Heparin binding protein-based transplant rejection risk early warning method

Abstract

The invention relates to the technical field of biochemistry, and particularly discloses a heparin binding protein-based transplant rejection risk early warning method, which comprises the following steps of: obtaining a biological sample of a transplant recipient; detecting the concentration of HBP in the biological sample; comparing the HBP concentration with a preset reference value; if the HBP concentration is higher than the reference value, prompting that a transplant rejection risk exists; by dynamically monitoring the concentration of the heparin-binding protein, the kit can prompt the occurrence of infection after organ transplantation earlier compared with a traditional inflammatory marker, has high sensitivity and specificity, and is beneficial to realizing evaluation and timely intervention of the immune state of a patient before typical clinical symptoms or microbiological evidences do not appear in the early stage of infection.

Description

Technical Field

[0001] This invention belongs to the field of biochemical technology, specifically relating to a method for early warning of transplant rejection risk based on heparin-binding proteins. Background Technology

[0002] Currently, the clinical monitoring and diagnosis of post-organ transplant infection mainly relies on non-specific inflammatory biomarkers, such as C-reactive protein (CRP) and procalcitonin (PCT). CRP is an acute-phase reactive protein synthesized by the liver, and its concentration increases after inflammation or tissue damage. PCT is widely secreted by various cells during bacterial infection. Both are widely used to help determine whether there are post-operative infectious complications and to guide the use of antibacterial drugs.

[0003] However, elevated CRP and PCT levels usually lag behind the onset of infection and neutrophil activation, making it difficult to achieve true early warning. Secondly, in the context of the widespread use of immunosuppressants in transplant patients, their immune response is suppressed, which may lead to elevated baseline levels of PCT and CRP or a sluggish response, further reducing sensitivity and specificity, resulting in diagnostic delays or misdiagnosis. Summary of the Invention

[0004] The purpose of this invention is to provide a method for early warning of transplant rejection risk based on heparin-binding proteins, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for predicting transplant rejection risk based on heparin-binding proteins includes the following steps:

[0007] S1. Obtain biological samples from the transplant recipient;

[0008] S2. Detect the concentration of HBP in the biological sample;

[0009] S3. Compare the HBP concentration with a preset reference value;

[0010] S4. If the HBP concentration is higher than the reference value, it indicates a risk of transplant rejection.

[0011] Preferably, the biological sample is selected from one of whole blood, plasma, serum, urine, bronchoalveolar lavage fluid (BALF), or transplant tissue biopsy fluid.

[0012] Preferably, the HBP concentration is detected using an immunoassay method, including enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), or immunoturbidimetric assay.

[0013] Preferably, the reference value is the median or mean HBP concentration in healthy individuals or patients in the stable post-transplant period, or a critical value determined based on the ROC curve.

[0014] Preferably, the HBP concentration is detected on day 1, day 3, day 7, day 14 post-transplantation and / or when clinical symptoms appear.

[0015] Preferably, the method further includes detecting at least one other inflammatory marker selected from procalcitonin (PCT), C-reactive protein (CRP), myeloperoxidase (MPO), interleukin-6 (IL-6), or neutrophil gelatinase-associated lipocalin (NGAL).

[0016] Preferably, the detection results of HBP and other inflammatory markers are integrated through a multivariate model to conduct a comprehensive assessment of rejection risk;

[0017] Specifically, the following steps are included:

[0018] a. The HBP concentration, along with the detection results of at least one other inflammatory marker and the patient's clinical parameters, constitute an input vector. The clinical parameters include, but are not limited to, transplant type, postoperative time, immunosuppressant blood concentration, renal function indicators, body temperature, and white blood cell count.

[0019] b. The weights of each biomarker and clinical parameter were dynamically calculated using an attention mechanism. The initial weight of HBP was set to 0.4–0.6, and the weights of the other biomarkers and parameters were dynamically adjusted based on their historical correlation with rejection events.

[0020] c. Calculate the comprehensive rejection risk score using the following formula. :

[0021] ;

[0022] in, and HBP and the Individualized baseline concentrations of each biomarker; , These are the feature weights obtained through training; This is an integrated clinical parameter term, output by the logistic regression sub-model;

[0023] d. Based on the RR value, the exclusion risk is divided into low risk (R<0.3), medium risk (0.3≤R<0.7) and high risk (R≥0.7), and a dynamic risk warning report is generated by combining the time series change trend.

