Diagnostic marker for graft versus host disease and hepatic minor vein occlusion, kit and application

Abstract

The invention is applicable to the technical field of biology, and provides a diagnostic marker for graft versus host disease and hepatic minor vein occlusion, a kit and application, and the diagnostic marker is the serological level of six factors of PAI-1, ST2, ANG-2, HA, VCAM-1 and L-Ficolin combination. The diagnostic kit for the graft-versus-host disease and the hepatic minor vein occlusion, provided by the invention, is used for detecting a plasma marker combination, and the detection result is used for assisting in diagnosing and predicting the child graft-versus-host disease and the hepatic minor vein occlusion and predicting the occurrence of the child graft-versus-host disease and the hepatic minor vein occlusion. The prediction result has high diagnosis sensitivity and specificity, and is expected to become a laboratory diagnosis index of the child graft versus host disease and the hepatic minor vein occlusion disease.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and in particular relates to diagnostic markers, kits and applications of graft-versus-host disease and hepatic veno-occlusive disease. Background Art

[0002] Graft-versus-host disease (GVHD) is one of the most common life-threatening complications of allogeneic hematopoietic cell transplantation. It is mainly caused by the T cells in the donor's graft being activated by immune response in the recipient's body after transplantation to acquire cytolytic ability and then attack cells carrying foreign antigens. Its characteristics are similar to the symptoms of autoimmune and other immune diseases, such as scleroderma, S-syndrome, immune cytopenia, etc. At present, the diagnosis of graft-versus-host disease is mainly based on pathological manifestations such as diarrhea and rash after transplantation and related laboratory tests.

[0003] Hepatic veno-occlusive disease (HVOD), also known as hepatic sinusoidal obstruction syndrome (HSOS), is a serious complication after hematopoietic stem cell transplantation. It is often associated with high-intensity chemotherapy and often occurs within 30 days after transplantation. Its main clinical manifestations include hepatomegaly, ascites, jaundice, weight gain, etc., and are often complicated by multiple organ failure syndrome or even death. The diagnosis of HVOD / HSOS mainly relies on clinical manifestations (including weight gain, fluid retention and ascites, tender hepatomegaly and jaundice). Imaging examinations such as liver ultrasound, CT and MRI can assist in diagnosis. If necessary, transjugular measurement of hepatic venous pressure gradient and liver puncture biopsy are required. However, the existing diagnosis is not unified. Transjugular measurement of hepatic venous pressure gradient and liver biopsy are more accurate diagnostic methods, but they are invasive and have a greater risk of bleeding and infection for patients whose hematopoietic function has not fully recovered in the early stage after HSCT, and are difficult to complete in routine practice.

[0004] Foreign studies have found that a variety of biomarkers can be used as potential predictive and early detection indicators for GVHD and HVOD / HSOS. PAI-1, ST2, ANG-2, HA, VCAM-1 and L-ficolin are broad-spectrum biomarkers that can participate in multiple mechanisms. Plasminogen activator inhibitor-1 (PAI-1): a single-chain globular glycoprotein, together with tissue-type plasminogen activator, is a key substance for regulating fibrinolytic activity, synthesized by vascular endothelial cells and released into the blood; tumor suppressor protein 2 (ST2): a member of the IL-1 family, it is essential for the maturation and stability of vascular development, and has become a biomarker for aGVHD and NRM; angiopoietin-2 (ANG2): a member of the angiopoietin family, it plays a role in promoting angiogenesis in the occurrence and development of tumors; hyaluronic acid (HA): an amino polysaccharide produced in mesenchymal cells, it is a good indicator of liver endothelial cell function and active fibrosis; vascular cell adhesion molecule-1 (VCAM1): usually has 7 C2-type immunoglobulin domains, and functions as a cell adhesion molecule; L-ficolin: an important initiator in the complement agglutination pathway, it plays an important role in resisting the occurrence of diseases. There are no reports on the use of a six-cytokine combination kit consisting of PAI-1, ST2, ANG-2, HA, VCAM-1 and L-Ficolin in GVHD and VOD.

[0005] To date, both GVHD and veno-occlusive disease lack characteristic pathological diagnostic markers, and GVHD lacks standard and widely used diagnostic guidelines. Summary of the invention

[0006] The purpose of the embodiments of the present application is to provide a diagnostic marker for graft-versus-host disease and hepatic veno-occlusive disease, aiming to make up for the deficiencies of the prior art.

