Biomarker for evaluating fibrosis degree of systemic sclerosis and application thereof

Abstract

The invention discloses a biomarker for evaluating the fibrosis degree of systemic sclerosis and application of the biomarker, and belongs to the technical field of disease diagnosis and evaluation. The research of the invention believes that Chi3L1 can be used as a biomarker for diagnosing diseases, monitoring disease activity and fibrosis degree and evaluating drug treatment effect, and the occurrence of fibrosis of SSc patients and the degree of skin and pulmonary fibrosis can be reflected by detecting the concentration level of the Chi3L1 in serum. The invention provides a key biomarker for evaluating SSc fibrosis occurrence and skin and pulmonary fibrosis degrees, so that the detection is simple, convenient and feasible.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease diagnosis and evaluation, and specifically relates to a biomarker for evaluating the degree of fibrosis in systemic sclerosis and an application thereof. Background Art

[0002] Systemic sclerosis (SSc), commonly known as "scleroderma", is a rare autoimmune disease characterized by fibrosis of multiple organs such as the skin and lungs. It has the highest mortality rate among all rheumatic diseases due to its irreversible and extensive fibrosis. It is estimated that there are about 2 million patients with systemic sclerosis worldwide, and up to 90% may develop varying degrees of pulmonary fibrosis, which is associated with a very poor prognosis and accounts for about 35% of all deaths.

[0003] Systemic sclerosis is characterized by fibrosis of the skin and internal organs such as the lungs, so the improvement of fibrosis is closely related to the patient's prognosis. The current gold standard for evaluating skin fibrosis is the modified Rodnan scoring scale, which is a trained physician's palpation of 17 parts of the patient (bilateral fingers, palms, forearms, upper arms, toes, calves, thighs, face, chest, and abdomen), and each part is scored from 0 to 3 points from mild to severe, with a total of 51 points. The higher the score, the more severe the skin fibrosis.

[0004] The lung is one of the organs with the highest incidence of visceral fibrosis and the highest cause of death in patients with systemic sclerosis. Currently, the means of evaluating pulmonary fibrosis are mostly through high-resolution CT scans of the lungs and pulmonary function tests. Among them, forced vital capacity (FVC) and carbon monoxide diffusion capacity (DLCO) less than 80% of the predicted value indicate the presence of restrictive ventilation dysfunction, indicating the occurrence of pulmonary fibrosis.

[0005] The current assessment methods for patients' skin and lung fibrosis require professional doctors trained in the modified Rodnan score and professional equipment such as lung function testers and CT machines. There is still a lack of suitable biomarkers to evaluate skin and lung fibrosis in patients with systemic sclerosis. Therefore, there is an urgent need for a key biomarker for evaluating the occurrence and degree of fibrosis in systemic sclerosis.

[0006] Chitinase-3-like-1 protein (Chi3L1) is secreted by a variety of immune cells and structural cells. A large number of studies have shown that Chi3L1 plays a vital role in various inflammatory reactions, tissue remodeling and fibrosis. At present, no study has explored the relationship between the serum Chi3L1 concentration and SSc fibrosis in patients with systemic sclerosis. The research team of the present invention found that the concentration of Chi3L1 protein in the serum of patients with systemic sclerosis was significantly higher than that in healthy subjects, and verified the relationship between the concentration of Chi3L1 protein in the serum of patients and the modified Rodnan scoring scale, the gold standard for skin fibrosis assessment, and the predicted FVC and DLCO values, the key indicators of pulmonary fibrosis. The research conclusion is: the serum concentration of Chi3L1 can be used to assess the degree of skin and pulmonary fibrosis in patients with systemic sclerosis. Summary of the invention

[0007] The research results of the present invention suggest that Chi3L1 can be used as a biomarker for diagnosing diseases, monitoring disease activity and fibrosis, and evaluating drug treatment effects. The occurrence and degree of fibrosis in SSc patients can be reflected by detecting the serum Chi3L1 concentration level. Based on this, one object of the present invention is to provide the use of Chi3L1 as a biomarker in the preparation of a product for assessing the risk of fibrosis and / or the degree of fibrosis in systemic sclerosis; the second object of the present invention is to provide a substance for detecting Chi3L1 for the preparation of a product for assessing the risk of fibrosis and / or the degree of fibrosis in systemic sclerosis; the third object of the present invention is to provide a kit for assessing the risk of fibrosis and / or the degree of fibrosis in systemic sclerosis; and another object of the present invention is to provide a method for assessing the risk of fibrosis and / or the degree of skin and lung fibrosis in systemic sclerosis.