[0024] Preferably, the transplant rejection risk includes acute rejection, antibody-mediated rejection (ABMR), or chronic graft vascular disease (CAV).

[0025] Preferably, the method further includes dynamically adjusting the immunosuppressive regimen or anti-infective treatment strategy based on the HBP concentration.

[0026] A kit for performing any of the above methods comprises an antibody, a standard, and a buffer for detecting HBP.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] (1) By dynamically monitoring the concentration of heparin-binding protein, the occurrence of post-organ transplant infection can be indicated earlier than traditional inflammatory markers. It has high sensitivity and specificity, which helps to assess the patient's immune status and intervene in a timely manner before typical clinical symptoms or microbiological evidence appear in the early stage of infection.

[0029] (2) It is applicable to postoperative infection monitoring of patients with different types of organ transplants, including high-risk groups such as relatives who have undergone kidney transplantation, combined pancreas and kidney transplantation, and those who have positive DSA. It helps to optimize postoperative anti-infection treatment strategies, reduce antibiotic abuse, and improve the prognosis and quality of life of transplant patients. Attached Figure Description

[0030] Figure 1 This is a flowchart of the present invention;

[0031] Figure 2 This is a multi-group trend graph of HBP levels in blood, urine, and drainage fluid during related kidney transplantation according to the present invention;

[0032] Figure 3 This is a multivariate time series monitoring graph of biomarkers in infected patients according to the present invention;

[0033] Figure 4 This is a multivariate time series monitoring graph of biomarkers in DSA-positive patients according to the present invention;

[0034] Figure 5 This is a multivariate time series monitoring graph of pathogen infection biomarkers in pancreas-kidney combined transplant patients according to the present invention. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Example 1:

[0037] Please see Figure 1 As shown, the method for early warning of transplant rejection risk based on heparin-binding proteins includes the following steps:

[0038] S1. Obtain biological samples from the transplant recipient;

[0039] Specifically, biological samples are collected from transplant recipients at designated post-transplant time points (e.g., day 1, day 3, day 7, and day 14 post-transplant) or when the patient exhibits clinical symptoms suggestive of rejection or infection (e.g., fever, transplant site pain, abnormal renal function indicators, etc.). Sample types can be selected based on clinical circumstances, including but not limited to:

[0040] Whole blood / plasma / serum: Collect 3–5 mL of peripheral venous blood using EDTA anticoagulant tubes or procoagulant tubes. Centrifuge at 2000–3000 rpm for 10–15 minutes at 4°C within 30 minutes after collection. Separate the supernatant plasma or serum, aliquot and store at -80°C to avoid repeated freeze-thaw cycles.

[0041] Urine: Collect 10–20 mL of midstream morning urine, centrifuge at 3000 rpm for 10 minutes at 4°C, and aliquot and freeze the supernatant.

[0042] Bronchoalveolar lavage fluid (BALF): During fiberoptic bronchoscopy, sterile saline is used for lavage. After the lavage fluid is recovered, it is immediately centrifuged at 4°C, and the supernatant is aliquoted and stored.

[0043] Transplanted tissue biopsy fluid or drainage fluid: Obtain puncture fluid or postoperative drainage fluid under ultrasound guidance, and process it as above after collection;

[0044] All sample collection procedures were conducted under aseptic conditions, and samples were processed as soon as possible after collection to minimize the impact of protein degradation on test results.

[0045] S2. Detect the concentration of HBP in the biological sample;

[0046] S3. Compare the HBP concentration with a preset reference value;

[0047] S4. If the HBP concentration is higher than the reference value, it indicates a risk of transplant rejection.

[0048] In one embodiment of the present invention, the biological sample is selected from one of whole blood, plasma, serum, urine, bronchoalveolar lavage fluid (BALF) or transplant tissue biopsy fluid.

[0049] In one embodiment of the present invention, the HBP concentration is detected by an immunoassay method, including enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), or immunoturbidimetric assay.

[0050] In one embodiment of the present invention, the reference value is the median or mean HBP concentration in healthy individuals or patients in the stable post-transplant period, or a critical value determined based on the ROC curve.

[0051] In one embodiment of the present invention, the HBP concentration is detected on day 1, day 3, day 7, day 14 post-transplantation and / or when clinical symptoms appear.

[0052] In one embodiment of the invention, the invention further includes the detection of at least one other inflammatory marker selected from procalcitonin (PCT), C-reactive protein (CRP), myeloperoxidase (MPO), interleukin-6 (IL-6), or neutrophil gelatinase-associated lipotransferase (NGAL).