[0007] The embodiment of the present application is achieved by providing a diagnostic marker for graft-versus-host disease and hepatic veno-occlusive disease, wherein the diagnostic marker is the serological level of six factors in combination of PAI-1, ST2, ANG-2, HA, VCAM-1 and L-Ficolin.

[0008] The CDS gene sequences of the above PAI-1 (gene name SERPINE1), ST2 (gene name IL1RL1), ANG-2 (gene name ANGPT2), VCAM-1 (gene name VCAM1) and L-Ficolin (gene name FCN2) are shown in SEQ ID NO. 1~5. HA is a metabolite, not a gene-encoded protein: Hyaluronic acid (HA) is a high molecular weight unbranched polysaccharide synthesized by a variety of organisms from bacteria to mammals, and is a component of the extracellular matrix. It is composed of alternating glucuronic acid and N-acetylglucosamine residues, which are linked by β-1-3 and β-1-4 glycosidic bonds. HA is synthesized by membrane-bound synthases on the inner surface of the plasma membrane, and the chains are extruded into the extracellular space through pore-like structures.

[0009] The present application also provides an application of the above-mentioned diagnostic markers for graft-versus-host disease and veno-occlusive disease in the preparation of a kit for diagnosing graft-versus-host disease and veno-occlusive disease.

[0010] The present application also provides a diagnostic kit for graft-versus-host disease and hepatic veno-occlusive disease, which is composed of several sub-kits for detecting PAI-1, ST2, ANG-2, HA, VCAM-1, and L-Ficolin, respectively. The diagnostic kit can be used to assist in the diagnosis and prediction of graft-versus-host disease and hepatic veno-occlusive disease in children.

[0011] Preferably, the diagnostic kit is composed of the following sub-kits: Human SERPINE1 ELISA Kit for detecting protein PAI-1, Human ST2 ELISA Kit for detecting protein ST2, Human Angiopoietin 2 ELISA Kit for detecting protein ANG2, Human Hyaluronic acid, HA ELISA Kit for detecting protein HA, Human VCAM-1 / CD106 ELISA Kit for detecting protein VCAM-1, and Human Ficolin-2 ELISA kit for detecting protein L-FICOLIN.

[0012] In one embodiment, the dosage of the antibody used in the diagnostic kit is the dosage recommended in the instructions of the purchased antibody.

[0013] In one embodiment, the test sample of the diagnostic kit is peripheral blood or bone marrow fluid.

[0014] The embodiments of the present application also provide a use of the above-mentioned diagnostic kit for graft-versus-host disease and veno-occlusive disease in the preparation of a reagent for auxiliary diagnosis of graft-versus-host disease and veno-occlusive disease in children.

[0015] Compared with the shortcomings and deficiencies of the prior art, the diagnostic kit for graft-versus-host disease and veno-occlusive disease provided in the embodiments of the present application is used to detect a combination of plasma markers. The test results are used to assist in the diagnosis and prediction of graft-versus-host disease and veno-occlusive disease in children, and to predict the occurrence of graft-versus-host disease and veno-occlusive disease in children. The prediction results have high diagnostic sensitivity and specificity, and are expected to become laboratory diagnostic indicators for graft-versus-host disease and veno-occlusive disease in children. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing the test results of ANG2, ST2, VCAM1, PAI-1, HA, and L-FICOLIN in the peripheral blood of patients with GVHD after transplantation provided in the examples of the present application; Figure 2 This is a graph showing the test results of ANG2, ST2, VCAM1, PAI-1, HA, and L-FICOLIN in the peripheral blood of patients with VOD after transplantation provided in the examples of the present application. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0018] 1. Selection of clinical cases In this example, 68 patients who underwent hematopoietic stem cell transplantation, 29 patients with graft-versus-host disease, 6 patients with veno-occlusive disease, 62 patients with infection, and 4 patients who did not develop graft-versus-host disease, veno-occlusive disease, or infection were randomly selected. The patients were under 18 years old, with no gender restriction.

