Detection reagent of lipid biomarker, application and screening method

Abstract

The invention relates to a detection reagent for lipid biomarkers, application and a screening method. The detection reagent comprises a lipid extraction protein precipitation reagent, a redissolving reagent and a chromatographic separation reagent, the lipid extraction protein precipitation reagent is a mixture of methyl tert-butyl ether and methanol in a ratio of 5: 1, and the redissolving reagent is methanol and dichloromethane in a ratio of 1: 1. Injecting the detection reagent into a detection device comprising a liquid chromatography-tandem mass spectrometer, and detecting through liquid chromatography-tandem mass spectrometry to obtain the content of each lipid in the lipid biomarker. The FH neonatal marker is represented through lipidomics characteristics for the first time, the lipid biomarker is screened out and used for evaluating the disease severity of the FH neonatal, and the obtained lipid biomarker has high value for early screening and timely intervention of FH. The lipid biomarker obtained through screening has good biological stability and representativeness, the biological activity of the lipid biomarker can be guaranteed after long-time storage, and the reliability of a detection result can be guaranteed.

Description

TECHNICAL FIELD

[0001] The technical field of the lipid biomarker of the present application, in particular relates to a detection reagent and application and screening method of lipid biomarker of familial hypercholesterolemia neonatal dried blood spot. BACKGROUND

[0002] Familial hypercholesterolemia (FH) is an autosomal dominant genetic disease, which leads to elevated plasma low-density lipoprotein cholesterol (LDL-C) levels in patients since childhood, and can lead to early events of atherosclerotic cardiovascular disease (ASCVD), such as myocardial infarction or stroke. The prevalence of heterozygous FH (hereinafter referred to as FH) in the general population is 1:313, in patients with ischemic heart disease is 1:31, and in patients with early-onset ischemic heart disease is 1:15. The prevalence of familial hypercholesterolemia (FH) in women and men is similar. Homozygous FH (HoFH) refers to the presence of double allelic pathogenic mutations in the FH gene, and its incidence is relatively low, estimated at 1 / 250,000-360,000. If not adequately treated with lipid-lowering therapy (LLT), heterozygous familial hypercholesterolemia patients have a 20-fold risk of ASCVD if their LDL-C levels exceed 5.5 mmol / l, compared with non-diseased adults with LDL-C levels below 3.5 mmol / l. For more severe homozygous familial hypercholesterolemia, some children have cardiovascular disease before the age of 10 without receiving lipid-lowering therapy. In untreated FH patients, 50% of men have atherosclerotic cardiovascular events (such as myocardial infarction, stroke) before the age of 50, and 30% of women have atherosclerotic cardiovascular events before the age of 60. It has been reported that the average death age of FH patients is 62 years old, which is 15 to 21 years earlier than the general population.

[0003] Early diagnosis of familial hypercholesterolemia is crucial, and through early diagnosis and timely initiation of lipid-lowering drug therapy (safe, effective, economical and accessible therapy), the risk of accelerated progression of atherosclerosis can be significantly improved. However, although FH is very common, the diagnosis and treatment of FH is severely insufficient in most countries. Only about 2% of FH patients can be effectively diagnosed before the age of 18, and most FH patients are diagnosed after the onset of cardiovascular events in adulthood (around the age of 44), and often do not receive timely and good treatment, indicating a lack of early screening programs worldwide.

[0004] The American Academy of Pediatrics recommends that all children aged 9 to 11 years undergo high cholesterol screening. Children under 2 years of age should be screened if there is a significant family history of high cholesterol or heart disease. Cost-effectiveness studies of familial hypercholesterolemia (FH) screening and timely statin therapy in children (average age 10 years) have shown that screening and treatment in children is both beneficial to health and cost-effective compared to not conducting cascade screening. However, FH screening has low adoption rates and cannot significantly improve FH detection rates due to the need for proactive medical visits, venous blood draws at ages with poor child compliance, and follow-up, as well as a worrying gap in public awareness and knowledge about FH care in children. Therefore, despite the widespread availability and effectiveness of LDL-C testing and treatment, only 10% of FH patients are currently aware of their condition, resulting in less than ideal treatment outcomes. Thus, more suitable methods for early FH screening are needed to prevent familial hypercholesterolemia patients from developing atherosclerotic cardiovascular disease (ASCVD) in later life.

