c) Define the absolute value of peripheral blood CD8 as a continuous variable c, with the unit being: cells;

[0013] d) The determination formula is: y1 = -1.228a + 0.008b – 0.009c;

[0014] i. When y1 > 0.5, it is determined to be acute rejection;

[0015] ii. When y1 < 0.5, it is determined that there is no acute rejection;

[0016] Step 5: When identifying the pathological type of acute rejection:

[0017] a) Define the proportion of CD8 in peripheral blood as a continuous variable a, with the unit being: %;

[0018] b) Define the absolute value of peripheral blood CD8 as a continuous variable b, with the unit being: cells;

[0019] c) Define the interlobar artery resistance index of the transplanted kidney as a continuous variable c, with a unit of 1;

[0020] d) Define the aortic resistance index of the transplanted kidney as a continuous variable d, with a unit of 1;

[0021] e) The determination formula is: y1 = 0.28a - 0.018b + 24.457c – 23.714d;

[0022] i. When y1>0.5, it is determined to be ABMR (antibody-mediated rejection).

[0023] ii. When y1 < 0.5, it is determined to be TCMR (T cell-mediated rejection).

[0024] Preferably, in step 2, the relative proportions and absolute counts of various immune cell populations in peripheral blood are obtained by separating peripheral blood mononuclear cells using Ficoll separation solution, resuspending these cells in phosphate buffer solution, centrifuging again to remove the supernatant, washing away extracellular components, staining with a 6-color TBNK kit, incubating with Trucount absolute counting tubes, and finally reading the relative proportions and absolute counts of various immune cell populations using a flow cytometer.

[0025] This invention provides a diagnostic kit for assessing transplant rejection status, wherein the diagnostic kit employs one of the methods described above for assessing transplant rejection status.

[0026] This invention provides a diagnostic kit for assessing the pathological type of transplanted kidney rejection, wherein the diagnostic kit employs one of the methods described above for assessing transplanted kidney rejection.

[0027] The present invention provides a detection system, including the diagnostic reagent kit described above.

[0028] The present invention provides a storage device that stores a plurality of instructions for performing the detection steps in the above-described method for assessing transplanted kidney rejection status.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The main purpose of this invention is to overcome the shortcomings of current methods for diagnosing the immune status of transplanted kidneys, which are invasive and cumbersome, so as to make the diagnosis of the immune status of transplanted kidneys non-invasive and have the potential to be routinely monitored.

[0031] This invention provides a diagnostic tool for diagnosing acute rejection in clinical kidney transplantation, optimizes the kidney transplantation process, and improves the quality of life for kidney transplant patients. Attached Figure Description

[0032] Figure 1 ROC curves for the sensitivity and specificity of using baseline and peripheral blood immune cells in combination to diagnose acute rejection of transplanted kidneys.

[0033] Figure A shows the ROC curve; Figure B shows the model predictions for the non-rejection group and the acute rejection group.

[0034] Figure 2 ROC curves for the sensitivity and specificity of using peripheral blood immune cells and transplanted kidney ultrasound in diagnosing transplant rejection pathological classification.

[0035] Figure A shows the ROC curve; Figure B shows the model predictions for the TCMR and ABMR groups. Detailed Implementation

[0036] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings:

[0037] The purpose of this invention is to provide a method for assessing transplanted kidney rejection status, comprising the following steps:

[0038] Step 1: For patients clinically suspected of kidney transplant rejection, draw 5ml of peripheral blood;

[0039] Step 2: Obtain the relative proportions and absolute counts of various immune cell populations in peripheral blood;

[0040] Step 3: Transplant kidney ultrasound. Clinical ultrasound was used to measure the ultrasound hemodynamic parameters of the transplant kidney on the same day as peripheral blood was drawn, including the renal interlobar artery resistance index and the renal aortic resistance index.