[0019] Patient inclusion criteria (patients who meet all of the following conditions will be included): (1) Under 18 years old (2) Hematopoietic stem cell transplant patients (first or second time) (3) ECOG score 0-2 (4) No history of neurological disease and / or mental illness (5) The subject patient (or his / her legal representative) signs the informed consent form (Excluded if any of the following conditions are met) (1) Life expectancy is less than 3 months (2) Patients with renal insufficiency, uncontrolled diabetes, severe cardiovascular disease, including myocardial infarction, uncontrolled angina, clinically significant pericardial disease, or grade III or IV heart failure 2. Peripheral blood samples were collected from all patients according to the routine ELISA procedure for subsequent experiments. The sampling time was before surgery and +5, +15, +25, and +35 days after surgery.

[0020] 3. ELISA test Six biomarkers were tested by ELISA platform to evaluate the risk and early diagnosis of postoperative GVHD and VOD. This procedure is applicable to all aspects of GVHD and VOD biomarker testing projects, standardizes the operating procedures, and ensures accurate and reliable test results.

[0021] 1) EDTA plasma separation: Centrifuge EDTA anticoagulated whole blood at 2000 rpm for 5 minutes, take the supernatant and store it at -20℃ for later use.

[0022] 2) ELISA detection of ANG2, ST2, VCAM1, and PAI-1 uses proteintech's ELISA kit, and HA and FICOLIN use Huamei's ELISA kit. The overall operation process is basically consistent with the standard process in the kit manual. The reagents used for detection are all the reagents in this kit. Do not mix other kit reagents without special instructions. Before the experiment, all buffer reagents need to be placed at room temperature for 30 minutes. Detection antibodies and labeled antibodies need to be configured into 1X working solution (the mother solution is 100X) with corresponding diluents, and configured according to the actual amount. Each time before using the steps, prepare it and use it. The washing solution is configured into 1X working solution with distilled water. The new kit needs to reconstruct the standard curve, repeat it twice, and configure 24 more copies of sd5 and sd2 at the same time when configuring the standard curve sample, each copy is 120μl, and frozen at -20℃ after packaging.

[0023] 3) Sample dilution: ANG2: Take a 1.5 ml centrifuge tube, add 24 μl plasma and 96 μl sample diluent (Sample Diluent PT3, 5-fold dilution), respectively, shake and mix, and then centrifuge briefly. ST2: Take a 1.5 ml centrifuge tube, add 12 μl plasma and 108 μl sample diluent (Sample Diluent PT3, 10-fold dilution), respectively, shake and mix, and then centrifuge briefly. VCAM1: Two-step dilution (200-fold dilution), take a 1.5 ml centrifuge tube, add 5 μl plasma and 95 μl sample diluent (Sample Diluent PT4), respectively, shake and mix, and then centrifuge briefly; take a new 1.5 ml centrifuge tube, add 12 μl of the dilution product in the previous step and 108 μl sample diluent (Sample Diluent PT4), shake and mix, and then centrifuge briefly.

[0024] 4) Dilution of standard: Add the standard powder to the corresponding kit sample diluent to prepare the mother solution, take 7 1.5ml centrifuge tubes (sd7-sd1) and arrange them in sequence, add 120μl sample diluent to each except sd7, and pipette 240μl standard sd7 into sd7 centrifuge tube. Take 120μl from sd7 tube to sd6 centrifuge tube, shake and mix, and centrifuge briefly. Take 120μl from sd6 tube to sd5 centrifuge tube, shake and mix, and centrifuge briefly. And so on to dilute the standard in multiples. Take another 1.5ml centrifuge tube sd0 and add only 120μl sample diluent. Shake and mix, centrifuge briefly, take all out into a 2ml centrifuge tube to obtain standard sd7, and set aside.

