Application of plasma marker as detection target in preparation of product for diagnosis or auxiliary diagnosis of liver-lung syndrome of liver cirrhosis patient

Abstract

The invention provides an application of a plasma marker as a detection target in preparation of a product for diagnosis or auxiliary diagnosis of liver-lung syndrome of a liver cirrhosis patient, and belongs to the technical field of medical diagnosis. The invention provides an application of a plasma marker as a detection target in preparation of a product for diagnosis or auxiliary diagnosis of liver-lung syndrome (HPS) of a liver cirrhosis patient. The plasma marker comprises vitamin A. Clinical research finds that the plasma vitamin A level of the liver cirrhosis patients accompanied with the liver-lung syndrome is obviously lower than that of the liver cirrhosis patients not accompanied with the liver-lung syndrome, and the plasma vitamin A level is in obvious negative correlation with the severity of the HPS. In addition, multi-factor analysis proves that the plasma vitamin A is an independent predictive factor of the HPS, and the diagnosis efficiency is remarkable. On the basis, the invention proposes that the plasma vitamin A is used as the HPS specific biomarker for the first time, and fills the blank of a non-invasive and early diagnosis technology of the HPS.

Description

Technical Field

[0001] This invention belongs to the field of medical diagnostic technology, and in particular relates to the application of a plasma biomarker as a detection target in the preparation of products for diagnosing or assisting in the diagnosis of hepatopulmonary syndrome in patients with cirrhosis. Background Technology

[0002] Hepatopulmonary syndrome (HPS) is the most common pulmonary complication of chronic liver disease, with an incidence of approximately 10% to 30% in patients with cirrhosis. The main pathological processes in HPS include intrapulmonary vasodilation (IPVD), intrapulmonary shunts, pulmonary vascular bed angiogenesis leading to alveolar ventilation-perfusion mismatch, arteriovenous shunts, and restricted gas exchange, ultimately resulting in severe hypoxemia.

[0003] Currently, the diagnosis of HPS relies on the formation of intrapulmonary shunts (IPVD) and abnormal gas exchange (arterial blood gas analysis showing alveolar-arterial oxygen partial pressure difference P(Aa)O2≥15mmHg). There is no non-invasive diagnostic method for HPS based on biomarkers in clinical practice. Summary of the Invention

[0004] The purpose of this invention is to provide an application of plasma biomarkers as detection targets in the preparation of products for the diagnosis or auxiliary diagnosis of hepatopulmonary syndrome in patients with cirrhosis, filling the gap in non-invasive and early diagnostic technology for hepatopulmonary syndrome in patients with cirrhosis.

[0005] This invention provides the application of a plasma biomarker as a detection target in the preparation of products for the diagnosis or auxiliary diagnosis of hepatopulmonary syndrome in patients with cirrhosis, wherein the plasma biomarker includes vitamin A.

[0006] Preferably, the plasma marker is vitamin A.

[0007] Preferably, the diagnosis includes early diagnosis.

[0008] The present invention also provides the application of a test reagent for measuring the amount of vitamin A in a sample in products for determining the risk of hepatopulmonary syndrome in patients with cirrhosis.

[0009] Preferably, the detection reagent for measuring the amount of vitamin A in the sample includes a high-performance liquid chromatography-mass spectrometry (HPLC-MS) detection reagent.

[0010] Preferably, the sample comprises blood, serum, or plasma from a patient with cirrhosis.

[0011] This invention also provides the application of vitamin A as a detection target in the preparation of products for guiding treatment and / or predicting prognosis of hepatopulmonary syndrome in patients with cirrhosis.

[0012] The present invention also provides a kit for determining the risk of hepatopulmonary syndrome in patients with cirrhosis, comprising a detection reagent for measuring the amount of vitamin A in a sample and a threshold reference description; the threshold reference description includes a condition indicating a high risk of hepatopulmonary syndrome when the level is below a preset threshold.

[0013] Preferably, when the detection reagent is a high-performance liquid chromatography-mass spectrometry (HPLC-MS) detection reagent, the threshold is 0.1750 mg / L.

