Hepatocellular carcinoma tumor lipidomics detection method and used tooling

Abstract

The application discloses a hepatocellular carcinoma tumor lipidomics detection method and a tool used in the method. The detection method comprises the following steps: a, obtaining a HCC sample, the HCC sample is obtained from the tumor site of a patient; b, using a high-frequency electrotome device to apply high-frequency current on the HCC sample in a cutting mode to generate tissue aerosol for lipidomics analysis; c, using the Venturi effect to send the continuously generated tissue aerosol during cutting into a mass spectrometer for mass spectrometry analysis, and processing the analysis results to obtain the mass-to-charge ratio parameters of FA18:0, FA20:4, FA16:0, FA18:1, PC36:1, PE38:3, PE(18:0 / 20:4), PA(O-36:1), PC(32:1), PC32:0, PE34:0 and PC(16:0 / 18:1); in the step b, the cutting power of the high-frequency electrotome is 20-50 W. The application has the advantages that lipid metabolites reacting with hepatocellular carcinoma can be accurately detected, and the safety is relatively high during use.

Description

Technical Field

[0001] This invention belongs to the field of hepatocellular carcinoma tumor lipidomics, and particularly relates to a method for detecting hepatocellular carcinoma tumor lipidomics and the tooling used therein. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a primary malignant tumor of the liver in patients with chronic liver disease or cirrhosis, accounting for more than 90% of all liver cancers. As a global health challenge, the incidence of liver cancer has been rising in recent years. The World Health Organization (WHO) predicts that more than one million people will die from liver cancer by 2030. As the second leading cause of cancer death, liver cancer has the second-highest 5-year survival rate after pancreatic cancer. According to the clinical practice guidelines of the European Association for the Study of the Liver (EASL) and the European Organisation for Research and Treatment of Cancer (EORTC) regarding HCC management, resection, liver transplantation, local ablation, systemic therapy, and chemoembolization, transcatheter therapy is a routine treatment based on the Barcelona Clinic's liver cancer (BCLC) staging results. Among these therapies, radiotherapy is a first-line treatment option for patients with solitary tumors and well-preserved liver function.

[0003] Non-invasive criteria and histopathological examination are the main methods for clinical diagnosis of HCC. Non-invasive criteria are based on identifying typical HCC markers using precise imaging techniques: phase-multidetector computed tomography (CT) scans and dynamic contrast-enhanced magnetic resonance imaging (MRI). Pathological diagnosis of early-stage HCC with highly dysplastic nodules is based on recommended immunostaining of phosphatidylinositol proteoglycan 3 (GPC3), heat shock protein 70 (HSP70), and glutamine synthase / gene expression profiles. Histopathological examination (including biopsy and surgical specimen examination) is of great significance in the diagnosis of hepatocellular carcinoma. It is often performed before or during hepatectomy or liver transplantation to confirm and verify preoperative diagnostic results, improving the level of clinical diagnosis. Although histopathological examination is considered the gold standard for diagnosing HCC, the processing of tissue sections on glass slides is time-consuming and labor-intensive, and the results are highly dependent on clinical diagnostic outcomes, potentially leading to false positives.

[0004] The etiology of hepatitis C (HCC) is a complex, multi-step process. The occurrence of HCC is largely considered a consequence of hepatitis B or C virus (HBV or HCV) infection, cirrhosis, and non-alcoholic steatohepatitis (NAH). In addition to these major causes, lipid metabolism has been shown to be associated with HCC via the c-Myc / sterol regulatory element binding protein 1 (SREBP1) or aberrant CAV1 / SREBP1 pathways. Current research suggests that lipid accumulation can exacerbate tumor development by promoting the proliferative growth of tumor cells. Lipidomics is the identification and quantification of lipids, which play crucial roles in many physiological processes in cells or organisms. Lipids are biomolecules, including sterols, glycerides, and phospholipids, most of which are composed of fatty acids. Polyunsaturated fatty acids (PUFAs), such as linoleic acid and alpha-linolenic acid, are considered essential fatty acids that the human body cannot synthesize and must obtain from food. Besides energy storage and biomembrane construction, some PUFAs and their metabolites act as signaling molecules in many biological processes, including cancer, and are known as lipid mediators. Increasing evidence suggests that altered lipid metabolism in cancer leads to rapid cancer cell growth and tumor formation. Using high-performance liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS), Patterson et al. discovered that certain plasma lipid levels in patients, including fatty acids and phospholipids, are associated with alpha-fetoprotein and hepatocellular carcinoma (HCC). Numerous reports have demonstrated abnormal lipid metabolism in HCC using clinical samples or mouse models. These findings provide new insights into the diagnosis and treatment of HCC. Due to its high specificity and sensitivity, mass spectrometry (MS) has been widely used in metabolomics and lipidomics. HPLC-MS and GC-MS are two common MS techniques. These traditional methods typically require labor-intensive sample pretreatment and time-consuming chromatographic separation prior to mass spectrometry analysis, which has significantly hindered progress in this field.