[0053] In one embodiment of the present invention, a multivariate model is used to integrate the detection results of HBP with other inflammatory markers to conduct a comprehensive assessment of rejection risk;

[0054] The multivariate model is a two-stage dynamic risk assessment model based on machine learning, including the following steps:

[0055] a. The HBP concentration, along with the detection results of at least one other inflammatory marker and the patient's clinical parameters, constitute an input vector. The clinical parameters include, but are not limited to, transplant type, postoperative time, immunosuppressant blood concentration, renal function indicators, body temperature, and white blood cell count.

[0056] Specifically,

[0057] The test data of each patient at each time point constitute an input vector X, including:

[0058] HBP concentration values ​​(logarithmically transformed to conform to a normal distribution);

[0059] Other inflammatory marker concentrations, including PCT, CRP, MPO, IL-6, and NGAL, were also standardized.

[0060] Clinical parameters: transplant type (coded as a categorical variable), postoperative time (days), immunosuppressant blood concentration (e.g., tacrolimus, cyclosporine A trough concentration), renal function indicators (serum creatinine, estimated glomerular filtration rate), body temperature (°C), white blood cell count (×10⁻⁶). 9 ( / L), whether or not diabetes is present, age, gender, etc.;

[0061] Temporal characteristics include the ratio of the current value to the baseline value, the moving average of the three most recent detection values, and the trend of change (slope of linear fitting).

[0062] Z-score standardization is applied to continuous variables: ,in and The mean and standard deviation are derived from the training set, respectively.

[0063] Categorical variables are encoded using one-hot encoding.

[0064] b. The weights of each biomarker and clinical parameter were dynamically calculated using an attention mechanism. The initial weight of HBP was set to 0.4–0.6, and the weights of the other biomarkers and parameters were dynamically adjusted based on their historical correlation with rejection events.

[0065] Specifically, a multi-head self-attention mechanism is used to weight the features of the input vector X. First, X is transformed through three different linear transformations to obtain the query, key, and value matrices:

[0066]

[0067]

[0068]

[0069] Calculate attention weights:

[0070] ;

[0071] in The dimension of the key vector, used for scaling;

[0072] Parallel computation is performed using a multi-head mechanism, and the results are concatenated and fused through a linear layer to obtain a weighted feature representation.

[0073] c. Calculate the comprehensive rejection risk score using the following formula. :

[0074] ;

[0075] in, and HBP and the Individualized baseline concentrations of each biomarker; , These are the feature weights obtained through training; This is an integrated clinical parameter term, output by the logistic regression sub-model;

[0076] Collect complete time-series data from at least 200 transplant patients, including HBP, other biomarkers, clinical parameters, and whether rejection events eventually occurred.

[0077] Using binary cross-entropy loss combined with a time-consistency regularization term:

[0078] ;

[0079] in The risk probability predicted by the model. For real labels, The regularization coefficient is used.

[0080] The Adam optimizer was used, with an initial learning rate of 0.001, which was decayed to 0.9 every 50 epochs. The data was divided into training and validation sets in a 7:3 ratio. 5-fold cross-validation was used to adjust the hyperparameters, and an early stopping strategy (patience=20) was used to prevent overfitting.

[0081] d. Based on the RR value, the exclusion risk is divided into low risk (R<0.3), medium risk (0.3≤R<0.7) and high risk (R≥0.7), and a dynamic risk warning report is generated by combining the time series change trend.

[0082] In one embodiment of the invention, the transplant rejection risk includes acute rejection, antibody-mediated rejection (ABMR), or chronic graft vascular disease (CAV).

[0083] In one embodiment of the present invention, the method further includes dynamically adjusting the immunosuppressive regimen or anti-infective treatment strategy based on the HBP concentration.

[0084] A kit for performing any of the above methods comprises an antibody, a standard, and a buffer for detecting HBP.

[0085] Example 2:

[0086] refer to Figure 2-3 As shown, HBP is used in the early diagnosis of infection after living donor kidney transplantation:

[0087] Forty patients who underwent living-donor kidney transplantation from their relatives were included, including 22 males and 18 females, with a mean age of 45±12 years. Peripheral venous blood (3 mL, anticoagulated with EDTA), midstream urine (10 mL), and peritoneal drainage (if any) (2 mL) were collected from the patients before surgery (baseline) and on postoperative days 1, 3, 5, and 7. If patients developed clinical symptoms of infection postoperatively, such as body temperature >38.3℃, abnormal white blood cell count, or turbid drainage, the sampling frequency was increased to once daily until the infection was controlled.