[0014] The present invention also provides a system for determining the risk of hepatopulmonary syndrome in patients with cirrhosis, including a detection module, an input module, an analysis module, and an output module;

[0015] The detection module is used to measure the amount of vitamin A in the sample;

[0016] The input module is used to input the numerical value of the amount of vitamin A in the sample;

[0017] The analysis module has a preset threshold. By comparing the amount of vitamin A in the sample with the threshold, it indicates the risk of hepatopulmonary syndrome. When the amount is below the threshold, it indicates a high risk of hepatopulmonary syndrome.

[0018] The output module is used to output the risk of hepatopulmonary syndrome in patients with cirrhosis.

[0019] This invention provides an application of a plasma biomarker as a detection target in the preparation of products for the diagnosis or auxiliary diagnosis of hepatopulmonary syndrome (HPS) in patients with cirrhosis. The plasma biomarker includes vitamin A. Clinical studies have found that plasma vitamin A levels in cirrhotic patients with hepatopulmonary syndrome are significantly lower than those in patients without hepatopulmonary syndrome, and plasma vitamin A levels are significantly negatively correlated with HPS severity (P(Aa)O2). Furthermore, multivariate analysis confirmed that plasma vitamin A is an independent predictor of HPS (OR = 0.994, p = 0.004), with significant diagnostic efficacy (AUC = 0.7422). Based on this, this invention is the first to propose using plasma vitamin A as a specific biomarker for HPS, filling the gap in non-invasive, early diagnosis technology for HPS and solving the problems of low sensitivity and complex operation in existing technologies. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a quality control chart for Vitamin A.

[0022] Figure 2 This is the standard curve for vitamin A;

[0023] Figure 3 A graph showing the comparison of plasma vitamin A levels in cohorts of cirrhotic patients with and without hepatopulmonary syndrome;

[0024] Figure 4 A scatter plot was created to reveal the negative correlation between plasma vitamin A levels and P(Aa)O2 in the cohort.

[0025] Figure 5 To discover the ROC curve of plasma vitamin A levels in the cohort;

[0026] Figure 6 To verify the comparison of plasma vitamin A levels in patients with and without hepatopulmonary syndrome in a cohort of cirrhotic patients;

[0027] Figure 7 A scatter plot to verify the negative correlation between plasma vitamin A levels and P(Aa)O2 in the cohort;

[0028] Figure 8 To verify the ROC curve of plasma vitamin A levels in the cohort. Detailed Implementation

[0029] This invention provides the application of a plasma biomarker as a detection target in the preparation of products for the diagnosis or auxiliary diagnosis of hepatopulmonary syndrome in patients with cirrhosis, wherein the plasma biomarker includes vitamin A.

[0030] In one implementation, the plasma marker is vitamin A.

[0031] As one implementation method, the diagnosis includes early diagnosis. Early stage of hepatopulmonary syndrome in patients with cirrhosis: PaO2 (mmHg) ≥ 80 mmHg, mild hypoxia, often asymptomatic, elevated alveolar-arterial oxygen partial pressure difference (P(Aa)O2), highly reversible, good prognosis, often missed. Mid-stage of hepatopulmonary syndrome in patients with cirrhosis: PaO2 (mmHg) = 60–79 mmHg, exertional dyspnea, some patients present with mild cyanosis, usually indicating significant pulmonary vasodilation. Late stage of hepatopulmonary syndrome in patients with cirrhosis: PaO2 (mmHg) = 50–59 mmHg, significant hypoxia, cyanosis, clubbing of fingers, decreased exercise tolerance; usually requires oxygen therapy, poor prognosis, affects liver transplant priority. Very severe hepatopulmonary syndrome in patients with cirrhosis: PaO2 (mmHg) < 50 mmHg, severe hypoxia at rest, extreme fatigue or respiratory failure; associated with high liver transplant risk and increased postoperative mortality.

[0032] The present invention also provides the application of a test reagent for measuring the amount of vitamin A in a sample in products for determining the risk of hepatopulmonary syndrome in patients with cirrhosis.

[0033] As one implementation, the detection reagent for measuring the amount of vitamin A in a sample includes a high-performance liquid chromatography-mass spectrometry (LC-MS / MS) detection reagent.