[0005] To facilitate the analysis of complex tissue samples, Takatas et al. developed a novel atmospheric pressure ionization mass spectrometry technique, Rapid Evaporation Ionization Mass Spectrometry (REIMS), also known as the "iKnife," which can directly extract tissue aerosols generated during high-frequency electrosurgical dissection for mass spectrometry-based lipidomics analysis, saving time and effort. The obtained mass spectra are mainly composed of information-rich fatty acid and phospholipid metabolites in the tissue. REIMS is suitable for real-time high-throughput lipidomics analysis of complex tissue samples and has been successfully applied to the identification or recognition of various tumor tissues, including breast tumors, cervical tumors, and colorectal lesions. Recently, we established and validated a REIMS-based lipidomics method for the analysis and diagnosis of glioblastoma multiforme (GBM), the most common brain tumor. Compared with normal brain tissue, 12 lipid biomarkers were found among the 42 identified lipid metabolites accumulated in GBM tumors. If mass spectrometry can be used to examine hepatocellular carcinoma, it has the advantages of saving time and effort due to the characteristics of mass spectrometry. Moreover, the clinical diagnostic results are not highly dependent and will not produce false positives, so it can be a good substitute for histopathological examination.

[0006] In 2013, Balog et al. attempted to establish an MS spectral database of human tissues, including HCC tumor tissues from five patients, for the purpose of characterizing and diagnosing tumor tissues using multivariate statistical methods. However, the sample size was very limited and the study was inadequate. Lipid metabolites in liver tissue were not fully characterized, and it was unclear whether the presence of each lipid metabolite was associated with hepatocellular carcinoma. As a result, mass spectrometry cannot yet replace existing histopathological examinations to help diagnose hepatocellular carcinoma.

[0007] Therefore, a new lipidomics detection method for hepatocellular carcinoma is needed to accurately detect lipid metabolites that react with hepatocellular carcinoma. Summary of the Invention

[0008] The purpose of this invention is to provide a method and tooling for the detection of lipidomics in hepatocellular carcinoma. This invention has the advantages of accurately detecting lipid metabolites in hepatocellular carcinoma and high safety during use.

[0009] The technical solution of this invention: a method for detecting lipidomics in hepatocellular carcinoma tumors, comprising the following steps:

[0010] a. Obtain HCC samples, which are obtained from the tumor site of the patient submitting the sample;

[0011] b. Using a high-frequency electrosurgical unit in cutting mode, a high-frequency current is applied to the HCC sample to generate tissue aerosols for lipidomics analysis.

[0012] c. Using the Venturi effect, the tissue aerosol continuously generated during cutting was sent to a mass spectrometer for mass spectrometry analysis. The analysis results were processed to obtain the mass-to-charge ratios of FA18:0, FA20:4, FA16:0, FA18:1, PC36:1, PE38:3, PE(18:0 / 20:4), PA(O-36:1), PC(32:1), PC32:0, PE34:0, and PC(16:0 / 18:1).

[0013] In the aforementioned hepatocellular carcinoma tumor lipidomics detection method, in step b, the cutting power of the high-frequency electrosurgical unit is 20-50W.

[0014] In the aforementioned method for detecting hepatocellular carcinoma tumor lipidomics, in step b, the cutting power of the high-frequency electrosurgical unit is 30W.

[0015] In the aforementioned method for detecting hepatocellular carcinoma tumor lipidomics, during step c, when performing mass spectrometry analysis, an isopropanol solution containing 200 ng / mL leucine phosphatidylcholine standard is continuously injected into the REIMS interface of the mass spectrometer, and mixed with tissue aerosol using a syringe pump at a flow rate of 0.1 ml / min.

[0016] In the aforementioned method for detecting hepatocellular carcinoma tumor lipidomics, in step c, the mass spectrometer operates in negative ionization mode, and the Q-Tof mass analyzer is set to high sensitivity.

[0017] The fixture used for lipidomics detection of hepatocellular carcinoma includes a base with an open top, a pressure plate inside the base, a first positioning mechanism at the bottom of the base, a pressure plate on the inner side of the base, a second positioning mechanism between the pressure plate and the base, a clamping mechanism connected to the base on the upper side of the pressure plate, and spiral through holes on the pressure plate.

[0018] In the aforementioned tooling, the base is fixed on the worktable of the CNC system, and the first positioning mechanism includes a positioning protrusion fixed to the base and a positioning ring located on the outside of the base. The protrusion is close to the positioning ring, and the positioning ring is fixed to the worktable.

[0019] In the aforementioned tooling, the second positioning mechanism includes a positioning groove located on the outside of the pressure plate, and a guide rib fixed to the inner wall of the base is provided in the positioning groove.