[0005] Newborn screening (NBS) is a successful public health program that aims to identify genetic, endocrine, and metabolic disorders that may affect the long-term health and survival of newborns by detecting these disorders in heel prick blood samples. Early detection, diagnosis, and intervention of serious diseases help prevent death and improve the quality of life for children with neonatal diseases. Currently, NBS primarily uses methods such as tandem mass spectrometry (MS-MS) to detect biochemical markers for various diseases. These methods are high-throughput and low-cost, but are limited by the number of biochemical markers and the types of diseases that can be detected; for example, fibrillary hemorrhage (FH) has not been included. If FH could be included in the newborn screening program, it could be detected in the neonatal period, which would not only directly benefit the affected child but also alert adult relatives who are unaware of the condition. However, the detection of traditional lipid markers (TC / LDL-C / apoB) in neonatal dried blood spots (DBS) is difficult, requiring complex preprocessing, making large-scale sample collection impossible and costly. Furthermore, there are currently no studies reporting FH-related lipid biochemical markers that can be included in NBS using MS-MS detection. With the rapid development of testing technology, some regions both domestically and internationally have begun to experiment with genome screening based on next-generation sequencing (NGS) technology. However, considering various factors such as public awareness, family income, testing costs, and the difficulty of operation and interpretation, widespread adoption remains a significant challenge. Furthermore, the cost of adding a single LDLR gene test is quite high, making it difficult to include in public health programs. Therefore, identifying biomarkers that can be effectively used for newborn fetal heart disease (FH) screening is the technical problem this invention aims to solve.

[0006] Currently known causes of febrile hemorrhage (FH) include mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein B (Apo B, APOB), or proprotein convertase subtilisin 9 (PCSK9) genes. Among these, FH cases caused by LDLR gene mutations account for the majority, approximately 85%–90%, with over 1600 LDLR mutation sites identified. We collected dried blood spot samples from 127 newborns with FH who were diagnosed with pathogenic LDLR mutations through newborn genetic screening and diagnosed with FH between March 2022 and June 2024 at our center, and 30 healthy control newborns. There were no significant differences in gestational age, birth weight, or delivery method between the two groups. The purpose of this invention is to provide a screening and application of lipid biomarkers for dried blood spots in newborns with FH, offering a new approach for early clinical screening of FH. Summary of the Invention

[0007] This invention provides a detection reagent, application, and screening method for lipid biomarkers. For the first time, lipidomics characteristics are used to characterize biomarkers in newborns with febrile hemorrhage (FH), and lipid biomarkers are screened to assess the severity of FH in newborns. The obtained lipid biomarkers enable early screening and timely intervention for FH.

[0008] A reagent for detecting lipid biomarkers, comprising a lipid extraction protein precipitation reagent, a reconstitution reagent, and a chromatographic separation reagent. The lipid extraction protein precipitation reagent is a 5:1 mixture of methyl tert-butyl ether and methanol; the reconstitution reagent is a 1:1 mixture of methanol and dichloromethane; the chromatographic separation reagent comprises a liquid chromatography phase A of 40% water and 60% acetonitrile solution containing 10 mmol / L ammonium formate; and a phase B of 10% acetonitrile and 90% isopropanol solution, wherein 50 mL of 10 mmol / L ammonium formate aqueous solution is added per 1000 mL.

[0009] Furthermore, the lipid biomarker is selected from one or any combination of PC (13:1 / 26:4), PC (15:1 / 26:4), PC (17:2 / 22:4), TAG (12:0 / 12:0 / 18:5), TAG (13:0 / 19:5 / 19:5), and TAG (16:5 / 16:5 / 21:4).

[0010] Furthermore, the detection reagent includes an internal standard mixture composed of 15:0-18:1-d7-PE and 18:1-d7Lyso PC.

[0011] The application of a detection reagent for lipid biomarkers involves injecting the detection reagent into a liquid chromatography system. The detection device of the tandem mass spectrometer uses liquid chromatography. The content of each lipid in the lipid biomarker was obtained by tandem mass spectrometry.

[0012] Furthermore, the detection device is used in conjunction with FH screening products to detect lipid biomarkers.

[0013] Furthermore, the test sample for the FH screening product is dried blood spots from the soles of newborns' feet.

