AFP蛋白芯片技术的检测流程优化

目的:通过优化现有的蛋白芯片检测过程,在保证检测准确性的同时缩短甲胎蛋白(Alpha Fetal protein,AFP)的检测时间,提高检测效率,为原发性肝癌的筛查提供经济、便捷、省时、有效的检测方法。方法:本研究在传统蛋白芯片检测流程(1-1.5小时)的基础上,通过优化检测流程将检测时间缩短至18分钟,并且通过和传统方法进行比较,评价该优化方法的检测效能。结果:与传统蛋白芯片检测方法相比,本优化方法的检测时间缩短至18分钟。重复检测同一样本,传统方法 AFP水平为16.50±1.172ng/m L,优化方法 AFP水平为18.33±1.029 ng/m L,结果无明显统计学差异(P=0.251)。结论:本研究成功地优化了AFP的蛋白芯片检测流程,在缩短检测时间的同时,保证了检测的准确率,是一种经济省时易操作的AFP检测方法。     

甲胎蛋白异质体在肝细胞癌中的诊断价值分析

目的:探讨甲胎蛋白异质体(AFP-L3)的检测对肝细胞肝癌(HCC)的诊断价值。方法:选取2009年2月至2012年11月在本院治疗的90例HCC患者为研究组,另选择同期接受治疗的慢性乙型肝炎患者30例和肝炎肝硬化患者30例作为对照组。观察三组患者的AFP、AFP-L3阳性率。结果:肝癌患者AFP和AFP-L3阳性率显著高于乙肝和肝硬化患者,差异具有统计学意义(P0.05)。乙肝和肝硬化两组患者的AFP及AFP-L3阳性率无显著差异(P0.05)。AFP400 ng/m L:肝癌组AFP-L3阳性率显著高于乙肝组和肝硬化组,差异具有统计学意义(P0.05)。结论:AFP-L3在HCC中的诊断价值较甲胎蛋白大,更适合在临床诊断中应用。     

MRI扫描联合血清4项肿瘤标志物诊断原发性肝癌的临床价值研究

目的:探究磁共振成像(MRI)扫描联合血清4项肿瘤标志物诊断原发性肝癌(PHC)的临床价值。方法:将我院从2017年9月~2019年8月收治的65例PHC患者(肝癌组)纳入研究,另选取同期我院收治的60例良性肝病患者作为对照组。比较两组MRI图像特征。此外,检测并比较两组血清甲胎蛋白(AFP)和甲胎蛋白异质体(AFP-L3)、高尔基体蛋白73(GP73)、磷脂酰肌醇蛋白聚糖-3(GPC-3)水平。以病理诊断为金标准,分析不同诊断方式应用于PHC患者诊断中的效能。结果:PHC患者的MRI图像表现为边界清晰,呈类圆形或不规则分叶状,肝癌组T1WI呈低或稍低信号人数占比高于对照组,且T2WI、DWI呈高信号强度人数占比高于对照组(均P<0.05)。肝癌组血清AFP、AFP-L3、GP73、GPC-3水平均高于对照组(均P<0.05)。以病理诊断为金标准,MRI联合血清AFP、AFP-L3、GP73、GPC-3诊断PHC的灵敏度、特异度以及准确度分别为95.24%(40/42)、95.65%(22/23)、95.38%(62/65),均高于MRI诊断的73.81%(31/42)、69.57%(16/23)、72.31%(47/65)(P<0.05),以及血清4项肿瘤标志物联合诊断的80.95%(34/42)、73.91%(17/23)、78.46%(51/65)(P<0.05)。结论:PHC患者血清AFP、AFP-L3、GP73、GPC-3水平升高,MRI扫描联合血清AFP、AFP-L3、GP73、GPC-3检测可作为诊断PHC的有效手段。     