[0025] 5) ELISA test: Take the corresponding number of samples + 2 wells to make SD4 and SD2 ELISA plates (add 16 ELISA plates for standard curves for the first test), add 100μl of standard and sample plasma to be tested respectively, ensure that the sample addition is uninterrupted, and the sample addition time is controlled within 10min. After the sample addition is completed, gently shake to mix, stick the sealing film, incubate at 37℃ for 120min, and when the incubation time is about to end, prepare 1X detection antibody (Detection Antibody), and add 1 portion of the total amount of working solution according to the number of tests. After the incubation is completed, remove the sealing film, shake out the liquid, cover with absorbent paper, and tap the ELISA plate downward several times. Add 300μl of 1X washing solution, shake out the liquid after each wash, cover with absorbent paper, and tap the ELISA plate downward several times, repeat 4 times, and ensure that there is no residual liquid in the plate for the last time. Add 100μl of 1X detection antibody to each well, cover with sealing film, and incubate at 37℃ for 60min. When the incubation time is almost up, prepare 1X HRP-conjugate antibody / HRP-conjugate streptavidin-HRP, and add 1 portion to prepare the total amount of working solution according to the number of tests. Repeat steps 4) and 5), and wash 4 times. Add 100μl of 1X HRP-conjugate antibody / HRP-conjugate streptavidin-HRP to each well, cover with sealing film, and incubate at 37℃ for 40min. Repeat steps 4) and 5), and wash 4 times. Add 100μl of TMB color development solution (Tetramethybenzidine Substrate) to each well, and color at 37℃ in the dark for 15min. Add 100μl of stop solution to each well, and the blue color turns to yellow. The order of adding stop solution is the same as that of adding TMB color development solution. The readings were taken using an enzyme-labeled instrument with a detection wavelength of 450 nm and a correction wavelength of 630 nm. The readings were taken 5 min after the addition of the stop solution.

[0026] 6) Data analysis: The net value was obtained by subtracting the 630nm reading from the 450nm reading from SD7 to SD1. A linear or binomial calibration curve was constructed with this net value and the corresponding standard concentration. The constructed calibration curve was qualified if the R2 was greater than 0.99. If there was obvious discrete data, it could be removed and the calibration curve was constructed. The net value was obtained by subtracting the 630nm reading from the 450nm reading of the sample. This net value was used as the Y value in the calibration curve equation, and the calculated X value was multiplied by the dilution factor to obtain the concentration of the six cytokines PAI-1, ST2, ANG-2, HA, VCAM-1 and L-Ficolin in the sample. The test results were expressed as (SD), and SPSS 19.0 (SPSS, Chicago, IL, USA) was used for data statistics and analysis, linear regression analysis, Pearson correlation analysis, etc. The Mann-Whitney U-test was used to compare the means between the two groups, and P<0.05 was considered statistically significant. The receiver operating characteristic (ROC) was calculated to evaluate the diagnostic performance of the candidate proteins.

[0027] 4. Experimental results Commercially available kits were searched for the above 6 proteins, and the Human SERPINE1ELISA Kit (KE00109) sub-kit for detecting protein PAI-1, the Human ST2 ELISA Kit (KE00055) sub-kit for detecting protein ST2, the Human Angiopoietin 2 ELISA Kit (KE00226) sub-kit for detecting protein ANG2, the Human Hyaluronic acid, HA ELISA Kit (CSB-E04805h) sub-kit for detecting protein HA, the Human VCAM-1 / CD106 ELISA Kit (KE00163) sub-kit for detecting protein VCAM-1, and the Human Ficolin-2 (FCN2) ELISA kit (CSB-EL008551HU) sub-kit for detecting protein L-FICOLIN were purchased. A total of 68 patients who underwent hematopoietic stem cell transplantation, 29 patients who underwent graft-versus-host disease, 6 patients who underwent veno-occlusive disease, 62 patients who developed infection, and 4 patients who did not develop graft-versus-host disease, veno-occlusive disease, or infection were tested. The specific results are shown in Tables 1 to 14. Figure 1-Figure 2 shown.

[0028] Compared with patients who did not develop GVHD before and after transplantation (eg Figure 1, Tables 1 to 6). The expression levels of ST2, PAI-1, and HA in the peripheral blood of patients with GVHD after transplantation were significantly increased (P<0.05), and the expression level of L-FICOLIN was decreased (P<0.1). There was no significant difference in the expression levels of ANG2 and VCAM1, suggesting that ST2, PAI-1, HA, and L-FICOLIN can be used as auxiliary diagnostic indicators for the occurrence of graft-versus-host disease.

[0029] Table 1 Serum ANG2 test results of four groups of subjects

[0030] Note: There was no statistically significant difference between before GVHD occurred after transplantation and before GVHD occurred after transplantation (p=0.514), and there was no statistically significant difference between after GVHD occurred after transplantation and before GVHD occurred after transplantation (p=0.956).

[0031] Table 2 Results of serum ST2 test in four groups of subjects

[0032] Note: The factor level in the group before GVHD occurred after transplantation was significantly higher than that in the group without GVHD after transplantation (p=0.043), and the factor level in the group after GVHD occurred after transplantation was significantly higher than that in the group without GVHD after transplantation (p=0.023).