[0020] In the aforementioned tooling, the clamping mechanism includes a pressure ring located on the upper side of the pressure plate. The lower end of the pressure ring abuts against the pressure plate, and the upper end of the pressure ring extends horizontally outward beyond the base to form a transition ring. The outer side of the transition ring extends downward to form a collar that is screwed onto the outer circumferential surface of the base.

[0021] The aforementioned tooling also includes an electric knife mounting bracket and a suction head mounting bracket, both of which are fixed to the moving end of the CNC system.

[0022] Compared with existing technologies, this invention, by limiting the cutting mode and power of the high-frequency electrosurgical unit, can accurately detect the mass-to-charge ratio parameters of 12 metabolites—FA18:0, FA20:4, FA16:0, FA18:1, PC36:1, PE38:3, PE(18:0 / 20:4), PA(O-36:1), PC(32:1), PC32:0, PE34:0, and PC(16:0 / 18:1)—on HCC samples using mass spectrometry. Through years of analysis and research by the applicant, the mass-to-charge ratio parameters of these 12 metabolites can reflect hepatocellular carcinoma, and the detection of these 12 metabolites can replace existing histopathological examinations to aid in the diagnosis of hepatocellular carcinoma. Furthermore, this invention also provides a fixture for cutting HCC samples. This fixture helps generate sufficient and uniformly stable tissue aerosols during high-frequency electrosurgical cutting of HCC samples, making the examination results more accurate. Moreover, through optimization of the fixture's structure and the materials of its components, the fixture also achieves high safety during use. Therefore, the present invention has the advantages of being able to accurately detect lipid metabolites that react to hepatocellular carcinoma and having high safety during use. Attached Figure Description

[0023] Figure 1 This is the front view of the tooling.

[0024] Figure 2 This is a top view of the tooling.

[0025] Figure 3 This is a top view of the pressure plate.

[0026] Figure 4 This is a schematic diagram showing the morphology of some specimens obtained in the comparative experimental case and the corresponding pathological microscopic examination results of the specimens.

[0027] Figure 5 This is a comparison of free fatty acids (m / z 255.2 and m / z 279.2) and phospholipids (m / z 742.5 and m / z 766.6) under two cutting modes in the comparative experimental examples.

[0028] Figure 6 This is a comparison of free fatty acids (m / z 255.2 and m / z 279.2) and phospholipids (m / z 742.5 and m / z 766.6) under different cutting powers in the comparative experiment.

[0029] Figure 7 These are the representative property spectra of the three groups of tissue specimens in the comparative experimental example.

[0030] Figure 8 This is the unsupervised PCA score plot from the comparative experimental example.

[0031] Figure 9 This is a supervised PCA score plot from the comparative experimental example.

[0032] Figure 10 This is a supervised OPLS-DA load diagram from the comparative experimental example.

[0033] Figure 11 This is the VIP plot from the comparative experimental example.

[0034] Figure 12 This is a box plot of the intensity of seven fatty acids in the comparative experiment.

[0035] Figure 13 This is a box plot of 10 important phospholipid metabolites verified by one-way ANOVA in a comparative experimental example.

[0036] Figure 14 This is a heatmap of the mass intensity of 43 identified lipid metabolites in the comparative experimental examples.

[0037] Figure 15 This is a diagram showing the tissue identification results of the OPLS-DA model used in the comparative experimental case.

[0038] The markings in the attached diagram are as follows: 1-base, 2-pressure plate, 3-through hole, 4-worktable, 5-positioning protrusion, 6-positioning ring, 7-positioning groove, 8-guide rib, 9-pressure ring, 10-adapter ring, 11-collar ring, 12-electric knife fixing bracket, 13-suction head fixing bracket. Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0040] Example 1. A method for detecting lipidomics in hepatocellular carcinoma tumors, comprising the following steps:

[0041] a. Obtain HCC samples. HCC samples are obtained from the tumor site of the patient submitting the sample.

[0042] b. Using a high-frequency electrosurgical device (SJ350B, EasternMagical, China) consisting of a power supply, a plate-shaped neutral electrode, and an electrosurgical blade, the cutting power was adjusted to 30W. In cutting mode, a high-frequency current was applied to the HCC sample. The electrosurgical blade handle was held to cut the HCC sample, generating tissue aerosols for lipidomics analysis.

[0043] c. Tissue aerosols continuously generated during dissection were extracted using the Venturi effect and transferred to a Waters quadrupole time-of-flight (Q-Tof) mass spectrometer (Xevo G2-XS, Wilmslow, UK). In this mass spectrometer, the traditional ESI (electrospray ionization) ion source was replaced by a unique REIMS interface for atmospheric pressure ionization and mass spectrometry analysis. Isopropanol solution (purchased from Merck, USA) containing 200 ng / mL leucine phosphatidylcholine standard (purchased from Sigma-Aldrich, USA) was continuously injected into the REIMS interface and mixed with the tissue aerosol at a flow rate of 0.1 ml / min using a syringe pump (Pump11 Elite, Harvard Apparatus, USA). The mass spectrometer was operated in negative ionization mode, and the Q-Tof mass analyzer was set to high sensitivity. The mass range covered was m / z 100-1000. The mass tank rate was set to 1 second per scan. In REIMS mass spectrometry analysis of tissue samples, three scans were acquired for each analysis, and each sample was analyzed three times.