化学发光蛋白芯片用于检测肝癌患者血清CA19-9水平的方法研究

目的:探讨一种简单、有效、低成本、低耗时的化学发光蛋白芯片方法用于检测血清中的糖链抗原19-9(Carbohydrate Antigen19-9,CA19-9),以有助于对原发性肝癌的早期辅助诊断。方法:预先在醛基芯片上包被鼠源CA19-9单克隆抗体,建立CA19-9抗体蛋白芯片,共筛选出46份肝癌血清和32份正常健康人血清,然后用蛋白芯片方法进行检测,并以化学发光成像对检测结果进行判定。结果:24份肝癌血清的CA19-9水平高于37 U/m L,22份肝癌血清的CA19-9水平低于37 U/m L;30份正常人血清CA19-9含量低于37 U/m L,2份正常人血清的CA19-9含量为37 U/m L;灵敏度为52.17%,特异性为93.75%,ROC曲线下面积0.688[95%CI:0.566,0.811]。结论:本研究成功的建立了血清CA19-9的化学发光蛋白芯片检测方法。     

甲胎蛋白异质体在原发性肝癌患者血清中的表达

为了揭示原发性肝癌患者血清中甲胎蛋白异质体(AFP-L3)的表达及意义,本研究选取2014年2月至2016年3月在本院治疗的原发性肝癌患者80例,同时选取肝硬化患者60例,慢性乙肝患者60例及健康志愿者60例,采用亲和吸附离心管法检测不同受试者血清中AFP-L3水平。研究显示,原发性肝癌患者的血清AFP-L3 (122.13 ng/mL)水平显著低于肝硬化患者,并且显著高于慢性乙肝患者和健康者,差异具有统计学意义(p0.05);原发性肝癌患者中,血清AFP-L3水平在肿瘤大小≤3 cm (171.14 ng/mL)和TNM分期为Ⅰ～Ⅱ期(147.53 ng/mL)患者中显著高于肿瘤大小3 cm和TNM分期为Ⅲ～Ⅳ期患者(p0.05);血清AFP-L3高表达(≥122.13 ng/mL)的肝癌患者中位生存时间(22.76月)与血清AFP-L3低表达的肝癌患者(122.13 ng/mL)(22.04月)差异不显著(p0.05)。本项研究表明原发性肝癌患者血清中AFP-L3水平明显上升,并且与患者的肿瘤大小和TNM分期具有相关性。     

脱-γ-羧基凝血酶原的Ba-蛋白芯片法的建立和应用

目的:研发建立一种快速、特异性高、低成本的检测血清中脱-γ-羧基凝血酶原(DCP)的蛋白芯片,并通过检测DCP阳性肝细胞肝癌(HCC)血清临床样本对其进行评价。方法:首先采用柠檬酸钡吸附沉淀法去除血清中正常凝血酶原,然后通过双抗体夹心法蛋白芯片检测血清中DCP含量。通过Ba-蛋白芯片法检测41例DCP阳性HCC患者血清和66例健康人血清,进行盲法验证。结果:通过Ecarin-显色多肽底物法证实柠檬酸钡吸附沉淀法能够完全除血清中正常凝血酶原,根据"平均数+3×标准差"公式设置蛋白芯片的Cut-off值作为阳性判定标准,Ba-蛋白芯片法检测DCP阳性HCC血清的灵敏度为95.1%(39/41),特异度为100%(66/66)。结论:成功建立检测血清中DCP浓度的Ba-蛋白芯片法,为多种标志物联合检测HCC的蛋白芯片的研发提供候选血清标志物。     