[0014] A method for screening lipid biomarkers includes the following steps: Sample pretreatment: used for the extraction of lipid compounds from dried blood spot samples; Lipidomics analysis of samples: for the detection of lipid compounds; Data quality control: Used for quality control of lipidomics data, determining the limit of quantitation and intra- and inter-batch coefficients of variation for each lipid compound; Differential lipid molecule screening and analysis: Lipids exhibiting concentration differences between the FH neonatal group and the healthy neonatal group, and lipids associated with disease risk, were screened using a two-independent-samples t-test, orthogonal partial least squares discriminant analysis, and false discovery rate correction. The predictive efficacy of the predictive model was evaluated using the area under the receiver operating characteristic (ROC) curve, and lipid biomarkers for dried blood spots in FH neonates were screened. These lipid biomarkers were one or any combination of PC(13:1 / 26:4), PC(15:1 / 26:4), PC(17:2 / 22:4), TAG(12:0 / 12:0 / 18:5), TAG(13:0 / 19:5 / 19:5), and TAG(16:5 / 16:5 / 21:4).

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is the first to characterize neonatal biomarkers for febrile hemorrhage (FH) using lipidomics features, and to screen out lipid biomarkers for assessing the severity of FH in newborns. The obtained lipid biomarkers have high value for early screening and timely intervention of FH.

[0016] The lipid biomarkers screened by this invention have good biological stability and representativeness, and their biological activity can be maintained even after long-term storage, thus ensuring the reliability of the detection results.

[0017] This invention is the first to characterize biomarkers for febrile hepatitis (FH) in newborns using lipidomics and to screen for lipid biomarkers to assess the severity of FH in newborns. It was found that TAG / PC molecules containing polyunsaturated fatty acid chains can serve as highly efficient biomarkers for FH newborn screening. This invention has good sensitivity: the combined biomarker AUC>0.85, which is significantly better than the detection of traditional indicators TC, LDL-C and apoB.

[0018] This invention is newborn-friendly: it requires only a heel prick blood DBS sample and is compatible with existing newborn screening systems (coverage >99%).

[0019] This invention enables high-throughput extraction and mass spectrometry detection of DBS samples, solving the problems of complex experimental procedures and difficult detection in traditional methods. This invention has a cost advantage: the cost of mass spectrometry detection is only 1 / 10 to 1 / 5 of that of gene sequencing; This invention has the function of family early warning: positive results can be linked to screening the parents of the child (50% of whom are undiagnosed FH patients), providing family members with FH with the opportunity for early detection and early intervention. Attached Figure Description

[0020] Figure 1 This is a schematic diagram illustrating the significant changes in lipid metabolites in FH newborns compared to normal newborns.

[0021] Figure 2 This is a schematic diagram of the lipid markers that were most significantly upregulated in the FH neonatal group.

[0022] Figure 3 This is a schematic diagram of ROC curve analysis for FH candidate lipid metabolites.

[0023] Figure 4 This is a schematic diagram of the ROC curve for the combined application of FH candidate lipids.

[0024] Figure 5 This diagram illustrates how candidate lipid metabolites can be used to identify the severity of FH. Detailed Implementation

[0025] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings: A reagent for detecting lipid biomarkers, comprising a lipid extraction protein precipitation reagent, a reconstitution reagent, and a chromatographic separation reagent. The lipid extraction protein precipitation reagent is a 5:1 mixture of methyl tert-butyl ether and methanol; the reconstitution reagent is a 1:1 mixture of methanol and dichloromethane; the chromatographic separation reagent comprises a liquid chromatography phase A of 40% water and 60% acetonitrile solution containing 10 mmol / L ammonium formate; and a phase B of 10% acetonitrile and 90% isopropanol solution, wherein 50 mL of 10 mmol / L ammonium formate aqueous solution is added per 1000 mL.

[0026] The lipid biomarkers of the present invention are selected from one or any combination of PC (13:1 / 26:4), PC (15:1 / 26:4), PC (17:2 / 22:4), TAG (12:0 / 12:0 / 18:5), TAG (13:0 / 19:5 / 19:5), and TAG (16:5 / 16:5 / 21:4).

[0027] The detection reagent of the present invention includes an internal standard mixture, which is composed of 15:0-18:1-d7-PE and 18:1-d7 Lyso PC.

[0028] This invention utilizes a combination of genomics and lipidomics to identify six lipid compounds with significantly different levels in dried blood spots of healthy and febrile newborns with familial hemorrhage (FH). These six lipid compounds exhibit excellent FH screening performance. Specifically, under unadjusted conditions, the AUCs of PC (13:1 / 26:4) were 0.83, PC (15:1 / 26:4) 0.79, PC (17:2 / 22:4) 0.75, TAG (12:0 / 12:0 / 18:5) 0.86, TAG (13:0 / 19:5 / 19:5) 0.82, and TAG (16:5 / 16:5 / 21:4) 0.82, indicating that these six lipid compounds are effective biomarkers for FH screening. Furthermore, the combined use of these six lipids maintains robust screening efficacy.