[0044] All MS data obtained were acquired and processed using MassLynx V4.1 (Waters, UK). After background subtraction and lockout mass correction (leucine enkephalin, m / z = 554.2615), the summarized mass spectrometry data were processed using SIMCA (version 14.1, MKSUmetrics, Sweden), SPSSStatistic (version 19, IBM, USA), and MetaboAnalyst (free online version 5.0, Canada) to obtain the mass-to-charge ratio parameters of the following lipid metabolites: FA18:0, FA20:4, FA16:0, FA18:1, PC36:1, PE38:3, PE(18:0 / 20:4), PA(O-36:1), PC(32:1), PC32:0, PE34:0, and PC(16:0 / 18:1). The aforementioned lipid metabolites were well characterized and associated with hepatocellular carcinoma, and can serve as prognostic markers to replace histopathological examination in the diagnosis of hepatocellular carcinoma.

[0045] Comparative experimental case. Ethical approval was obtained from the Medical Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University (Reference No.: 2021-K-32-01). Informed consent was obtained from 12 HCC patients who underwent surgery in the Department of Hepatobiliary Surgery regarding the use of resected liver tissue specimens for research purposes. HCC tumor tissue, along with paired PT (less than 1 cm from the tumor site) and NCT (at least 2 cm from the tumor site), was separated and verified by routine histopathological examination in the pathology department. Figure 4 (As shown), the residual tissue specimens were stored at -80°C and completely thawed before REIMS mass spectrometry analysis.

[0046] A high-frequency electrosurgical unit (SJ350B, EasternMagical, China), consisting of a power supply, a plate-shaped neutral electrode, and an electrosurgical blade, was used to apply a high-frequency current to tissue specimens and generate tissue aerosols for lipidomics analysis. Tissue specimens were placed on the neutral plate electrode and cut with the electrosurgical blade in both cutting and coagulation modes to generate tissue aerosols. These aerosols were directly extracted using the Venturi effect and transferred to a Waters quadrupole time-of-flight (Q-Tof) mass spectrometer (Xevo G2-XS, Wilmslow, UK). The traditional ESI (electrospray ionization) source was replaced by a unique REIMS interface for atmospheric pressure ionization and mass spectrometry analysis. An isopropanol solution containing 200 ng / mL leucine phosphatidylcholine standard was continuously injected into the REIMS interface and mixed with the tissue aerosols using a syringe pump (Pump11Elite, Harvard Apparatus, USA) at a flow rate of 0.1 ml / min. The mass spectrometer was operated in negative ionization mode, and the Q-Tof mass analyzer was set to high sensitivity. The mass range covered was m / z 100-1000. The mass tank rate was set to 1 second per scan. In REIMS mass spectrometry analysis of tissue samples, three scans were acquired per analysis, and each sample was analyzed three times.

[0047] For REIMS mass spectrometry analysis, a high-frequency electrosurgical unit, also known as diathermy, was used. High-frequency current is applied to a small area on the tissue surface, creating a focusing effect that rapidly heats the tissue for cutting or coagulation. Therefore, the high-frequency electrosurgical unit has two modes: cutting mode and coagulation mode. The cutting mode uses a low-voltage continuous waveform, while the coagulation mode uses a high-voltage pulsed waveform. Both modes are widely used in surgery to rapidly evaporate water and lipids from small tissue sites. Initially, a small piece of NCL tissue was used to study and compare the two high-frequency electrosurgical modes. Figure 5 As shown, the average intensities of the four dominant representative peaks in the MS spectra of free fatty acids (m / z 255.2 and m / z 279.2) and phospholipids (m / z 742.5 and m / z 766.6) were calculated and presented. All four peaks showed higher intensities in cutting mode. This is because the heat is more concentrated in cutting mode, while the actual heat from cutting tissue is dispersed, and cogulation mode is insufficient to generate enough tissue aerosols for mass spectrometry analysis in coagulation mode. Therefore, cutting mode was selected and applied in subsequent REIMS mass spectrometry analyses.

[0048] Once the high-frequency electrosurgical operation mode is determined, the cutting power can be optimized. For example... Figure 6As shown, the intensities of the four representative lipid peaks increased significantly when the cleavage power increased from 10 W to 30 W, reaching their highest values ​​at 30 W. Subsequently, the intensities of the four representative lipid peaks decreased when the cleavage power increased from 30 W to 60 W. In short, higher cleavage power generates more tissue aerosols, including lipids and matrix, but excessive matrix can cause interference and reduce the signal intensity of lipids. Therefore, in subsequent REIMS mass spectrometry analysis of human liver tissue specimens, the cleavage power was set at 30 W.