血清AFP、CEA与肝癌患者临床病理分期和预后的关系及其诊断价值分析

目的:探讨血清甲胎蛋白(AFP)、癌胚抗原(CEA)与肝癌患者临床病理分期和预后的关系及其诊断价值。方法:选取2010年3月至2014年4月期间我院就诊的90例肝癌患者作为肝癌组,并选择同期在我院进行体检的90例健康体检者作为健康组。统计两组的血清AFP、CEA水平及阳性率,比较不同TNM分期肝癌患者的血清AFP和CEA水平。分析肝癌患者的5年无复发生存率以及血清AFP和CEA水平对肝癌的诊断价值。结果:与健康组相比,肝癌组血清AFP、CEA水平及阳性率明显升高(P0.05)。Ⅳ期血清AFP和CEA水平最高,其次Ⅲ期,Ⅱ期次之,Ⅰ期最低(P0.05)。血清AFP、CEA阳性患者5年无复发生存率明显低于血清AFP、CEA阴性患者(P0.05),血清AFP、CEA联合诊断具有较高的敏感性和特异性。结论:肝癌患者的血清AFP和CEA水平升高,并随TNM分期增加而上调。血清AFP阳性以及CEA阳性患者的5年无复发生存率明显下降,血清AFP联合血清CEA检测在肝癌诊断中具有一定的临床价值。     

甲胎蛋白异质体分离方法的优化

目的:优化现有的甲胎蛋白异质体(AFP-L3)分离和检测方法,找到一个最有效的AFP-L3的提取方法,为今后的AFP-L3研究奠定前期基础。方法:应用北京热景生物技术公司的甲胎蛋白异质体(AFP-L3)亲和吸附离心管,通过改变亲和介质使用量和洗脱液体积的配伍梯度变化来优化实验流程,排除提取AFP-L3的影响因素,分离血清中的AFP-L3,并用罗氏公司的甲胎蛋白检测试剂盒检测分离的效果。结果:发现在不改变亲和介质体积的情况下,随着洗脱液体积的减少AFP-L3回收浓度逐渐增加,且在75 ul洗脱液时能够获得最高AFP-L3回收浓度。结论:成功地优化了北京热景生物技术公司的甲胎蛋白异质体(AFP-L3)亲和吸附离心管及其配套试剂的使用方法。     

乙型肝炎病毒核心抗体IgM的蛋白芯片检测法研究

目的:探索蛋白芯片技术检测乙型肝炎病毒(HBV)抗HBc-IgM的可行性。方法:应用Nano-Plotter TM-压电式微量喷墨点阵制备系统自制的核心抗原蛋白芯片在经10%山羊血清封闭后加入待测血清37℃孵育,PBST清洗晾干后后再次加入检测抗体即HRP-抗人Ig M抗体,芯片用伯乐成像仪检测有无信号。结果:24份HBs Ag和抗HBc阳性血清中经蛋白芯片检测抗HBc-IgM阳性率为83.3%(20/24);24份健康志愿者血清中经蛋白芯片检测抗HBc-IgM阳性率为4.1%(1/24)。乙肝血清与健康志愿者血清化学发光信号差异明显。结论:蛋白芯片技术可较好地应用于定性检测HBV抗HBc-IgM,为临床快速判断是否存在HBV感染和监测慢性肝炎HBV活动性提供新的辅助诊断方法。     

寨卡病毒感染者血液、尿液和唾液中IgA抗体水平初步研究

为研究寨卡病毒(ZIKV)感染者血清、尿液和唾液样本中特异性IgA抗体水平,进一步了解ZIKV感染免疫机制,本研究重组表达制备寨卡病毒NS1蛋白,对其浓度、纯度进行鉴定,初步评估了NS1蛋白抗原性,并建立间接酶联免疫(Indirect-ELISA)方法,检测患者临床血清、尿液和唾液样本中的寨卡特异性IgA抗体。利用健康人群血清、尿液和唾液标本,评估检测方法的特异性,并确定以阴性对照的均值加3倍标准差作为判定检测结果的阈值,特异性为100%。对经病毒核酸检测所确诊病例的33份血清样本、4份尿液样本和3份唾液样本进行检测。选择3份具有较高IgA抗体水平的血清,通过2倍系列稀释的方法评价了血清样本中特异性IgA抗体的滴度,可有效检出经3 200倍以上稀释的血清样本中的IgA抗体。重复性检测实验显示板间变异系数为(3.0±0.8)%,板内变异系数为(2.7±1.0)%。寨卡患者血清、尿液和唾液样本中的特异性IgA抗体的检出率分别为75.8%(25/33)、100%(4/4)和33.3%(1/3),提示IgA抗体在3种体液标本中均具有显著的存在,具有充当体外诊断指标的意义。同时尿液、唾液标本中特异性IgA抗体的存在,提示潜在的粘膜免疫效应,有助于增强对病毒体内播散和体外传播的理解,也初步提示了寨卡病毒NS1蛋白可用于相关免疫学诊断试剂的研发。     