[0029] The application of a detection reagent for lipid biomarkers involves injecting the detection reagent into a liquid chromatography system. The detection device of the tandem mass spectrometer uses liquid chromatography. The content of each lipid in the lipid biomarker was obtained by tandem mass spectrometry.

[0030] The detection device of this invention is used in conjunction with FH screening products to detect lipid biomarkers.

[0031] The test sample for the FH screening product of this invention is dried blood spots from the soles of newborns' feet.

[0032] A method for screening lipid biomarkers includes the following steps: Sample pretreatment: used for the extraction of lipid compounds from dried blood spot samples; Lipidomics analysis of samples: for the detection of lipid compounds; Data quality control: Used for quality control of lipidomics data, determining the limit of quantitation and intra- and inter-batch coefficients of variation for each lipid compound; Differential lipid molecule screening and analysis: Lipids exhibiting concentration differences between the FH neonatal group and the healthy neonatal group, and lipids associated with disease risk, were screened using a two-independent-samples t-test, orthogonal partial least squares discriminant analysis, and false discovery rate correction. The predictive efficacy of the predictive model was evaluated using the area under the receiver operating characteristic (ROC) curve, and lipid biomarkers for dried blood spots in FH neonates were screened. These lipid biomarkers were one or any combination of PC(13:1 / 26:4), PC(15:1 / 26:4), PC(17:2 / 22:4), TAG(12:0 / 12:0 / 18:5), TAG(13:0 / 19:5 / 19:5), and TAG(16:5 / 16:5 / 21:4).

[0033] Example 1: Sample collection and data processing Dried blood spot samples were collected from 127 newborns diagnosed with fetal heart disease (FH) and 30 healthy control newborns with pathogenic LDLR variants at Nanjing Maternal and Child Health Hospital. The samples were collected 48–72 hours after birth, air-dried naturally, and stored at 4°C. There were no significant differences in gestational age, birth weight, or delivery method between the two groups. The FH group contained 48 LDLR variant sites. We plan to use a combination of genomics and lipidomics to identify novel biochemical markers for FH newborn screening and to preliminarily assess the possible genotype and disease severity of FH infants by observing changes in these markers.

[0034] Example 2: FH biomarker detection procedure for DBS samples 1. Kit Components Lipid extract (MTBE:MeOH = 5:1); Standard solutions (TAG / PC series concentration gradient); LC-MS / MS dedicated calibration plate.

[0035] 2. Testing Procedures (1) Sample preparation Place the newborn's DBS (3.2 mm in diameter) into a 1.5 mL ep tube; Add 200 μL of water to the sample, sonicate in an ice-water bath for 30 min, and then add 480 μL of extraction buffer (MTBE:MeOH=5:1). Vortex mix for 30 seconds, then sonicate in an ice-water bath for 10 minutes; The sample was left to stand at -40 ℃ for 1 h.

[0036] Centrifuge the sample at 4 ℃, 3000 rpm (centrifugal force 900 (×g), radius 8.6 cm) for 15 min; Take 300 μL of the supernatant and dry it under low temperature and vacuum. Add 100 μL of extraction buffer (DCM:MeOH = 1:1, v / v) to the dried sample. The extraction buffer contains an isotope-labeled internal standard for reconstitution. Vortex mix for 30 seconds, then sonicate in an ice-water bath for 10 minutes. Centrifuge the sample at 4 ℃, 12000 rpm (centrifugal force 16200 (×g), radius 8.6 cm) for 15 min; Take 75 μL of the supernatant into a sample vial for instrumental analysis; Take 20 μL of supernatant from each sample and mix them together to form a QC sample for instrument testing.