[0033] Table 3 Serum VCAM1 test results of the four groups of subjects

[0034] Note: There was no statistically significant difference between before GVHD occurred after transplantation and before GVHD occurred after transplantation (p=0.960), and there was no statistically significant difference between after GVHD occurred after transplantation and before GVHD occurred after transplantation (p=0.872).

[0035] Table 4 Serum PAI-1 test results of the four groups of subjects

[0036] Note: There was no statistically significant difference between before GVHD after transplantation and before GVHD after transplantation (p=0.219). The factor level in the group after GVHD after transplantation was significantly higher than that in the group without GVHD after transplantation (p=0.013).

[0037] Table 5 Serum HA test results of the four groups of subjects

[0038] Note: There was no statistically significant difference between before GVHD occurred after transplantation and before GVHD occurred after transplantation (p=0.494), and there was no statistically significant difference between after GVHD occurred after transplantation and before GVHD occurred after transplantation (p=0.160).

[0039] Table 6 Serum L-FICOLIN test results of the four groups of subjects

[0040] Note: There was no statistically significant difference between before GVHD occurred after transplantation and before GVHD occurred after transplantation (p=0.397), and there was no statistically significant difference between after GVHD occurred after transplantation and before GVHD occurred after transplantation (p=0.098).

[0041] Compared with patients without VOD before and after transplantation (eg Figure 2 , Tables 7-12), the expression level of ANG2 in the peripheral blood of patients with VOD after transplantation was increased (P<0.05), and there was no significant difference in the expression levels of L-FICOLIN, HA, VCAM1, ST2 and PAI-1, suggesting that ANG2 can be used as an auxiliary diagnostic indicator for the occurrence of hepatic veno-occlusive disease.

[0042] Table 7 Serum ANG2 test results of the four groups of subjects

[0043] Note: The factor levels in the group with GVHD after transplantation were significantly higher than those in the group without GVHD after transplantation (p=0.016).

[0044] Table 8 Serum ST2 test results of four groups of subjects

[0045] Note: There was no statistically significant difference between the occurrence of GVHD after transplantation and the non-occurrence of GVHD after transplantation (p=0.851).

[0046] Table 9 Serum VCAM1 test results of four groups of subjects

[0047] Note: There was no statistically significant difference between the occurrence of GVHD after transplantation and the non-occurrence of GVHD after transplantation (p=0.614).

[0048] Table 10 Serum PAI-1 test results of the four groups of subjects

[0049] Note: There was no statistically significant difference between the occurrence of GVHD after transplantation and the non-occurrence of GVHD after transplantation (p=0.782).

[0050] Table 11 Serum HA test results of the four groups of subjects

[0051] Note: There was no statistically significant difference between the occurrence of GVHD after transplantation and the non-occurrence of GVHD after transplantation (p=0.226).

[0052] Table 12 Results of serum L-FICOLIN test in four groups of subjects

[0053] Note: There was no statistically significant difference between the occurrence of GVHD after transplantation and the non-occurrence of GVHD after transplantation (p=0.145).

[0054] Compared with patients without GVHD before and after transplantation (as shown in Table 13), the AUCs of ANG-2, ST2, VCAM-1, PAI-1, HA and L-Ficolin in the peripheral blood of patients with GVHD after transplantation were 0.57 [95% confidence interval (CI), 0.4623-0.6762], 0.596 (95% CI, 0.4634-0.7353), 0.656 (95% CI, 0.5765-0.7437), 0.67 (95% CI, 0.5774-0.7602), 0.663 (95% CI, 0.5612-0.7605) and 0.592 (95% CI, 0.4963-0.6917), respectively. The area under the combined diagnostic curve (ROC curve area) was 0.722 for ST2+ PAI-1+HA, 0.752 for ST2+ PAI-1+HA+L-FICOLIN, and 0.76 for ANG2+ST2+VCAM1+PAI-1+HA+L-FICOLIN. The area under the curve of the combined diagnosis of six proteins (ANG2+ST2+VCAM1+PAI-1+HA+L-FICOLIN) was the largest. However, PAI-1 had the best diagnostic sensitivity (88.80%), and ST2 had the highest diagnostic specificity, which was 89.8%, as shown in Table 13.