[0088] For testing, enzyme-linked immunosorbent assay (ELISA) was used to determine the concentration of HBP in plasma, urine and drainage fluid supernatant. At the same time, serum procalcitonin (PCT) and C-reactive protein (CRP) levels were measured, and the microbial culture results of various samples were used as the gold standard for infection diagnosis.

[0089] The results showed that HBP concentration was significantly elevated in patients with infection before their blood and urine cultures were positive. The increase in HBP occurred earlier than the increase in CRP, indicating that HBP has a better early warning ability in the early stage of infection. In the drainage fluid samples, the change in HBP concentration can also reflect the local infection status.

[0090] Example 3:

[0091] refer to Figure 4 As shown, HBP is used in the diagnosis of infection after pancreas-kidney transplantation (SPK):

[0092] Twenty patients who underwent pancreas-kidney transplantation (SPK) were selected, and plasma, urine, and peritoneal drainage were collected on postoperative days 1, 3, 5, and 7.

[0093] The detection method is the same as in Example 2, detecting the HBP concentration in each body fluid;

[0094] In patients undergoing combined pancreas and kidney transplantation, the HBP concentration in the infected group was significantly higher than that in the uninfected group. The increase in HBP in blood and urine preceded the appearance of clinical infection symptoms and was consistent with the changes in HBP levels in drainage fluid. HBP showed high sensitivity and specificity in identifying post-transplant infection.

[0095] Example 4:

[0096] refer to Figure 5 As shown, HBP is used for infection surveillance in DSA-positive transplant patients:

[0097] For kidney transplant or pancreas-kidney combined transplant patients who have positive donor-specific antibodies (DSA) before or after surgery, blood samples are collected at routine postoperative time points and when signs of rejection or infection appear.

[0098] The concentration of HBP in plasma was detected by ELISA, and the results were analyzed in combination with indicators such as DSA titer, CRP, and PCT.

[0099] The results showed that if DSA-positive patients had concurrent infections, their HBP levels were significantly elevated, and earlier than traditional markers such as CRP. Changes in HBP levels can help differentiate between rejection reactions and inflammatory states caused by infection.

[0100] Example 5:

[0101] Please see Figure 1 As shown, plasma HBP concentration is used to predict the risk of acute rejection after kidney transplantation:

[0102] Sixty patients who underwent allogeneic kidney transplantation were selected as the study subjects. Peripheral venous blood was collected from the patients on the 1st, 3rd, and 7th day after surgery, and when rejection was suspected (such as fever, swelling and pain in the transplanted kidney area, and elevated serum creatinine levels).

[0103] Blood samples were collected using EDTA anticoagulant tubes and centrifuged at 3000 rpm for 15 minutes at 4°C within 2 hours after collection. The supernatant plasma was separated, aliquoted, and stored at -80°C for later testing.

[0104] The detection was performed using a commercially available human heparin-binding protein (HBP) enzyme-linked immunosorbent assay (ELISA) kit;

[0105] Frozen plasma samples were thawed at room temperature. 100 μL of the sample was added to the wells of an antibody-coated ELISA plate and incubated at 37°C for 2 hours. After washing the plate, biotin-labeled detection antibody was added and incubated for 1 hour. After washing the plate again, horseradish peroxidase-labeled streptavidin was added and incubated for 30 minutes. Finally, the substrate TMB was added for color development, and the reaction was terminated with stop solution.

[0106] The absorbance (OD value) of each well was measured at 450 nm using a microplate reader. The HBP concentration of each sample was calculated based on the standard concentration curve, in ng / mL. The detection results are shown in the table below:

[0107] Group Number of examples (n) Preoperative (baseline) Day 1 after surgery Day 3 after surgery 7 days after surgery At the time of clinical diagnosis (peak) Acute rejection group (AR) 10 8.2 ± 2.1 15.3 ± 4.5 28.7 ± 8.9** 45.6 ± 12.3 58.2 ± 15.7 Non-AR group 50 7.9 ± 2.5 12.8 ± 3.7 10.1 ± 3.2 8.7 ± 3.5 -

[0108] The results showed that in patients (n=10) who developed acute rejection confirmed by puncture biopsy after surgery, their plasma HBP concentration was significantly elevated 1-3 days before the onset of clinical symptoms, with a peak concentration of 45.6 ± 12.3 ng / mL, which was much higher than the reference value.