[0041] Step 4: Determine if there is acute rejection / if there is no acute rejection:

[0042] a) Define the type of donor kidney as variable a, where: living donor kidney = 0, deceased donor kidney = 1;

[0043] b) Define the absolute value of peripheral blood CD4 as a continuous variable b, with the unit being: cells;

[0044] c) Define the absolute value of peripheral blood CD8 as a continuous variable c, with the unit being: cells;

[0045] d) The determination formula is: y1 = -1.228a + 0.008b – 0.009c;

[0046] i. When y1 > 0.5, it is determined to be acute rejection;

[0047] ii. When y1 < 0.5, it is determined that there is no acute rejection;

[0048] Step 5: When identifying the pathological type of acute rejection:

[0049] a) Define the proportion of CD8 in peripheral blood as a continuous variable a, with the unit being: %;

[0050] b) Define the absolute value of peripheral blood CD8 as a continuous variable b, with the unit being: cells;

[0051] c) Define the interlobar artery resistance index of the transplanted kidney as a continuous variable c, with a unit of 1;

[0052] d) Define the aortic resistance index of the transplanted kidney as a continuous variable d, with a unit of 1;

[0053] e) The determination formula is: y1 = 0.28a - 0.018b + 24.457c – 23.714d;

[0054] i. When y1 > 0.5, it is determined to be ABMR;

[0055] ii. When y1 < 0.5, it is determined to be TCMR.

[0056] In step 2 above, the relative proportions and absolute counts of various immune cell populations in peripheral blood were obtained by separating peripheral blood mononuclear cells using Ficoll separation solution, resuspending these cells in phosphate buffer solution, centrifuging again to remove the supernatant, washing away extracellular components, staining with a 6-color TBNK kit, incubating with Trucount absolute counting tubes, and finally reading the relative proportions and absolute counts of various immune cell populations using flow cytometry.

[0057] This invention provides a diagnostic kit for assessing transplant rejection status, wherein the diagnostic kit employs one of the methods described above for assessing transplant rejection status.

[0058] This invention provides a diagnostic kit for assessing the pathological type of transplanted kidney rejection, wherein the diagnostic kit employs one of the methods described above for assessing transplanted kidney rejection.

[0059] The present invention provides a detection system, including the diagnostic reagent kit described above.

[0060] The present invention provides a storage device that stores a plurality of instructions for performing the detection steps in the above-described method for assessing transplanted kidney rejection status.

[0061] Example

[0062] This invention provides a method for assessing the rejection status of a transplanted kidney, the specific steps of which are as follows:

[0063] Step 1: For patients clinically suspected of kidney transplant rejection, draw 5ml of peripheral blood;

[0064] Step 2: Peripheral blood mononuclear cells were separated using Ficoll separation medium. These cells were aspirated, resuspended in phosphate-buffered saline, centrifuged again to remove the supernatant, and the extracellular components were washed away. Subsequently, they were stained using a 6-color TBNK kit from BD (BioDx) and incubated in Trucount absolute counting tubes. Finally, the relative proportions and absolute counts of each immune cell population were obtained using flow cytometry.

[0065] Step 3: Transplant kidney ultrasound. Clinical ultrasound was used to measure the ultrasound hemodynamic parameters of the transplant kidney, including the interlobar artery resistance index, on the same day that peripheral blood was drawn.

[0066] Step 4: The specific calculation method for the probability value is as follows:

[0067] a) Definition

[0068] b)y=logit(P)=β0+β1X1+β2X2+……+β n X n

[0069] Step 5: Determine if there is acute rejection - If there is no acute rejection:

[0070] a) Define the type of donor kidney as variable a, where: living donor kidney = 0, deceased donor kidney = 1;

[0071] b) Define the absolute value of peripheral blood CD4 as a continuous variable b, with the unit being: cells;

[0072] c) Define the absolute value of peripheral blood CD8 as a continuous variable c, with the unit being: cells;