[0008] The present invention includes the following technical solutions:

[0009] In a first aspect of the present invention, the present invention provides a use of Chi3L1 as a biomarker in at least one of the following:

[0010] a1) Use in the preparation and / or screening of products for assessing or assisting in assessing the risk of developing systemic sclerosis fibrosis;

[0011] a2) Use in the preparation and / or screening of products for evaluating or assisting in the evaluation of the degree of skin and / or pulmonary fibrosis in systemic sclerosis.

[0012] The products include but are not limited to reagents, test kits, chips, test strips, membrane strips or detection platforms.

[0013] In a second aspect of the present invention, the present invention provides a use of a substance for detecting Chi3L1 in at least one of the following:

[0014] b1) Use in the preparation and / or screening of products for assessing or assisting in assessing the risk of developing systemic sclerosis fibrosis;

[0015] b2) Use in the preparation and / or screening of products for evaluating or assisting in the evaluation of the degree of skin and / or pulmonary fibrosis in systemic sclerosis.

[0016] The products include but are not limited to reagents, test kits, chips, test strips, membrane strips or detection platforms.

[0017] The evaluation or auxiliary evaluation described in the present invention comprises the following steps:

[0018] c1) using a substance for detecting Chi3L1 to measure the Chi3L1 level of the subject;

[0019] c2) When the Chi3L1 level is higher than the critical value, it indicates that the person being tested has a high risk of developing systemic sclerosis fibrosis;

[0020] c3) When Chi3L1 levels were above the critical value, Chi3L1 levels were positively correlated with the degree of skin and / or lung fibrosis.

[0021] The substance for detecting Chi3L1 is any reagent required for detecting Chi3L1 protein level or gene expression level.

[0022] The technicians of the present invention can select one or a combination of two or more of antibody chip detection, flow cytometer analysis, radioisotope immunoassay, enzyme-linked immunosorbent assay (ELISA), protein blotting (Western Blot), immunohistochemistry (IHC), immunofluorescence, immunoenzyme labeling, immunochromatography, transmission immunoturbidimetry, and scattering immunoturbidimetry to detect the Chi3L1 protein level.

[0023] The technicians of the present invention can select one or a combination of two or more of RT-PCR, RT-qPCR, gene chip detection, Southern blotting, in situ hybridization, and spatial transcriptome technology to detect the expression level of Chi3L1 gene.

[0024] The substance for detecting Chi3L1 is any reagent required by the detection method selected for detecting Chi3L1 protein level or gene expression level.

[0025] In a preferred embodiment of the present invention, the substance for detecting Chi3L1 is any reagent required for detecting the concentration of Chi3L1 protein by enzyme-linked immunosorbent assay (ELISA).

[0026] The Chi3L1 level of the present invention includes the Chi3L1 protein concentration in the serum of the subject and / or the level of Chi3L1 transcription product in the skin tissue or peripheral blood of the subject.

[0027] In a preferred embodiment of the present invention, the Chi3L1 level is the Chi3L1 protein concentration in the serum of the subject.

[0028] The critical value of the present invention is the average value of Chi3L1 protein concentration in the serum of healthy subjects + 2SD.

[0029] Specifically, the critical value is 85.42 ng / ml.

[0030] Therefore, in the method provided by the present invention for assessing the risk of systemic sclerosis fibrosis and the degree of skin and / or pulmonary fibrosis in a subject, when the Chi3L1 protein level in the serum of the subject is higher than the set critical value (Cut-off, i.e. 85.42 ng / ml), it indicates that the subject is at high risk of systemic sclerosis fibrosis; when the Chi3L1 level in the serum of the subject is lower than the set critical value (Cut-off, i.e. 85.42 ng / ml), it indicates that the subject is at low risk of systemic sclerosis fibrosis.