Clinical evaluation of lentil lectin-reactive alpha-fetoprotein-L3 in histology-proven hepatocellular carcinoma

INTRODUCTION: Serum alpha-fetoprotein (AFP) is a useful marker of hepatocellular carcinoma (HCC), although the serum AFP concentration is also increased in patients with chronic liver diseases (CLD). The analysis of AFP glycoforms has been known to be of diagnostic value. We applied the lectin-affinity electrophoresis and antibody-affinity blotting techniques to HCC patients in Vietnam in order to better understand the role of lentil lectin-affinity AFP-L3 in the diagnosis and differential diagnosis of HCC, and its relationship with the biological characteristics of HCC. METHODS: Lens culinaris agglutinin-reactive AFP (AFP-L3) was measured in 65 patients with histologically proven HCC and 25 patients with CLD. All patients had serum AFP levels above 54 ng/mL. AFP-L3 levels were determined by lectin affinity electrophoresis coupled with antibody-affinity blotting. The diagnosis of HCC was confirmed histologically by ultrasound-guided biopsy. RESULTS: The mean value of AFP-L3 in the HCC patients was 49.6 +/- 21.6%, which was significantly higher (p<0.001) than that in the 25 CLD patients (10.7 +/- 4.3%). When the cutoff level for AFP-L3 was set at 15% (mean +/- SD), the sensitivity was 96.9%, the specificity 92.0% and the accuracy 95.5% in the 65 HCC patients. There was no clear correlation between serum AFP level and AFP-L3 percentage (r=0.16). There was no correlation between AFP-L3 and the maximum diameter of HCC nodules (r=0.05). However, the mean AFP-L3 value was higher in moderately or poorly differentiated HCC than in well differentiated tumors (p<0.001). CONCLUSIONS: AFP-L3 is potentially a clinically useful marker for the differentiation of increased AFP levels in hepatocellular carcinoma and chronic liver diseases. The AFP-L3 percentage is closely related to HCC differentiation. We consider the analysis of AFP-L3 a useful adjunct in the diagnosis of HCC.     

Evaluation of serum alpha fetoprotein-L3 as an accuracy novel biomarker for the early diagnosis of hepatocellular carcinoma in Egyptian patients

BackgroundMost Hepatocellular Carcinomas (HCCs) are diagnosed at an advanced stage. However, HCC early diagnosis is complicated by the coexistence of inflammation and cirrhosis. The unsatisfactory sensitivity and specificity of Alpha-fetoprotien (AFP) for screening of early-stage HCC paved the way for new novel biomarkers to complement AFP such as AFP-L3. The aim of this study was the Evaluation of alpha fetoprotein-L3 (AFP-L3) as earlier marker in diagnosis of hepatocellular carcinoma in Egyptian patients. This study was conducted on 80 patients categorized into 2 groups; group 2 (40 patients with chronic active hepatitis) and group 3 (40 patients with HCC). HCC diagnosis was done by clinical, triphasic CT and positive US for focal lesion, in addition to 20 healthy individuals as controls (group 1).ResultsThe median range of AFP and AFP-L3 were highly statistically significant difference between HCC group and other groups [p < 0.001]. In this study ALT, AST, Total & direct bilirubin and albumin results showed highly significant differences between HCC group and other groups. Serum AFP-L3 shows sensitivity 100%, specificity 100%, positive predictive value 100% and negative predictive value 100% with AUC = 1 in HCC cases.ConclusionSerum AFP-L3 may serve as a diagnostic biomarker for the detection of early stage of HCC and show higher sensitivity than AFP.     