[0073] d) The determination formula is: y1 = logit(P1) = -1.228a + 0.008b – 0.009c

[0074] i. When y1 > 0.5, it is determined to be acute rejection;

[0075] ii. When y1 < 0.5, it is determined that there is no acute rejection;

[0076] Step 6: When identifying the pathological type of acute rejection:

[0077] a) Define the proportion of CD8 in peripheral blood as a continuous variable a, with the unit being: %;

[0078] b) Define the absolute value of peripheral blood CD8 as a continuous variable b, with the unit being: cells;

[0079] c) Define the interlobar artery resistance index of the transplanted kidney as a continuous variable c, with a unit of 1;

[0080] d) Define the aortic resistance index of the transplanted kidney as a continuous variable d, with a unit of 1;

[0081] e) The determination formula is: y1 = logit(P1) = 0.28a - 0.018b + 24.457c – 23.714d

[0082] i. When y1 > 0.5, it is determined to be ABMR;

[0083] ii. When y1 < 0.5, it is determined to be TCMR;

[0084] Given the complexity of transplant rejection, non-invasive diagnosis using a single indicator is extremely difficult. Peripheral blood immune cell detection and multimodal ultrasound examination of the transplanted kidney have wide clinical applications and can better reflect the immune and functional status of the transplanted kidney. Considering the scientific rigor, accessibility, and patient compliance of clinical testing, these two sets of indicators were selected as the screening sources for the diagnostic model.

[0085] The two main pathological types of acute rejection in transplanted kidneys include antibody-mediated rejection (ABMR) and T cell-mediated rejection (TCMR), both of which are closely related to lymphocyte immunity. TCMR is characterized by lymphocyte infiltration in the tissue, while ABMR is a specific immune response triggered by antibodies produced by B lymphocytes. Therefore, T cells play an important pathological role in acute rejection in kidney transplantation and have shown a close correlation with its occurrence in clinical studies. Furthermore, in some clinical studies, the total number and subtype of B cells have also shown a strong correlation with the outcome of acute rejection. Weimer et al. demonstrated that CD4+ T cell deficiency can predict post-transplant rejection. Rovira et al. completed a similar study in 2022, but focused on acute rejection of the transplanted pancreas in pancreas-kidney transplant patients. Their work confirmed that peripheral immune cells, including CD3+, CD4+, CD8+, and CD19+ cells, have predictive ability for acute rejection of transplanted pancreas. Specifically, CD4+ T cells showed a diagnostic AUC of 0.94 for acute rejection, while CD3+ T cells achieved 100% diagnostic sensitivity. However, their predictive ability was limited to the first 3 months post-transplantation. Similar results were observed in the study by Heidt et al., where B-cell subtypes based on functional molecular markers were used to identify the immune activation status of the transplanted kidney. However, current research on transplant rejection using immune cells largely focuses on the predictive ability of a specific single cell type for acute rejection. Including data from all relevant lymphocytes could provide a better description of the immune status of transplant patients, helping to determine whether the patient's immune system has been activated.

[0086] Multimodal ultrasound offers advantages such as being non-invasive, convenient, and accurate, thus its application in transplant kidney ultrasound examination is more widespread in clinical practice. Changes in blood flow parameters in the transplant kidney often indicate increased tissue inflammation, providing clues for immune rejection. Among the ultrasound data of the transplant kidney, the resistance index (RI) is of primary concern. RI is a hemodynamic parameter calculated based on peak systolic velocity (PSV) and end-diastolic velocity (EDV), indicating the presence of factors increasing resistance ahead of blood flow, such as stenosis or thrombosis. Some studies have reported that intrarenal RI has a predictive role in acute transplant kidney rejection and can differentiate between T-cell and non-T-cell rejection. However, a prospective study involving over 300 participants showed that RI did not show a correlation with the histopathology of acute rejection in procedural biopsy data of the transplant kidney. The role of RI alone in diagnosing transplant kidney rejection is often ambiguous and difficult to differentiate between acute tubular necrosis and transplant kidney rejection. Shear wave elastography (SWE) can quantitatively detect soft tissue elasticity in real time, thereby obtaining quantitative information on the mechanical properties related to soft tissue injury and healing. In diagnosing acute rejection of transplanted kidneys, SWE technology can achieve a sensitivity of 73.68% and a specificity of 80%. Ultrasound-based elastography and contrast-enhanced ultrasound have shown promising performance in recent years, providing a more comprehensive depiction of the physical properties and biomarkers of transplanted kidney tissue; however, their diagnostic specificity for acute rejection still needs improvement.