[0049] For lipidomics studies, the REIMS method described above was used to analyze a total of 36 tissue specimens from three groups (12 HCCs, 12 PTs, and 12 NCTs). Figure 7 Representative spectra for each group are provided. Mass peaks were characterized based on their extract mass number and / or tandem MS (MS / MS) spectra. All characterized lipids are listed in Tables 1 and 2. Within the low mass range (m / z 200-400), based on their precise mass, are FA16:0 (palmitic acid, m / z 255.2), FA18:2 (linoleic acid, m / z 279.2), FA18:1 (oleic acid, m / z 281.2), FA18:0 (stearic acid, m / z 283.3), FA20:4 (arachidonic acid, m / z 303.2), FA20:0 (arachidic acid, m / z 311.3), FA22:7 (sepiacetic acid, m / z 325.2), FA22:6 (docosahexaenoic acid, m / z 327.2), and FA22:0 (betainic acid, m / z 339.3).

[0050] In the high-quality range (m / z 600-900), 34 ion peaks were preliminarily characterized as deprotonated [MH]-, demethylated [M-CH3]-, or deamination [M-NH4]- ions of phospholipids. Based on the fragment ions obtained from the collision-induced dissociation (CID) MS / MS spectra, the major ion peaks at m / z 697.5, 699.5, 742.5, 744.5, 766.5, 790.5, and 885.6 were identified as PE (16:0 / 18:2), PE (16:0 / 18:1), PC (16:0 / 18:2), PC (16:0 / 18:1), PE (18:0 / 20:4), PE (18:0 / 22:6), and PI (18:0 / 20:4), respectively. For example, the high-mass spectrum of HCC showed a rich ion peak at m / z 766.5383, which had a difference of 0.0004 Da (0.5 ppm) compared to the theoretical m / z of the [MH]– ion of PE (38:4) (C43H78NO8P). In the MS / MS spectrum of the parent ion m / z 766.5383, the two major fragment ions at m / z 283.3 (FA18:0) and m / z 303.3 (FA20:4) were identified as fatty acid chains of PE (18:0 / 20:4).

[0051] To simplify the analysis of the multivariate dataset, REIMS mass spectrometry data obtained from 12 HCC, 12 PT, and 12 NCT specimens were exported for dimensionality reduction using unsupervised principal component analysis (PCA), a classic statistical method used to highlight variation and identify biomarkers in a given sample dataset. Figure 8 The PCA score plot with two components is shown. Most data points from the three different sample groups are distributed within the 95% confidence interval (CI) and cluster in different regions. To establish an identification model for HCC tumor tissue, supervised orthogonal partial least squares discriminant analysis (OPLS-DA) was performed, and the score plot is shown below. Figure 9 As shown in the OPLS-DA score plot, most data points from HCC, PT, or NCT fall within the 95% CI range and are well separated from the other two groups.

[0052] To identify the lipid metabolites responsible for group separation, the variable importance of the OPLS-DA loading map and projection map was determined. Figure 10 and 11The loading plots show the relationship between metabolite variables and the model for each group. Variables closer to the predicted group model point typically play a major role in distinguishing that group from others. It is readily apparent that variables m / z 325.22, 311.29, and 339.32 are helpful in identifying PT tissues, while variables m / z 327.23, 762.51, and 790.55 are helpful in identifying NCT tissues. The VIP score plots summarize the importance of the variables in relation to group differences. Generally, a VIP value greater than 1 indicates an important variable. Therefore, the 24 lipid metabolites detected in the loading plots (including these six identified contributing metabolites) were found to be important variables in the discriminant analysis. One-way ANOVA was performed as a statistical method to test for differences in means among three or more groups to validate the identified lipid metabolites with VIP values ​​greater than 1. Apart from the fact that most of the five metabolites (PEO-38:5, PEO-38:6, PEO-36:5, PC34:0, PE34:0) had relatively low abundance in the three groups, the 17 lipid metabolites, including seven fatty acids and ten phospholipids, showed significant differences among the HCC, NCT and PT groups in the ANOVA analysis (p<0.05).

[0053] The box plots of the strength of the above 7 fatty acids are as follows: Figure 12 As shown. Compared to HCC and NCT, FA22:0, FA22:7, and FA20:0 exhibit higher strength in PT, while the most common fatty acids FA18:0, FA20:4, FA16:0, and FA18:1 show lower strength in PT. Therefore, PT is a different tissue from HCC and NCT and cannot be simply considered a cross between HCC and NCT. Regarding FA22:0, FA22:7, and FA20:0, HCC and NCT show similar mass strength. However, for the most common fatty acids FA18:0, FA20:4, FA16:0, and FA18:1, HCC shows higher strength compared to NCT.