Glycomic analysis of alpha-fetoprotein L3 in hepatoma cell lines and hepatocellular carcinoma patients

The N-glycan structures of the Lens culinaris agglutinin (LCA)-reactive fraction of alpha-fetoprotein (AFP-L3), a tumor marker of hepatocellular carcinomas (HCC), were analyzed in relationship to glycosyltransferases and LCA-affinity electrophoresis. Using HPLC and MALDI-TOF MS, we determined the N-glycan structures of AFP from HCC cell lines, and demonstrated they were affected by N-acetylglucosaminyltransferase III and fucosyltransferase VIII, but not by N-acetylglucosaminyltransferase V. Moreover, we identified the N-glycan structures of AFP in HCC patients.     

Retraction Note: miR-195-5p/NOTCH2-mediated EMT modulates IL-4 secretion in colorectal cancer to affect M2-like TAM polarization

Background

Hepatocellular carcinoma (HCC) generally arises from a background of liver cirrhosis (LC). Patients with cirrhosis and suspected HCC are recommended to undergo serum biomarker tests and imaging diagnostic evaluation. However, the performance of routine diagnostic methods in detecting early HCC remains unpromising.

Methods

Here, we conducted a large-scale, multicenter study of 1675 participants including 490 healthy controls, 577 LC patients, and 608 HCC patients from nine clinical centers across nine provinces of China, profiled gene mutation signatures of cell-free DNA (cfDNA) using Circulating Single-Molecule Amplification and Resequencing Technology (cSMART) through detecting 931 mutation sites across 21 genes.

Results

An integrated diagnostic model called “Combined method” was developed by combining three mutation sites and three serum biomarkers. Combined method outperformed AFP in the diagnosis of HCC, especially early HCC, with sensitivities of 81.25% for all stages and 66.67% for early HCC, respectively. Importantly, the integrated model exhibited high accuracy in differentiating AFP-negative, AFP-L3-negative, and PIVKA-II-negative HCCs from LCs.

     

Prognostic Role of Pre-Treatment Serum AFP-L3% in Hepatocellular Carcinoma: Systematic Review and Meta-Analysis

Background

Serum lens culinaris agglutinin-reactive fraction of α-fetoprotein (AFP-L3%) has been widely used for HCC diagnosis and follow-up surveillance as tumor serologic marker. However, the prognostic value of high pre-treatment serum AFP-L3% in patients with hepatocellular carcinoma (HCC) remains controversial. We therefore conduct a meta-analysis to assess the relationship between high pre-treatment serum AFP-L3% and clinical outcome of HCC.

Methods

Eligible studies were identified through systematic literature searches. A meta-analysis of fifteen studies (4,465 patients) was carried out to evaluate the association between high pre-treatment serum AFP-L3% and overall survival (OS) and disease-free survival (DFS) in HCC patients. Sensitivity and subgroup analyses were also conducted in this meta-analysis.

Results

Our analysis results showed that high pre-treatment serum AFP-L3% implied poor OS (HR: 1.65, 95%CI: 1.45–1.89 p<0.00001) and DFS (HR: 1.80, 95% CI: 1.49–2.17 p<0.00001) of HCC. Subgroup analysis revealed that there was association between pre-treatment serum AFP-L3% and endpoint (OS and DFS) in low AFP concentration HCC patients (HR: 1.96, 95% CI: 1.24–3.10, p = 0.004; HR: 2.53, 95% CI: 1.09–5.89, p = 0.03, respectively).

Conclusion

The current evidence suggests that high pre-treatment serum AFP-L3% levels indicated a poor prognosis for patients with HCC and AFP-L3% may have significant prognostic value in HCC patients with low AFP concentration.     