[0087] The aim of this study was to establish a novel non-invasive method for diagnosing transplant rejection. To test the reliability of this method, we first selected the gold standard. The currently accepted best method for diagnosing transplant rejection in clinical practice is transplant biopsy; therefore, we used transplant biopsy as the gold standard for this diagnostic method. This study collected data from 447 patients who underwent transplant biopsy at the Department of Urology, Zhongshan Hospital Affiliated to Fudan University between 2012 and 2020 and had standardized records, and analyzed and modeled their data.

[0088] Based on the biopsy pathology results of these patients, we further categorized them to obtain the pathological diagnoses of interest in this study. These included a control group of 133 patients with negative biopsy results (NP) and an experimental group of 117 patients with cell-mediated acute rejection (TCMR) and 88 patients with antibody-mediated acute rejection (ABMR). A total of 338 patients meeting the study criteria were ultimately included in this study. For the analysis of this type of clinical data, we selected Logistic regression analysis as the statistical method. However, Logistic regression only allows for statistical analysis of binary classification results. Therefore, we first grouped the ABMR and TCMR patients into the acute rejection (AR) group and analyzed the differences between the NP and AR groups; further, within the AR group, we analyzed the differences between ABMR and TCMR patients. This study used Logistic regression analysis, therefore the EPV (events pervariable) method was used to estimate the sample size. The transplanted kidney biopsies at our center were not procedural but rather conservative, based on clinician judgment; therefore, the proportion of acute rejection observed during biopsies was higher than the epidemiological average. For the NP-AR group, the probability of diagnosing acute rejection with each transplanted kidney biopsy was approximately 1 / 3; while for the ABMR-TCMR group, the probability of diagnosing acute rejection with either ABMR or TCMR was approximately 1 / 2. For caution, the EPV for both groups was defined as 10. In diagnosing NP-AR, this method included 3 variables, therefore requiring a sample size of 10 * 3 / (1 / 3) = 90 cases (<338 cases); while in diagnosing ABMR-TCMR, this method included 4 variables, therefore requiring a sample size of 10 * 4 / (1 / 2) = 80 cases (<195 cases). Evaluation indicators of this method, including sensitivity, specificity, and ROC curves, as well as related data and graphs, are described later.

[0089] We first compared the basic medical history data of kidney transplant patients in the NP and AR groups. This data included patient age, sex, post-operative puncture time, pre-operative dialysis time, donor age and sex, PRA level and HLA matching at transplantation, donor kidney type, secondary transplantation, creatinine, blood urea nitrogen, and proteinuria levels at puncture, as well as pre-transplant induction and post-transplant immunosuppression protocols. Among these, in diagnosing acute rejection (AR) and non-rejection transplanted kidney (NP), the patient's baseline data (including post-operative puncture time, donor kidney type, and creatinine at puncture) and peripheral blood immune cells (including absolute CD4+ T cell and CD8+ T cell counts) showed certain diagnostic power (Table 1). Furthermore, combining the data from both groups could establish a more potent diagnostic model. Figure 1In differentiating between TCMR and ABMR rejection in acute rejection, peripheral blood immune cells (including the proportion and absolute number of CD8+ T cells) and transplanted kidney ultrasound data (including the transplanted kidney arterial resistance index and the transplanted kidney aortic resistance index) showed good diagnostic efficacy (Table 2). Furthermore, combining these two sets of data can establish a more effective diagnostic model. Figure 2 ).