[0037] (2) LC-MS / MS analysis Chromatographic conditions: The Orbitrap Exploris 120 mass spectrometer can perform primary and secondary mass spectrometry data acquisition under the control of software (Xcalibur, version 4.4, Thermo). Detailed parameters are as follows: Sheath gas flow rate: 30 Arb, Aux gas flow rate: 10 Arb, Capillary temperature: 320 ℃ (positive) or 320 ℃ (negative), Full ms resolution: 60000, MS / MS resolution: 15000; Chromatograph: Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph; Column: Phenomenex Kinetex C18 (2.1 mm × 100 mm, 2.6 μm); Mobile phase: A (40% water + 60% acetonitrile, containing 10 mmol / L ammonium formate), B (10% acetonitrile + 90% isopropanol solution, with 50 mL of 10 mmol / L ammonium formate aqueous solution added per 1000 mL); Injection volume: 2 μL.

[0038] Mass spectrometry conditions: Mass spectrometer: Orbitrap Exploris 120 mass spectrometer; Control software: Xcalibur, version 4.4, Thermo; Detailed parameters: Sheath gas flow rate: 30 Arb, Aux gas flow rate: 10 Arb, Capillary temperature: 320 ℃ (positive) or 320 ℃ (negative), Full msresolution: 60000, MS / MS resolution: 15000, Collision energy: 15 / 30 / 45 in NCEmode, Spray Voltage: 3.8 kV (positive) or -3.4 kV (negative).

[0039] (3) Results processing The raw mass spectrometry data was converted to mzXML format using ProteoWizard software. XCMS was then used for retention time correction, peak identification, peak extraction, peak integration, and peak alignment, with minfrac set to 0.5 and cutoff set to 0.3. Lipid identification was performed using XCMS software, a self-written R package, and the lipidblast database.

[0040] Example 3: Currently, there are no biochemical screening programs for neonatal fibrillation (FH) in clinical practice. Through a neonatal genetic screening program including the LDLR gene, we compared lipidomics analysis of newborns diagnosed with FH and healthy controls. We found that, compared to healthy newborns, FH newborns exhibited upregulation of 123 lipid metabolites and downregulation of 12 metabolites (P<0.05, VIP>1). Figure 1 (Volcano plot). Six lipids were significantly elevated in the DBS of FH neonates, including three PCs and three TAGs (P<0.001, FDR<0.05, log2 Fold change>1.5). Figure 2 As shown, Figure 2 In the diagram, A corresponds to PC (13:1 / 26:4), B corresponds to PC (15:1 / 26:4), C corresponds to PC (17:2 / 22:4), D corresponds to TAG (12:0 / 12:0 / 18:5), E corresponds to TAG (13:0 / 19:5 / 19:5), and F corresponds to TAG (16:5 / 16:5 / 21:4). Positive = 96, Negative = 30.

[0041] ROC curves were plotted, revealing three superior indicators: TAG (12:0 / 12:0 / 18:5), TAG (13:0 / 19:5 / 19:5), and TAG (16:5 / 16:5 / 21:4), with AUC > 0.8, specificity > 85%, and sensitivity > 70%. Among these three lipid metabolites, TAG (12:0 / 12:0 / 18:5) performed best, with an AUC of 0.86, specificity of 86.36%, and sensitivity of 76.60%. Figure 3 As shown, Figure 3 In the above, A corresponds to PC (13:1 / 26:4), B corresponds to PC (15:1 / 26:4), C corresponds to PC (17:2 / 22:4), D corresponds to TAG (12:0 / 12:0 / 18:5), E corresponds to TAG (13:0 / 19:5 / 19:5), and F corresponds to TAG (16:5 / 16:5 / 21:4). Positive = 96, Negative = 30. To further improve screening efficiency, we also conducted a combined analysis of these six indicators that were significantly elevated in FH newborns, selecting three relatively good combined indicators. The optimal indicator was the combination of TAG (12:0 / 12:0 / 18:5) + TAG (16:5 / 16:5 / 21:4) + PC (13:1 / 26:4), with AUC = 0.88, specificity 90.91%, and sensitivity 78.72%. Figure 4 As shown, Figure 4 In the example, A corresponds to TAG(12:0 / 12:0 / 18:5) + TAG(13:0 / 19:5 / 19:5) + TAG(16:5 / 16:5 / 21:4), B corresponds to TAG(12:0 / 12:0 / 18:5) + PC(13:1 / 26:4), and C corresponds to TAG(12:0 / 12:0 / 18:5) + TAG(16:5 / 16:5 / 21:4) + PC(13:1 / 26:4). Positive = 96, Negative = 30.

[0042] Therefore, based on the combined application and analysis of our genome and lipidomics, we have invented a biochemical screening kit for newborns with FH based on specific lipid metabolite indicators. This kit can detect FH children by detecting the combined metabolite indicators of TAG(12:0 / 12:0 / 18:5)+TAG(16:5 / 16:5 / 21:4)+PC(13:1 / 26:4).