[0034] This invention uses LC-MS / MS to detect plasma vitamin A levels in patients with cirrhosis (including those with HPS and those without HPS). When the levels are below a preset threshold, it indicates a high risk of hepatopulmonary syndrome (HPS). This allows for the diagnosis or auxiliary diagnosis of hepatopulmonary syndrome in subjects with cirrhosis, i.e., determining whether a patient with cirrhosis has developed hepatopulmonary syndrome. This method is simple and easy to implement, the diagnostic process is safe and effective, it is easily accepted by patients, the diagnostic criteria are standardized, and it is less affected by subjective factors.

[0035] In one embodiment, the sample includes blood, serum, or plasma from a patient with cirrhosis.

[0036] This invention also provides the application of vitamin A as a detection target in the preparation of products for guiding treatment and / or predicting prognosis of hepatopulmonary syndrome in patients with cirrhosis.

[0037] In one implementation, the product includes reagents, kits, systems, or devices.

[0038] The present invention also provides a kit for determining the risk of hepatopulmonary syndrome in patients with cirrhosis, comprising a detection reagent for measuring the amount of vitamin A in a sample and a threshold reference description; the threshold reference description includes a condition indicating a high risk of hepatopulmonary syndrome when the level is below a preset threshold.

[0039] In one embodiment, the sample includes blood, serum, or plasma from a patient with cirrhosis.

[0040] As one implementation method, when the detection reagent is a high-performance liquid chromatography-mass spectrometry (HPLC-MS) reagent, the threshold is 0.1750 mg / L. Patients with cirrhosis whose plasma samples have a vitamin A level ≤0.1750 mg / L are considered to be at high risk for hepatopulmonary syndrome.

[0041] In one embodiment, the kit further includes at least one of a standard, an internal standard, an extraction reagent, and a detection procedure description; the extraction reagent includes at least one of methanol, pure water, and formic acid.

[0042] The present invention also provides a system for determining the risk of hepatopulmonary syndrome in patients with cirrhosis, including a detection module, an input module, an analysis module, and an output module;

[0043] The detection module is used to measure the amount of vitamin A in the sample;

[0044] The input module is used to input the numerical value of the amount of vitamin A in the sample;

[0045] The analysis module has a preset threshold. By comparing the amount of vitamin A in the sample with the threshold, it indicates the risk of hepatopulmonary syndrome. When the amount is below the threshold, it indicates a high risk of hepatopulmonary syndrome.

[0046] The output module is used to output the risk of hepatopulmonary syndrome in patients with cirrhosis.

[0047] In one embodiment, the sample includes blood, serum, or plasma from a patient with cirrhosis.

[0048] As one implementation method, when the detection reagent is a high-performance liquid chromatography-mass spectrometry (HPLC-MS) reagent, the threshold is 0.1750 mg / L. Patients with cirrhosis whose plasma samples have a vitamin A level ≤0.1750 mg / L are considered to be at high risk for hepatopulmonary syndrome.

[0049] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes the application of a plasma biomarker provided by the present invention as a detection target in the preparation of products for diagnosing or assisting in the diagnosis of hepatopulmonary syndrome in patients with cirrhosis. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0050] Example 1

[0051] Discovery queue diagnostic methods

[0052] 1) Sample Collection: This study was approved by the Ethics Committee of Beijing You'an Hospital (LL-2022-141-K) and conducted in accordance with relevant guidelines and regulations. After obtaining written informed consent from all participants, from April to December 2023, peripheral venous blood (3-5 ml) was collected from 220 patients with cirrhosis (180 without hepatopulmonary syndrome and 40 with hepatopulmonary syndrome) at the Department of Liver Transplantation and Respiratory and Critical Care Medicine, Beijing You'an Hospital, meeting the inclusion criteria. The blood was placed in EDTA anticoagulant tubes and centrifuged (3000 rpm, 10 min) to separate plasma. This study was registered in the ClinicalTrials.gov database under the identifier NCT 05932927.

[0053] 2) Vitamin A detection: Mass spectrometry (LC-MS / MS)

[0054] Testing steps:

[0055] a. Sample pretreatment

[0056] Transfer 50 μl of the sample to be tested into a 96-well plate, add 250 μl of methanol (containing vitamin A acetate internal standard), vortex at 1000 rpm for 10 min; centrifuge at 4000 rpm for 10 min, and take the supernatant for testing.