[0031] In the third aspect of the present invention, the present invention provides a kit for assessing the risk of occurrence of systemic sclerosis fibrosis and / or assessing the degree of skin and / or pulmonary fibrosis, wherein the kit comprises the substance for detecting Chi3L1 as described in the second aspect of the present invention.

[0032] In a preferred embodiment of the present invention, the kit is an ELISA kit for detecting Chi3L1 protein concentration, and the kit comprises an anti-Chi3L1 specific antibody.

[0033] The anti-Chi3L1 specific antibody is selected from a monoclonal antibody or a polyclonal antibody. Further, the antibody can be directly or indirectly labeled with a marker, and the marker is selected from 1. fluorescent substances, such as FITC and rhodamine; 2. radioactive substances, such as 32P, 35S, 14C, 3H; 3. enzymes, such as alkaline phosphatase and peroxidase; 4. colored particles, such as metal colloidal particles and colored latex; 5. one or a combination of two or more of biotin

[0034] Furthermore, the kit also includes conventional reagents required in the operation process of enzyme-linked immunosorbent assay (ELISA).

[0035] In a fourth aspect of the present invention, the present invention provides a method for assessing the risk of systemic sclerosis fibrosis and / or assessing the degree of skin and / or pulmonary fibrosis for non-diagnostic and non-therapeutic purposes, the method comprising the following steps:

[0036] d1) using a substance for detecting Chi3L1 to measure the Chi3L1 level of the subject;

[0037] d2) When the Chi3L1 level is higher than the critical value, it indicates that the person being tested has a high risk of developing systemic sclerosis fibrosis;

[0038] d3) When Chi3L1 levels were higher than the critical value, Chi3L1 levels were positively correlated with the degree of skin and / or lung fibrosis.

[0039] The substance for detecting Chi3L1 is any reagent required for detecting Chi3L1 protein level or gene expression level.

[0040] In a specific embodiment of the present invention, the substance for detecting Chi3L1 is any reagent required for detecting the concentration of Chi3L1 protein by enzyme-linked immunosorbent assay (ELISA).

[0041] The Chi3L1 level of the present invention includes the Chi3L1 protein concentration in the serum of the subject and / or the level of Chi3L1 transcription product in the skin tissue or peripheral blood of the subject.

[0042] In a preferred embodiment of the present invention, the Chi3L1 level is the Chi3L1 protein concentration in the serum of the subject.

[0043] The critical value of the present invention is the average value of Chi3L1 protein concentration in the serum of healthy subjects + 2SD, specifically 85.42 ng / ml.

[0044] In a specific embodiment of the present invention, the method for assessing the risk of occurrence of systemic sclerosis fibrosis and / or assessing the degree of skin and / or pulmonary fibrosis comprises:

[0045] e1) detecting the Chi3L1 protein concentration in the serum of the subject by enzyme-linked immunosorbent assay (ELISA);

[0046] e2) When the Chi3L1 protein concentration is higher than 85.42 ng / ml, it indicates that the subject is at high risk of developing systemic sclerosis fibrosis; when the Chi3L1 protein concentration is lower than 85.42 ng / ml, it indicates that the subject is at low risk of developing systemic sclerosis fibrosis.

[0047] e3) When Chi3L1 protein concentration was higher than 85.42 ng / ml, Chi3L1 level was positively correlated with the level of skin and / or lung fibrosis in patients with systemic sclerosis.

[0048] The assessment of the present invention includes using the method provided in the fourth aspect of the present invention to judge the risk of occurrence and / or degree of fibrosis in systemic sclerosis, and also includes combining with other conventional technical means in the field for joint assessment. The other conventional technical means in the field include but are not limited to assessing the skin fibrosis of the subject by using a modified Rodnan scoring scale, and / or assessing the pulmonary fibrosis of the subject by using a pulmonary function tester and a CT device.