[0043] Example 4: Different LDLR variant sites can lead to varying degrees of fetal hemorrhage (FH). We categorized FH neonatal samples into positive and weakly positive groups based on clinical biochemical findings. Cluster analysis revealed that different variant sites resulted in significantly different degrees of lipid metabolite alterations, and the various indicators we selected effectively distinguished between positive and weakly positive samples, such as… Figure 5 The diagram shown illustrates the candidate lipid metabolites used to identify the severity of FH, with an AUC > 0.9 and both sensitivity and specificity greater than 80%. Among them, PC (13:1 / 26:4) has a sensitivity greater than 95%, while TAG (12:0 / 12:0 / 18:5) and TAG (16:5 / 16:5 / 21:4) have specificities greater than 90%. Figure 5 In the diagram, A represents a heatmap of lipid metabolism differences between the positive and weakly positive groups; B, C, and D are box plots corresponding to PC (13:1 / 26:4), TAG (12:0 / 12:0 / 18:5), and AG (16:5 / 16:5 / 21:4), respectively; E, F, and G are ROC curves corresponding to PC (13:1 / 26:4), TAG (12:0 / 12:0 / 18:5), and TAG (16:5 / 16:5 / 21:4), respectively. The positive value is 96, and the weakly positive value is 31. These changes in metabolite levels allow for a preliminary assessment of the possible genotype and disease severity in children with FH.

Claims

1. A reagent for detecting lipid biomarkers, characterized in that... The detection reagents include lipid extraction protein precipitation, reconstitution reagent, and chromatographic separation reagent. The lipid extraction protein precipitation reagent is a 5:1 mixture of methyl tert-butyl ether and methanol, and the reconstitution reagent is a 1:1 mixture of methanol and dichloromethane. The chromatographic separation reagent consists of a liquid chromatography phase A of 40% water and 60% acetonitrile solution containing 10 mmol / L ammonium formate; and a phase B of 10% acetonitrile and 90% isopropanol solution, with 50 mL of 10 mmol / L ammonium formate aqueous solution added per 1000 mL.

2. The detection reagent for lipid biomarkers according to claim 1, characterized in that... The lipid biomarker is selected from one or any combination of PC (13:1 / 26:4), PC (15:1 / 26:4), PC (17:2 / 22:4), TAG (12:0 / 12:0 / 18:5), TAG (13:0 / 19:5 / 19:5), and TAG (16:5 / 16:5 / 21:4).

3. The detection reagent for lipid biomarkers according to claim 1, characterized in that... The detection reagent includes an internal standard mixture consisting of 15:0-18:1-d7-PE and 18:1-d7 Lyso PC.

4. The application of the detection reagent for lipid biomarkers according to any one of claims 1-3, characterized in that... Inject the detection reagent into a liquid chromatography system. The detection device of the tandem mass spectrometer uses liquid chromatography. The content of each lipid in the lipid biomarker was obtained by tandem mass spectrometry.

5. The application according to claim 4, characterized in that... The detection device is used in conjunction with FH screening products to detect lipid biomarkers.

6. The application according to claim 5, characterized in that... The FH screening product uses dried blood spots from the soles of newborns as test samples.

7. A method for screening lipid biomarkers, characterized in that... Includes the following steps: Sample pretreatment: used for the extraction of lipid compounds from dried blood spot samples; Lipidomics analysis of samples: for the detection of lipid compounds; Data quality control: Used for quality control of lipidomics data, determining the limit of quantitation and intra- and inter-batch coefficients of variation for each lipid compound; Differential lipid molecule screening and analysis: Lipids exhibiting concentration differences between the FH neonatal group and the healthy neonatal group, and lipids associated with disease risk, were screened using a two-independent-samples t-test, orthogonal partial least squares discriminant analysis, and false discovery rate correction. The predictive efficacy of the predictive model was evaluated using the area under the receiver operating characteristic (ROC) curve, and lipid biomarkers for dried blood spots in FH neonates were screened. These lipid biomarkers were one or any combination of PC(13:1 / 26:4), PC(15:1 / 26:4), PC(17:2 / 22:4), TAG(12:0 / 12:0 / 18:5), TAG(13:0 / 19:5 / 19:5), and TAG(16:5 / 16:5 / 21:4).

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