[0054] As a precursor to many lipid mediators in inflammation, n-6 fatty acid FA20:4 can be converted into prostaglandins (PGs) and leukotrienes (LTs), which are associated with a number of diseases, including the enhancement of chronic inflammation and tumor extension. Based on previous studies, higher levels of FA20:4, as well as FA16:0 and FA18:1 in the serum or plasma of HCC patients have been identified as potential biomarkers compared to control subjects. In addition to fat accumulation, non-alcoholic fatty liver disease (NAFLD) is generally considered a risk factor for HCC development. In a study on changes in lipid metabolism during NAFLD progression, high proportions of FA18:0, FA16:0, and FA18:1 were found to be significantly associated with advanced liver fibrosis, a major predictor of NAFLD. These results suggest that FA16:0 and FA18:0 have significant cytotoxic potential in the progression of hepatocellular injury, death, and fibrosis. As the most abundant fatty acids in liver triglycerides in healthy individuals and NAFLD patients, FA16:0 and FA18:1 have been shown to cause detrimental effects of excessive hepatic fat accumulation when a mixture of saturated and unsaturated FA16:0 and FA18:1 is incubated with human hepatocytes and HepG2 cells. Meanwhile, higher concentrations of monounsaturated fatty acid 18:1 in human feces have also been identified as a potential new screening biomarker for male colorectal cancer patients.

[0055] Figure 13Box plots of the mass intensity of 10 phospholipids, validated by one-way ANOVA, are presented. Compared to NCT, PE38:6, PE(18:0 / 22:6), and PC(16:0 / 18:2), which showed lower mass intensity in HCC, PC36:1, PE38:3, PE(18:0 / 20:4), PA(O-36:1), PC(32:1), and in PCHCC 32:0, PE34:0, and PC(16:0 / 18:1) showed higher mass intensity. In previous studies, downregulation of PE38:6, PE40:6, and PC34:2 and upregulation of PC36:1, PE38:3, PC32:1, PC32:0, and PC34 were also detected in HCC tissue compared to healthy liver tissue. Therefore, these results are largely consistent with the current study. The upregulation of PE (18:0 / 20:4), a phospholipid composed of FA18:0 and 20:4, is consistent with the results regarding the composition of free fatty acids in HCC. Except for PE (18:0 / 20:4), the results are consistent with the conclusion that saturated and monounsaturated fatty acids accumulate in tumors, given the limited ability of mammalian cells to synthesize polyunsaturated fatty acids. Compared to PT, except for PC36:1, PE38:3, PE (18:0 / 20:4), and PC32:1, the mean mass intensity of validated phospholipid biomarkers in HCC showed similar mass intensity. As the first and second most abundant phospholipids in mammalian cell membranes, particularly eukaryotic cell membranes, the abundance and distribution of PC and PE species can significantly influence liver health. Comprehensive lipidomics studies have identified a large number of PC and PE biomarkers in HCC tissues.

[0056] Mass intensity visualization heatmap of 43 identified lipid metabolites, as shown below Figure 14 As shown in the dendrogram (dendrogram) used for hierarchical clustering analysis (HCA), the lipid composition of HCC was found to be closer to PT than that of NCT. In the heatmap, color changes indicate an increase in the mass intensity of the corresponding metabolite. Most lipid metabolites showed different intensities between HCC and NCT.

[0057] The tissue identification results of the OPLS-DA model used are as follows: Figure 15 As shown, the sensitivity of real-time HCC identification technology, calculated using the ratio of true positives to the sum of true positives and false negatives, is 100%. The specificity, calculated using the ratio of true negatives to the sum of false positives and true negatives, is 90.5%. The overall accuracy for HCC tissue identification is 88.89%. Furthermore, no lipid metabolites are currently used as prognostic biomarkers for HCC. Therefore, real-time HCC identification based on REIMS spectral data may be a promising technology for future tumor identification.

[0058] To better investigate lipid composition changes in HCC tumors and their paired PT and NCT samples, atmospheric pressure ionization mass spectrometry (REIMS) was optimized for lipidomics studies. Surgical high-frequency electrosurgical units were used to generate sufficient tissue aerosols for real-time ex vivo lipidomics analysis; therefore, the operating mode and cutting power of the high-frequency electrosurgical unit were investigated and optimized to obtain abundant lipid metabolite peaks in the mass spectrometer. A total of 12 HCC tumor samples, along with paired PT and NCT samples, were analyzed using the described REIMS method. Nine free fatty acids and 34 phospholipid metabolites were identified based on extract mass numbers and / or MS / MS spectra. With the aid of statistical methods, it was found that seven free fatty acids and ten phospholipids exhibited different distributions in HCC and their paired PT and NCT samples. Among these metabolites, free fatty acids FA18:0, FA20:4, FA16:0, and FA18:1, and phospholipids PC36:1, PE38:3, PE(18:0 / 20:4), PA(O-36:1), PC(32:1), PC32:0, PE34:0, and PC(16:0 / 18:1) showed sufficiently high signal intensities in HCC tissue samples to serve as prognostic biomarkers. PT samples showed different lipid compositions compared to HCC and NCT samples. As a real-time identification technique for HCC tumors, the sensitivity and specificity of the REI MS method were calculated to be 100% and 90.5%, respectively.