Automated immunoassay system for AFP-L3% using on-chip electrokinetic reaction and separation by affinity electrophoresis

Implementation of the on-chip immunoassay for α-fetoprotein (AFP)-L3% was achieved using a fully automated microfluidic instrument platform that will prepare the chip and run the assay with a total assay time of less than 10 min. Reagent/sample mixing, concentration, and reaction in microfluidic channels occur by the electrokinetic analyte transport assay (EATA) technique, enabling the integration of all assay steps on-chip. The determination of AFP-L3%, a biomarker for hepatocellular carcinoma, was achieved by the presence of Lens culinaris agglutinin in the separation channel, causing separation of the fucosylated isoform, AFP-L3, from the nonfucosylated AFP-L1 by lectin affinity electrophoresis. Laser-induced-fluorescence (LIF) detection was used to quantitate the labeled immunocomplexes. The limit of detection (LOD) was 0.1 ng/ml AFP, and assay precision of less than 2% coefficient of variation (CV) was obtained for quantitation from 24 to 922 ng/ml total AFP in spiked serum samples. Assay precision of less than 3% CV was obtained for AFP-L3% measurements from 8.5 to 81%. Furthermore, good correlation of test results for 68 patient serum samples with a commercially available reference method (LiBASys assay for AFP-L3%) was obtained, with r² = 0.981 and slope = 1.03.     

复方苦参注射液联合TACE对中晚期肝癌患者预后、生存质量和血清AFP、AFP-L3水平的影响

摘要 目的：观察肝动脉化疗栓塞术（TACE）联合复方苦参注射液对中晚期肝癌患者预后、生存质量和血清甲胎蛋白（AFP）、甲胎蛋白异质体（AFP-L3）水平的影响。方法：选择我院于2016年3月~2019年10月期间收治的98例中晚期肝癌患者,经随机数字表法分为对照组（49例,TACE治疗）和研究组（49例,复方苦参注射液联合TACE治疗）。对比两组疗效、生存率、生存质量改善率、血清AFP、AFP-L3水平及不良反应发生率。结果：与对照组比较,研究组的临床总有效率明显升高（P<0.05）。研究组的1年生存率高于对照组（P<0.05）。与对照组比较,研究组的生存质量改善率明显升高（P<0.05）。两组治疗后血清AFP、AFP-L3水平较治疗前下降,且研究组低于对照组（P<0.05）。两组不良反应总发生率组间对比无统计学差异（P>0.05）。结论：TACE基础上联合复方苦参注射液治疗中晚期肝癌患者,可改善其短期预后及生存质量,降低其血清AFP、AFP-L3水平。     

脱-R-羧基凝血酶原的Ba- 蛋白芯片法的建立和应用

目的：研发建立一种快速、特异性高、低成本的检测血清中脱-r- 羧基凝血酶原（DCP）的蛋白芯片,并通过检测DCP 阳性肝 细胞肝癌（HCC）血清临床样本对其进行评价。方法：首先采用柠檬酸钡吸附沉淀法去除血清中正常凝血酶原,然后通过双抗体夹 心法蛋白芯片检测血清中DCP 含量。通过Ba- 蛋白芯片法检测41 例DCP阳性HCC患者血清和66 例健康人血清,进行盲法验 证。结果：通过Ecarin- 显色多肽底物法证实柠檬酸钡吸附沉淀法能够完全除血清中正常凝血酶原,根据" 平均数+3× 标准差" 公式设置蛋白芯片的Cut-off值作为阳性判定标准,Ba-蛋白芯片法检测DCP阳性HCC血清的灵敏度为95.1 %（39/41）,特异度 为100 %（66/66）。结论：成功建立检测血清中DCP浓度的Ba- 蛋白芯片法,为多种标志物联合检测HCC 的蛋白芯片的研发提供 候选血清标志物。