[0049] The present invention determines that the Chi3L1 protein concentration in the serum of SSc patients is significantly higher than that of healthy subjects by enzyme-linked immunosorbent assay (ELISA), indicating that Chi3L1 plays an important role in the occurrence of SSc fibrosis. Further, the present invention performs Spearman correlation analysis on the gold standard modified Rodnan score for evaluating SSc skin fibrosis and the key indicators FVC predicted value and DLCO predicted value in evaluating pulmonary fibrosis with the patient's serum Chi3L1 protein concentration. The analysis results show that the patient's serum Chi3L1 protein concentration is significantly positively correlated with the gold standard modified Rodnan score, and significantly negatively correlated with the FVC predicted value and DLCO predicted value. Therefore, the Chi3L1 serum concentration can be used to evaluate the risk of SSc fibrosis and the degree of skin and pulmonary fibrosis.

[0050] The present invention creatively uses the Chi3L1 protein concentration in the patient's serum as an indicator for predicting the occurrence of systemic sclerosis fibrosis and evaluating the degree of fibrosis, providing a key biomarker for evaluating the occurrence and degree of fibrosis. Compared with the conventional methods currently used to evaluate patient skin and pulmonary fibrosis, the detection method is simple and easy, has low detection cost, low requirements on detection personnel, and saves labor costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 is the serum Chi3L1 concentration in SSc patients and healthy subjects.

[0052] Figure 2 This is the result of Spearman correlation analysis between serum Chi3L1 concentration and modified Rodnan score in SSc patients.

[0053] Figure 3 This is the result of Spearman correlation analysis between serum Chi3L1 concentration and predicted FVC value in SSc patients.

[0054] Figure 4This is the result of Spearman correlation analysis between serum Chi3L1 concentration and predicted DLCO value in SSc patients. DETAILED DESCRIPTION

[0055] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0056] The research group of the present invention recruited 48 SSc patients and 24 healthy subjects for the experiment. First, the Chi3L1 concentration in the serum of the patients and healthy subjects was detected. Then, trained physicians were asked to perform modified Rodnan scores and pulmonary function tests on the SSc patients, and the modified Rodnan scores, predicted FVC values, and predicted DLCO values ​​were collected. Spearman correlation analysis was performed on the peripheral blood Chi3L1 concentration of the SSc patients with the modified Rodnan scores, predicted FVC values, and predicted DLCO values ​​to obtain the relationship between the serum Chi3L1 concentration of the SSc patients and the current gold standard for skin and pulmonary fibrosis assessment.

[0057] The experimental design of the present invention includes the following parts:

[0058] 1. Detection of Chi3L1 protein concentration in the serum of SSc patients

[0059] Purpose of the experiment: To detect the concentration of Chi3L1 protein in the serum of SSc patients and healthy subjects by ELISA (enzyme-linked immunosorbent assay).

[0060] Experimental method: Peripheral blood was drawn from 48 SSc patients and 24 healthy subjects to determine the Chi3L1 concentration in serum. Specifically, the steps for determining the Chi3L1 concentration in serum are as follows:

[0061] S1) Draw peripheral blood from the subject, centrifuge the blood sample at 3000×g for 10 minutes, and take the supernatant;

[0062] S2) Prepare 8 tubes of standard protein according to the instructions of the kit (ABCAM, catalog number ab255719), and reduce the concentration by half from the first tube to the seventh tube. Only add the sample diluent to the eighth tube, that is, the standard protein concentration is 0 pg / mL;

[0063] S3) Take out the ELISA plate from the kit, add 8 different concentrations of standard protein solutions and samples, set up two replicate wells, stick a sealing film, and incubate in a 37°C oven for 1 hour;

[0064] S4) Pour out the liquid in the wells, tap on absorbent paper several times without washing, prepare biotin antibody working solution according to the instructions, add 100 μL to each well, apply sealing film, and incubate in a 37°C oven for 1 hour;

[0065] S5) Pour out the liquid in the wells, prepare washing solution according to the instructions, add 200 μL of washing solution to each well, pour out after 2 minutes, and gently tap on absorbent paper to dry, and wash three times in this way;

[0066] S6) Add HRP-coupled avidin working solution to each well, apply sealing film, and incubate in a 37°C oven for 30 minutes. Then add 200 μL of washing solution to each well, pour it out after 2 minutes, and gently tap it on absorbent paper to dry it. Wash it 3 times in this way;

[0067] S7) adding the reaction substrate 3,3',5,5'-tetramethylbenzidine (TBM), incubating at 37°C for about 15 minutes in the dark, and adding the stop solution to terminate the reaction after sufficient reaction;

[0068] S8) The absorbance value of each well at 450 nm was measured using an ELISA instrument, and a concentration-absorbance standard curve was drawn according to the concentration and absorbance of the standard, and finally the protein concentration of Chi3L1 in the serum sample was calculated according to the standard curve.