[0059] Comparative experiments have demonstrated that identifying FA18:0, FA20:4, FA16:0, FA18:1, PC36:1, PE38:3, PE(18:0 / 20:4), PA(O-36:1), PC(32:1), PC32:0, PE34:0, and PC(16:0 / 18:1) in HCC samples as prognostic markers can accurately determine the lesion status of submitted samples and can replace histopathological examination of hepatocellular carcinoma. To more accurately and sensitively detect these prognostic markers, the main improvement made to the existing mass spectrometry method in Example 1 is the limitation of the cutting mode and cutting power of the high-frequency electrosurgical unit.

[0060]

[0061]

[0062]

[0063] Table 1

[0064]

[0065]

[0066] Table 2

[0067] Example 2. Extensive experiments revealed that due to the small volume of HCC samples, coupled with unstable cutting speed and uneven cutting thickness during manual operation, the rate of tissue aerosol formation during the cutting process is unstable. Especially after actual use in hospitals, the samples submitted for testing are generally obtained by puncture needles, which are very small in diameter and elongated. In step b of Example 1, these samples are difficult to fix and continuously and stably cut, and tissue aerosol cannot be generated at a uniform speed and stably. This increases the error fluctuation of the final detection results. Therefore, a tooling for hepatocellular carcinoma tumor lipidomics detection is needed.

[0068] like Figures 1 to 3 As shown, the fixture includes a base 1 with an open top, a pressure plate 2 inside the base 1, a first positioning mechanism at the bottom of the base 1, a pressure plate 2 on the inner side of the base 1, a second positioning mechanism between the pressure plate 2 and the base 1, a clamping mechanism connected to the base 1 on the upper side of the pressure plate 2, and a spiral through hole 3 on the pressure plate 2. The width of the through hole 3 is about 0.5 mm, and the distance between the inner and outer rings of the through hole 3 is 2 mm, which is slightly larger than the thickness of the electric knife tip (generally 0.2 mm).

[0069] The base 1 is fixed to the worktable 4 of the CNC system. The first positioning mechanism includes a positioning protrusion 5 fixed to the base 1 and a positioning ring 6 located on the outside of the base 1. The protrusion is close to the positioning ring 6, and the positioning ring 6 is fixed to the worktable 4. The base 1 can be removed from the worktable 4 for easy cleaning.

[0070] The second positioning mechanism includes a positioning groove 7 located on the outside of the pressure plate 2, and a guide rib 8 fixed to the inner side wall of the base 1 is provided in the positioning groove 7. The function of the positioning groove 7 and the guide rib 8 is to determine the position of the two ends of the through hole 3 on the pressure plate 2 on the worktable 4 after the pressure plate 2 is placed into the base 1.

[0071] The clamping mechanism includes a clamping ring 9 located on the upper side of the clamping plate 2. The lower end of the clamping ring 9 abuts against the clamping plate 2. The upper end of the clamping ring 9 extends horizontally outward beyond the base 1 to form a transition ring 10. The outer side of the transition ring 10 extends downward to form a collar 11 that is screwed to the outer circumferential surface of the base 1.

[0072] It also includes an electric knife mounting bracket 12 and a suction head mounting bracket 13, both of which are fixed to the moving end of the CNC system.

[0073] Since the electrosurgical knife's movement trajectory does not require very high precision, the CNC system can use an FTS40XYZ-L modular worktable plus a corresponding controller, keeping the cost within 5000 yuan. The controller drives the electrosurgical knife's cutting head to rise and fall, and to move horizontally along the shape of the through-hole 3.

[0074] The base 1 is made of aluminum alloy and connects to the neutral electrode of the high-frequency electrosurgical unit. The pressure plate 2 is made of high-strength, wear-resistant plastic, such as polysulfone. The worktable 4 is plate-shaped and made of plastic. The pressure ring 9, adapter ring 10, and collar 11 are an integral structure made of plastic. The combination of these materials improves safety during use.

[0075] How to use the tooling: Vertically fix the electric knife of the high-frequency electric knife device on the electric knife fixing bracket 12, with the electric knife tip facing downward.

[0076] On the syringe pump of an existing mass spectrometer, there is a branch tube on the side wall of the tubing connected to the REIMS interface. The end of the branch tube is a pipette tip. When the syringe pump pumps isopropanol solution into the REIMS interface, a negative pressure is formed in the branch tube due to the Venturi effect. Tissue aerosols then enter the tubing through the pipette tip and the branch tube, mixing with the isopropanol solution. The pipette tip is fixed on the pipette tip holder 13, facing and close to the tip of the electrosurgical cutter. This allows the aerosol fumes generated during electrosurgical cutting to largely enter the pipette tip, avoiding loss and reducing fluctuations in detection errors. The electrosurgical cutter holder 12 and the pipette tip holder 13 only serve a fixing and transition function; their structures are not limited, and those skilled in the art should be able to design them according to their functions, so they will not be described in detail.