[0057] b. Pretreatment of standard curves and quality control samples

[0058] Measure 250 μl of ultrapure water into the standard curve and quality control reagent bottle at room temperature, vortex mix for 5 min until the lyophilized powder is completely dissolved, and then set aside for use (if there is powder on the stopper, please stopper and invert several times until the lyophilized powder dissolves). Bottles should not be mixed.

[0059] c. Instruments used

[0060] Detection system: SCIEX Triple Quad 4500MD liquid chromatography-tandem mass spectrometry detection system;

[0061] Chromatographic column: DISIGNS column-004 (50mm×2.1mm) (manufacturer: Diagnostics).

[0062] The laboratory prepared its own quality control data, and the batch number of the quality control product is: RQ2408002.

[0063] The laboratory quality control data for Vitamin A are shown in Table 1, and the quality control chart is shown below. Figure 1 .

[0064] Table 1. Laboratory quality control data for Vitamin A

[0065]

[0066] Standard curve:

[0067] Plotting compound concentrations on the x-axis and the peak area ratio of the compound to the internal standard on the y-axis, a weighted least squares regression (weight 1 / X) was performed to obtain a linear regression equation. The correlation coefficient R > 0.99 for each compound's regression equation. The linear regression equation is: y = 0.0114x - 0.0268 (r = 0.9997). The standard curve results are shown below. Figure 2 As shown.

[0068] 3) Threshold determination: A plasma vitamin A concentration ≤0.1750 mg / L (LC-MS / MS method) is diagnosed as HPS positive.

[0069] 4) Data analysis and statistical methods

[0070] Continuous variables in a normally distributed system were expressed as mean ± standard deviation (SD), and t-tests were used to compare differences between the two groups. Continuous variables in a non-normally distributed system were expressed as median and interquartile range (IQR, 25th–75th percentile), and Mann-Whitney U tests were used to compare differences between the two groups. Pearson correlation analysis was used for normally distributed variables, and Spearman correlation analysis was used for non-normally distributed and categorical variables. Receiver operating characteristic (ROC) curves were plotted, and the area under the curve [AUC (with 95% confidence interval CI)] was used to assess the diagnostic value of plasma vitamin A levels in patients with hepatopulmonary syndrome due to cirrhosis. A two-sided p-value <0.05 was considered statistically significant. All analyses were performed using SPSS (version 20.0, SPSS, Chicago, IL, USA) and GraphPadPrism 9.

[0071] Figure 3 The graph shows the comparison of plasma vitamin A levels in patients with and without hepatopulmonary syndrome (HPS) in the cohort. It can be seen that the vitamin A level in the HPS group was significantly lower than that in the non-HPS group.

[0072] Figure 4 A scatter plot was used to identify the negative correlation between plasma vitamin A levels and P(Aa)O2 in the cohort; it can be seen that there is a significant negative correlation between plasma vitamin A (VitA) levels and alveolar-arterial oxygen partial pressure difference.

[0073] Figure 5 The ROC curve of plasma vitamin A levels in the cohort was used to identify HPS. It can be seen that the area under the ROC curve for plasma vitamin A levels in identifying HPS is 0.7468 (95% CI: 0.6097–0.8840, p = 0.0019), suggesting that it has moderate discriminative ability in predicting HPS.

[0074] Table 2 shows the receiver operating characteristic (ROC) curves for patients with cirrhosis and hepatopulmonary syndrome in the cohort.

[0075]

[0076] Table 2 shows the ROC curve analysis results, indicating that plasma vitamin A concentration has certain diagnostic value in predicting hepatopulmonary syndrome (HPS) in patients with cirrhosis. When the cut-off value is <0.1750 mg / L, the sensitivity is 92.5% (95% CI: 80.14–97.42), the specificity is 48.33% (95% CI: 41.14–55.59), the Youden's index is 0.4083, and the area under the curve (AUC) is 0.7422 (95% CI: 0.6649–0.8196), which are statistically significant (p<0.0001).

[0077] Table 3 shows the univariate and multivariate logistic regression analyses of predictors of hepatopulmonary syndrome in cohorts of patients with cirrhosis.