[0109] In patients who did not experience rejection (n=50), although the HBP concentration fluctuated at different time points after surgery, the average level remained at 8.7 ± 3.5 ng / mL.

[0110] Plasma HBP levels were measured in patients whose function had been stable for more than 6 months post-transplantation, and the upper limit of the reference range for HBP in our center was determined to be 15 ng / mL (mean + 2 standard deviation).

[0111] As shown above, dynamic monitoring of plasma HBP concentration in kidney transplant recipients can effectively predict the occurrence of acute rejection before typical clinical symptoms and changes in biochemical indicators appear. When HBP concentration remains above 15 ng / mL or shows a sharp increase, the risk of rejection should be highly suspected, and early transplant kidney biopsy is recommended to confirm the diagnosis and adjust the immunosuppressive regimen.

[0112] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for early warning of transplant rejection risk based on heparin-binding proteins, characterized in that, Includes the following steps: S1. Obtain biological samples from the transplant recipient; S2. Detect the concentration of HBP in the biological sample; S3. Compare the HBP concentration with a preset reference value; S4. If the HBP concentration is higher than the reference value, it indicates a risk of transplant rejection.

2. The method for early warning of transplant rejection risk based on heparin-binding protein according to claim 1, characterized in that: The biological sample is selected from one of the following: whole blood, plasma, serum, urine, bronchoalveolar lavage fluid (BALF), or transplant tissue biopsy fluid.

3. The method for early warning of transplant rejection risk based on heparin-binding protein according to claim 1, characterized in that: The HBP concentration was detected using immunoassay methods, including enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA), or immunoturbidimetric assay.

4. The method for early warning of transplant rejection risk based on heparin-binding protein according to claim 1, characterized in that: The reference values ​​are the median or mean HBP concentrations in healthy individuals or patients in the stable post-transplant period, or the critical values ​​determined based on the ROC curve.

5. The method for early warning of transplant rejection risk based on heparin-binding protein according to claim 1, characterized in that: The HBP concentration was measured on days 1, 3, 7, and 14 post-transplant and / or when clinical symptoms appeared.

6. The method for early warning of transplant rejection risk based on heparin-binding protein according to claim 1, characterized in that: It also includes the detection of at least one other inflammatory marker selected from procalcitonin (PCT), C-reactive protein (CRP), myeloperoxidase (MPO), interleukin-6 (IL-6), or neutrophil gelatinase-associated lipocalin (NGAL).

7. The method for early warning of transplant rejection risk based on heparin-binding protein according to claim 6, characterized in that: By integrating the detection results of HBP with other inflammatory markers through a multivariate model, a comprehensive assessment of rejection risk can be conducted. Specifically, the following steps are included: a. The HBP concentration, along with the detection results of at least one other inflammatory marker and the patient's clinical parameters, constitute an input vector. The clinical parameters include, but are not limited to, transplant type, postoperative time, immunosuppressant blood concentration, renal function indicators, body temperature, and white blood cell count. b. The weights of each biomarker and clinical parameter were dynamically calculated using an attention mechanism. The initial weight of HBP was set to 0.4–0.6, and the weights of the other biomarkers and parameters were dynamically adjusted based on their historical correlation with rejection events. c. Calculate the comprehensive rejection risk score using the following formula. : ; in, and HBP and the Individualized baseline concentrations of each biomarker; , These are the feature weights obtained through training; This is an integrated clinical parameter term, output by the logistic regression sub-model; d. Based on the RR value, the exclusion risk is divided into low risk (R<0.3), medium risk (0.3≤R<0.7) and high risk (R≥0.7), and a dynamic risk warning report is generated by combining the time series change trend.

8. The method for early warning of transplant rejection risk based on heparin-binding protein according to claim 1, characterized in that: The transplant rejection risks include acute rejection, antibody-mediated rejection (ABMR), or chronic graft vascular disease (CAV).

9. The method for early warning of transplant rejection risk based on heparin-binding protein according to claim 1, characterized in that: The method also includes dynamically adjusting the immunosuppressive regimen or anti-infective treatment strategy based on HBP concentration.

10. A kit for implementing the method of any one of claims 1 to 9, characterized in that, It contains antibodies, standards, and buffer solutions for detecting HBP.

Images