[0090] To validate the accuracy of this model, we included data from 11 patients who underwent kidney transplant biopsy at the Department of Urology, Zhongshan Hospital in 2023, and used pathological biopsy diagnosis as the gold standard to test the efficacy of the diagnostic model. The validation results are shown in Table 3. Among them, the accuracy rate of AR-NP diagnosis was 10 / 11 (90.9%), and the accuracy rate of TCMR-ABMR diagnosis was 6 / 8 (75.0%).

[0091] Table 1. Diagnosis of acute kidney transplant rejection using patient baseline and peripheral blood immune cell counts alone.

[0092]

[0093] Table 2. Diagnosis of transplant rejection pathological types using peripheral blood immune cells and transplanted kidney ultrasound alone

[0094]

[0095] Table 3. Validation Set

[0096]

[0097]

[0098] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A diagnostic kit for evaluating the state of rejection of a transplanted kidney, characterized by comprising: The diagnostic kit uses the following steps: Step 1: For patients clinically suspected of kidney transplant rejection, draw 5ml of peripheral blood; Step 2: Obtain the relative proportions and absolute counts of various immune cell populations in peripheral blood; Step 3: Transplant kidney ultrasound. Clinical ultrasound was used to measure the ultrasound hemodynamic parameters of the transplant kidney on the same day as peripheral blood was drawn, including the renal interlobar artery resistance index and the renal aortic resistance index. Step 4: Determine if there is acute rejection - If there is no acute rejection: a) Define the type of donor kidney as variable a, where: living donor kidney = 0, deceased donor kidney = 1; b) Define the absolute value of peripheral blood CD4 as a continuous variable b, with the unit being: cells; c) Define the absolute value of peripheral blood CD8 as a continuous variable c, with the unit being: cells; d) The determination formula is: y1 = -1.228a + 0.008b – 0.009c; i. When y1 > 0.5, it is determined to be acute rejection; ii. When y1 < 0.5, it is determined that there is no acute rejection; Step 5: When identifying the pathological type of acute rejection: a) Define the peripheral blood CD8 ratio as a continuous variable 'a', with units of % . b) Define the absolute value of peripheral blood CD8 as a continuous variable b, with the unit being: cells; c) Define the interlobar artery resistance index of the transplanted kidney as a continuous variable c, with a unit of 1; d) Define the aortic resistance index of the transplanted kidney as a continuous variable d, with a unit of 1; e) The determination formula is: y1 = 0.28a - 0.018b + 24.457c – 23.714d; i. When y1 > 0.5, it is determined to be ABMR; ii. When y1 < 0.5, it is determined to be TCMR; In step 2, the relative proportions and absolute counts of various immune cell populations in peripheral blood are obtained by separating peripheral blood mononuclear cells using Ficoll separation solution, resuspending these cells in phosphate buffer solution, centrifuging again to remove the supernatant, washing away extracellular components, staining with a 6-color TBNK kit, incubating with Trucount absolute counting tubes, and finally reading the relative proportions and absolute counts of various immune cell populations using flow cytometry.