[0055] Table 13 Diagnostic performance of PAI-1, ST2, ANG-2, HA, VCAM-1 and L-Ficolin detection levels for GVHD

[0056] a Abbreviations: AUC, area under the curve; 95% CI, 95% confidence interval. Compared with patients without VOD before and after transplantation (as shown in Table 14), the AUCs of ANG-2, ST2, VCAM-1, PAI-1, HA and L-Ficolin in the peripheral blood of patients with VOD after transplantation were 0.403 [95% confidence interval (CI), 0.1275-0.6778], 0.463 (95% CI, 0.1983-0.7287), 0.508 (95% CI, 0.3041-0.7113), 0.472 (95% CI, 0.2458-0.6985), 0.641 (95% CI, 0.510-0.870) and 0.446 (95% CI, 0.3041-0.6143), respectively, and the area under the combined diagnostic curve (ROC curve area) was 0.505 The area under the curve for HA diagnosis was the largest and had the best diagnostic sensitivity (79.6%), while ANG2+HA had the highest diagnostic specificity of 77.20%, as shown in Table 14.

[0057] Table 14 Diagnostic performance of PAI-1, ST2, ANG-2, HA, VCAM-1 and L-Ficolin detection levels for VOD

[0058] a Abbreviations: AUC, area under the curve; 95% CI, 95% confidence interval. In summary, the above results indicate that PAI-1, ST2, ANG-2, HA, VCAM-1 and L-Ficolin can be used as auxiliary indicators for the diagnosis and prediction of graft-versus-host disease and hepatic veno-occlusive disease in children. They are simple and easy to operate and have high clinical application value.

[0059] It is worth noting that this application first discovered that the combination of PAI-1, ST2, ANG-2, HA, VCAM-1 and L-Ficolin markers in the peripheral blood of children with graft-versus-host disease and hepatic veno-occlusive disease has significant differences between GVHD and VOD and hematopoietic stem cell transplant patients without GVHD and VOD (GVHD: AUC=0.784, VOD: AUC=0.510), which can be used as one of the laboratory immune-related detection indicators for auxiliary diagnosis of GVHD and VOD, and the operation is simple and feasible, with high clinical application value. In addition, this application also provides a detection method for PAI-1, ST2, ANG-2, HA, VCAM-1 and L-Ficolin markers, which can be used to quantitatively analyze the content of the above markers. The present application collects peripheral blood from children who have received hematopoietic stem cell transplantation before and after transplantation, and determines the clinical application value of various markers and their combinations by detecting the peripheral serum of the children using the ELISA method. On this basis, the present application uses a combination kit to detect the combination of peripheral blood markers, and uses the test results to assist in the diagnosis of graft-versus-host disease and veno-occlusive disease in children.

[0060] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the attached claims.

[0061] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A diagnostic marker for graft-versus-host disease and hepatic veno-occlusive disease, characterized in that: The diagnostic markers are the serological levels of six factors in combination of PAI-1, ST2, ANG-2, HA, VCAM-1 and L-Ficolin.

2. Use of the diagnostic markers for graft-versus-host disease and hepatic veno-occlusive disease according to claim 1 in the preparation of a kit for diagnosing graft-versus-host disease and hepatic veno-occlusive disease.

3. A diagnostic kit for graft-versus-host disease and hepatic veno-occlusive disease, characterized in that: The diagnostic kit is composed of several sub-kits for detecting PAI-1, ST2, ANG-2, HA, VCAM-1 and L-Ficolin respectively.

4. The diagnostic kit for graft-versus-host disease and hepatic veno-occlusive disease according to claim 3, characterized in that: The diagnostic kit is composed of the following sub-kits: Human SERPINE1 ELISA Kit for detecting protein PAI-1, Human ST2 ELISA Kit for detecting protein ST2, Human Angiopoietin 2 ELISA Kit for detecting protein ANG2, Human Hyaluronic acid, HA ELISA Kit for detecting protein HA, Human VCAM-1 / CD106 ELISA Kit for detecting protein VCAM-1, and Human Ficolin-2 ELISA kit for detecting protein L-FICOLIN.

5. Use of the diagnostic kit for graft-versus-host disease and veno-occlusive disease according to claim 3 in the preparation of reagents for auxiliary diagnosis of graft-versus-host disease and veno-occlusive disease in children.

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