[0069] The statistical results of Chi3L1 concentration in the serum of 48 SSc patients and 24 healthy subjects measured by the above method are as follows Figure 1 As shown, the results showed that the Chi3L1 concentration in the serum of SSc patients was significantly higher than that in healthy subjects, with a cut-off value of 85.42 ng / ml (the average serum Chi3L1 concentration of healthy subjects + 2SD).

[0070] 2. Evaluation of skin fibrosis in SSc patients and correlation analysis with serum Chi3L1 concentration

[0071] Professional physicians were asked to evaluate the skin fibrosis of 48 SSc patients using the gold standard - the modified Rodnan scoring scale. Specifically, the physician palpated 17 parts of the patient (bilateral fingers, palms, forearms, upper arms, toes, calves, thighs, face, chest, and abdomen), and scored each part from 0 to 3 points from mild to severe, with a total score of 51 points. The higher the score obtained by the patient, the more severe the skin fibrosis.

[0072] Spearman correlation analysis was performed with the peripheral blood Chi3L1 concentration of SSc patients as the ordinate and the modified Rodnan scoring scale score of SSc patients as the abscissa. The linear curve is shown in Figure 2 As shown in the results, there was a significant positive correlation between serum Chi3L1 concentration and modified Rodnan score in SSc patients.

[0073] 3. Evaluation of pulmonary fibrosis in SSc patients and correlation analysis with serum Chi3L1 concentration

[0074] Professional physicians were asked to evaluate the pulmonary fibrosis of 48 SSc patients and obtain the estimated forced vital capacity (FVC) of the patients.

[0075] Spearman correlation analysis was performed with the peripheral blood Chi3L1 concentration of SSc patients as the ordinate and the predicted FVC value of SSc patients as the abscissa. The linear curve is as follows Figure 3 As shown in the results, there was a significant negative correlation between serum Chi3L1 concentration and predicted FVC in SSc patients.

[0076] 4. Evaluation of pulmonary fibrosis in SSc patients and correlation analysis with serum Chi3L1 concentration

[0077] Professional physicians were asked to evaluate the pulmonary fibrosis of 48 SSc patients and obtain the estimated values ​​of the patients' diffusion capacity for carbon monoxide (DLCO).

[0078] Spearman correlation analysis was performed with the peripheral blood Chi3L1 concentration of SSc patients as the ordinate and the predicted DLCO value of SSc patients as the abscissa. The linear curve is as follows Figure 4 As shown in the results, there was a significant negative correlation between serum Chi3L1 concentration and DLCO predicted value in SSc patients.

[0079] The present invention measures the Chi3L1 level in the serum of SSc patients by ELISA method, and obtains a Cut-off value of 85.42ng / ml, which reflects the possibility and level of fibrosis in SSc patients. Further, the present invention obtains the modified Rodnan score, FVC predicted value, and DLCO predicted value of the SSc patients, and performs correlation analysis on the Chi3L1 level in the serum with the modified Rodnan score, FVC predicted value, and DLCO predicted value, respectively. The results show that the serum Chi3L1 concentration of SSc patients is positively correlated with the modified Rodnan score, and negatively correlated with the FVC predicted value and DLCO predicted value.

[0080] The research results of the present invention reveal that the occurrence and degree of fibrosis in SSc patients can be reflected by detecting the concentration level of serum Chi3L1, and Chi3L1 can be used as a biomarker for diagnosing diseases, monitoring disease activity, and evaluating drug treatment effects. Compared with the conventional method currently used to evaluate the skin and lung fibrosis of patients, the method of reflecting the degree of fibrosis in SSc patients by detecting the concentration of Chi3L1 in serum is simple and easy, saves detection costs, and saves manpower.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of Chi3L1 as a biomarker in at least one of the following: a1) Use in the preparation and / or screening of products for assessing or assisting in assessing the risk of developing systemic sclerosis fibrosis; a2) Use in the preparation and / or screening of products for evaluating or assisting in the evaluation of the degree of skin and / or pulmonary fibrosis in systemic sclerosis; The products include but are not limited to reagents, test kits, chips, test strips, membrane strips or detection platforms.