[0077] Rotate the collar 11 to remove the pressure plate 2. Place the obtained HCC sample into the base 1, align the positioning groove 7 with the guide rib 8, and place the pressure plate 2 back into the base 1. Rotate the collar 11 to make the pressure ring 9 press the pressure plate 2 tightly until the HCC sample is squeezed out of the through hole 3. Use a scraper to remove the portion of the HCC sample that exceeds the top surface of the pressure plate 2. If there is an unfilled portion in the through hole 3, take a portion of the hanging HCC sample to fill it. For some severely diseased and hardened HCC samples, in order to facilitate smooth extrusion, they can be broken into a mud-like state before being placed into the base 1.

[0078] Then, the outer helical end of through-hole 3 is used as the cutting start point, and the inner helical end of through-hole 3 is used as the cutting end point. The CNC system moves the electrosurgical unit above the cutting start point, lowers the height, inserts the electrosurgical tip into through-hole 3, and then cuts at a constant speed along the helical direction of through-hole 3 to the cutting end point. The electrosurgical unit is then raised to complete the cutting operation. During the cutting start and end phases, the injection pump remains operational, allowing tissue aerosols to be drawn into the mass spectrometer.

[0079] Compared to Example 1, Example 2 extruded a smaller HCC sample into a spiral shape, approximating a mosquito coil, allowing for a longer cutting path and uniform thickness. A CNC system was used to maintain a constant speed for the electrosurgical unit, generating sufficient and stable tissue aerosol, thus reducing error fluctuations. Furthermore, further structural improvements to the tooling prevented accidental collisions between the electrosurgical unit and the tooling, enhancing safety.

Claims

1. A method for detecting lipidomics in hepatocellular carcinoma tumors, characterized in that: Includes the following steps, a. Obtain HCC samples, which are obtained from the tumor site of the patient submitting the sample; b. Using a high-frequency electrosurgical unit in cutting mode, a high-frequency current is applied to the HCC sample to generate tissue aerosols for lipidomics analysis. c. Using the Venturi effect, the tissue aerosol continuously generated during cutting was sent to a mass spectrometer for mass spectrometry analysis. The analysis results were then processed to obtain the mass-to-charge ratio parameters of FA18:0, FA20:4, FA16:0, FA18:1, PC36:1, PE38:3, PE(18:0 / 20:4), PA(O-36:1), PC(32:1), PC32:0, PE34:0, and PC(16:0 / 18:1). In step b, the cutting power of the high-frequency electric knife is 30W. The tooling used for testing includes a base (1) with an open top, a pressure plate (2) inside the base (1), a first positioning mechanism at the bottom of the base (1), a pressure plate (2) on the inner side of the base (1), a second positioning mechanism between the pressure plate (2) and the base (1), a clamping mechanism connected to the base (1) on the upper side of the pressure plate (2), and a spiral through hole (3) on the pressure plate (2). In step c, during mass spectrometry analysis, an isopropanol solution containing 200 ng / mL leucine phosphatidylcholine standard is continuously injected into the REIMS interface of the mass spectrometer and mixed with tissue aerosol at a flow rate of 0.1 ml / min using a syringe pump. The mass spectrometer operates in negative ionization mode, and the Q-Tof mass analyzer is set to high sensitivity.

2. The method for detecting hepatocellular carcinoma tumor lipidomics according to claim 1, characterized in that: The base (1) is fixed on the worktable (4) of the CNC system. The first positioning mechanism includes a positioning protrusion (5) fixed to the base (1) and a positioning ring (6) located on the outside of the base (1). The protrusion is close to the positioning ring (6), and the positioning ring (6) is fixed to the worktable (4).

3. The method for detecting hepatocellular carcinoma tumor lipidomics according to claim 1, characterized in that: The second positioning mechanism includes a positioning groove (7) located outside the pressure plate (2), and a guide rib (8) fixed to the inner wall of the base (1) is provided in the positioning groove (7).

4. The method for detecting hepatocellular carcinoma tumor lipidomics according to claim 3, characterized in that: The clamping mechanism includes a clamping ring (9) located on the upper side of the clamping plate (2). The lower end of the clamping ring (9) abuts against the clamping plate (2). The upper end of the clamping ring (9) extends horizontally outward beyond the base (1) to form a transition ring (10). The outer side of the transition ring (10) extends downward to form a collar (11) screwed to the outer circumference of the base (1).

5. The method for detecting hepatocellular carcinoma tumor lipidomics according to claim 1, characterized in that: It also includes an electric knife mounting bracket (12) and a suction head mounting bracket (13), both of which are fixed to the moving end of the CNC system.

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