2. A diagnostic kit for assessing the pathological type of transplanted kidney rejection, characterized in that, The diagnostic kit uses the following steps: Step 1: For patients clinically suspected of kidney transplant rejection, draw 5ml of peripheral blood; Step 2: Obtain the relative proportions and absolute counts of various immune cell populations in peripheral blood; Step 3: Transplant kidney ultrasound. Clinical ultrasound was used to measure the ultrasound hemodynamic parameters of the transplant kidney on the same day as peripheral blood was drawn, including the renal interlobar artery resistance index and the renal aortic resistance index. Step 4: Determine if there is acute rejection - If there is no acute rejection: a) Define the type of donor kidney as variable a, where: living donor kidney = 0, deceased donor kidney = 1; b) Define the absolute value of peripheral blood CD4 as a continuous variable b, with the unit being: cells; c) Define the absolute value of peripheral blood CD8 as a continuous variable c, with the unit being: cells; d) The determination formula is: y1 = -1.228a + 0.008b – 0.009c; i. When y1 > 0.5, it is determined to be acute rejection; ii. When y1 < 0.5, it is determined that there is no acute rejection; Step 5: When identifying the pathological type of acute rejection: a) Define the peripheral blood CD8 ratio as a continuous variable 'a', with units of % . b) Define the absolute value of peripheral blood CD8 as a continuous variable b, with the unit being: cells; c) Define the interlobar artery resistance index of the transplanted kidney as a continuous variable c, with a unit of 1; d) Define the aortic resistance index of the transplanted kidney as a continuous variable d, with a unit of 1; e) The determination formula is: y1 = 0.28a - 0.018b + 24.457c – 23.714d; i. When y1 > 0.5, it is determined to be ABMR; ii. When y1 < 0.5, it is determined to be TCMR; In step 2, the relative proportions and absolute counts of various immune cell populations in peripheral blood are obtained by separating peripheral blood mononuclear cells using Ficoll separation solution, resuspending these cells in phosphate buffer solution, centrifuging again to remove the supernatant, washing away extracellular components, staining with a 6-color TBNK kit, incubating with Trucount absolute counting tubes, and finally reading the relative proportions and absolute counts of various immune cell populations using flow cytometry.

3. A detection system, characterized in that, Includes the diagnostic kit according to claim 1 or claim 2.

4. A storage device, characterized in that, The storage device stores multiple instructions, which are used to perform the following detection steps: Step 1: For patients clinically suspected of kidney transplant rejection, draw 5ml of peripheral blood; Step 2: Obtain the relative proportions and absolute counts of various immune cell populations in peripheral blood; Step 3: Transplant kidney ultrasound. Clinical ultrasound was used to measure the ultrasound hemodynamic parameters of the transplant kidney on the same day as peripheral blood was drawn, including the renal interlobar artery resistance index and the renal aortic resistance index. Step 4: Determine if there is acute rejection - If there is no acute rejection: a) Define the type of donor kidney as variable a, where: living donor kidney = 0, deceased donor kidney = 1; b) Define the absolute value of peripheral blood CD4 as a continuous variable b, with the unit being: cells; c) Define the absolute value of peripheral blood CD8 as a continuous variable c, with the unit being: cells; d) The determination formula is: y1 = -1.228a + 0.008b – 0.009c; i. When y1 > 0.5, it is determined to be acute rejection; ii. When y1 < 0.5, it is determined that there is no acute rejection; Step 5: When identifying the pathological type of acute rejection: a) Define the peripheral blood CD8 ratio as a continuous variable 'a', with units of % . b) Define the absolute value of peripheral blood CD8 as a continuous variable b, with the unit being: cells; c) Define the interlobar artery resistance index of the transplanted kidney as a continuous variable c, with a unit of 1; d) Define the aortic resistance index of the transplanted kidney as a continuous variable d, with a unit of 1; e) The determination formula is: y1 = 0.28a - 0.018b + 24.457c – 23.714d; i. When y1 > 0.5, it is determined to be ABMR; ii. When y1 < 0.5, it is determined to be TCMR; In step 2, the relative proportions and absolute counts of various immune cell populations in peripheral blood are obtained by separating peripheral blood mononuclear cells using Ficoll separation solution, resuspending these cells in phosphate buffer solution, centrifuging again to remove the supernatant, washing away extracellular components, staining with a 6-color TBNK kit, incubating with Trucount absolute counting tubes, and finally reading the relative proportions and absolute counts of various immune cell populations using flow cytometry.

Images