2. Use of a substance for detecting Chi3L1 in at least one of the following: b1) Use in the preparation and / or screening of products for assessing or assisting in assessing the risk of developing systemic sclerosis fibrosis; b2) Use in the preparation and / or screening of products for evaluating or assisting in the evaluation of the degree of skin and / or pulmonary fibrosis in systemic sclerosis; The evaluation or auxiliary evaluation comprises the following steps: c1) using a substance for detecting Chi3L1 to measure the Chi3L1 level of the subject; c2) When the Chi3L1 level is higher than the critical value, it indicates that the person being tested has a high risk of developing systemic sclerosis fibrosis; c3) When Chi3L1 levels were higher than the critical value, Chi3L1 levels were positively correlated with the degree of skin and / or lung fibrosis; The substance for detecting Chi3L1 is any reagent required for detecting Chi3L1 protein level or gene expression level.

3. The use according to claim 2, characterized in that: The method for detecting Chi3L1 protein level is selected from one or a combination of two or more of antibody chip detection, flow cytometer analysis, radioisotope immunoassay, enzyme-linked immunosorbent assay, protein blotting, immunohistochemistry, immunofluorescence, immunoenzyme labeling, immunochromatography, transmission immunoturbidimetry, and scattering immunoturbidimetry; The method for detecting the expression level of the Chi3L1 gene is selected from one or a combination of two or more of RT-PCR, RT-qPCR, gene chip detection, Southern blotting, in situ hybridization, and spatial transcriptome technology.

4. The use according to claim 2, characterized in that: The substance for detecting Chi3L1 is any reagent required for detecting the concentration of Chi3L1 protein by enzyme-linked immunosorbent assay.

5. The use according to claim 2, characterized in that: The Chi3L1 level includes the Chi3L1 protein concentration in the serum of the subject and / or the level of Chi3L1 transcription products in the skin tissue or peripheral blood of the subject, preferably the Chi3L1 protein concentration in the serum of the subject.

6. The use according to claim 2, characterized in that: The critical value is the average value of Chi3L1 protein concentration in the serum of healthy subjects + 2SD. Specifically, the critical value is 85.42 ng / ml.

7. A kit for assessing the risk of developing systemic sclerosis fibrosis and / or assessing the degree of skin and / or pulmonary fibrosis, the kit comprising the substance for detecting Chi3L1 as described in any one of claims 2 to 6.

8. The kit according to claim 7, characterized in that The kit is an ELISA kit for detecting Chi3L1 protein concentration, and the kit comprises anti-Chi3L1 specific antibodies.

9. The kit according to claim 8, characterized in that The anti-Chi3L1 specific antibody is selected from a monoclonal antibody or a polyclonal antibody, and can be directly or indirectly labeled with a marker; Furthermore, the kit also includes conventional reagents required during the operation of the enzyme-linked immunosorbent assay.

10. A method for assessing the risk of developing systemic sclerosis fibrosis and / or assessing the degree of skin and / or pulmonary fibrosis for non-diagnostic and non-therapeutic purposes, the method comprising the following steps: d1) using a substance for detecting Chi3L1 to measure the Chi3L1 level of the subject; d2) When the Chi3L1 level is higher than the critical value, it indicates that the person being tested has a high risk of developing systemic sclerosis fibrosis; d3) When Chi3L1 levels were higher than the critical value, Chi3L1 levels were positively correlated with the degree of skin and / or lung fibrosis; The substance for detecting Chi3L1 is any reagent required for detecting Chi3L1 protein level or gene expression level; the Chi3L1 level includes the Chi3L1 protein concentration in the serum of the subject and / or the level of Chi3L1 transcription product in the skin tissue or peripheral blood of the subject; the critical value is the average value of Chi3L1 protein concentration in the serum of healthy subjects + 2SD.

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