[0078]

[0079]

[0080] OR: Ratio.

[0081] Table 3 shows the results of multivariate logistic regression analysis, which indicates that plasma vitamin A level is an independent predictor of hepatopulmonary syndrome in patients with cirrhosis (OR = 0.994, p = 0.004).

[0082] Example 2

[0083] 1. Collection and processing of validation queue samples

[0084] From January to April 2024, peripheral venous blood (3-5 ml) was collected from 95 patients with cirrhosis who met the inclusion criteria at the Department of Liver Transplantation and Respiratory and Critical Care Medicine, Beijing You'an Hospital. The blood was placed in EDTA anticoagulant tubes and centrifuged (3000 rpm, 10 min) to separate plasma.

[0085] 2. Detection of biomarkers

[0086] The vitamin A was detected using LC-MS / MS, and details can be found in the vitamin A detection method in Example 1.

[0087] 3. For data analysis and statistical methods, please refer to Example 1.

[0088] 4. Results

[0089] Figure 6 To verify the comparison of plasma vitamin A levels in patients with and without hepatopulmonary syndrome (HPS) in a cohort. Results showed that plasma vitamin A levels were significantly lower in the HPS group compared to the non-HPS group (p<0.01).

[0090] Figure 7 To verify the negative correlation between plasma vitamin A levels and P(Aa)O2 in the cohort, a scatter plot was created. The results showed that plasma vitamin A levels were significantly negatively correlated with P(Aa)O2 (r = -0.2245, p = 0.0287).

[0091] Figure 8 ROC curves of plasma vitamin A levels in the validation cohort were plotted. Results showed that plasma vitamin A had some diagnostic efficacy in identifying HPS in the validation cohort.

[0092] Table 4. Receiver Operating Characteristic (ROC) curves for patients with cirrhosis and hepatopulmonary syndrome in the validation cohort.

[0093]

[0094] Table 4 shows that plasma vitamin A levels also demonstrated good predictive ability in the validation cohort (AUC = 0.7468, p = 0.0019), supporting its feasibility as a potential diagnostic biomarker for HPS.

[0095] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of a plasma biomarker as a detection target in the preparation of products for diagnosing or assisting in the diagnosis of hepatopulmonary syndrome in patients with cirrhosis, characterized in that, The plasma markers include vitamin A.

2. The application according to claim 1, characterized in that, The plasma marker is vitamin A.

3. The application according to claim 1, characterized in that, The diagnosis includes early diagnosis.

4. Application of assay reagents for measuring the amount of vitamin A in a sample in products used to determine the risk of hepatopulmonary syndrome in patients with cirrhosis.

5. The application according to claim 4, characterized in that, The detection reagents used to measure the amount of vitamin A in a sample include high-performance liquid chromatography-mass spectrometry (HPLC-MS) reagents.

6. The application according to claim 4 or 5, characterized in that, The samples include blood, serum, or plasma from patients with cirrhosis.

7. Application of Vitamin A as a detection target in the preparation of products for guiding treatment and / or predicting prognosis of hepatopulmonary syndrome in patients with cirrhosis.

8. A kit for determining the risk of hepatopulmonary syndrome in patients with cirrhosis, characterized in that, It includes a detection reagent for measuring the amount of vitamin A in a sample and a threshold reference; the threshold reference includes a condition that indicates a high risk of hepatopulmonary syndrome when the level is below a preset threshold.

9. The reagent kit according to claim 8, characterized in that, When the detection reagent is a high-performance liquid chromatography-mass spectrometry (HPLC-MS) detection reagent, the threshold is 0.1750 mg / L.

10. A system for determining the risk of hepatopulmonary syndrome in patients with cirrhosis, characterized in that, It includes a detection module, an input module, an analysis module, and an output module; The detection module is used to measure the amount of vitamin A in the sample; The input module is used to input the numerical value of the amount of vitamin A in the sample; The analysis module has a preset threshold. By comparing the amount of vitamin A in the sample with the threshold, it indicates the risk of hepatopulmonary syndrome. When the amount is below the threshold, it indicates a high risk of hepatopulmonary syndrome. The output module is used to output the risk of hepatopulmonary syndrome in patients with cirrhosis.

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