CEA、NSE、CYFRA21-1联合检测在肺癌诊断中的价值

[目的]了解血清肿瘤标志物癌胚抗原(CEA)、神经元特异性烯醇化酶(NSE)和细胞角蛋白19片段(CYFRA21-1)在肺癌诊断中的应用价值.[方法]应用电化学发光技术测定58例肺癌患者,34例肺良性疾病患者和31例正常对照者血清CEA、NSE、CYFRA21-1水平.[结果]肺癌组CEA、NSE、CYFRA21-1水平明显高于肺良性疾病组和对照组(P<0.01).CEA、NSE、CYFRA21-1对肺癌的敏感度分别为51.7%、56.9%、55.2%.NSE对小细胞肺癌敏感度为82.6%,CYFRA21-1对肺鳞癌敏感度为83.3%.三者联合检测对肺癌敏感度为93.1%.[结论]血清CEA、NSE、CYFRA21-1联合检测对肺癌的诊断及分型有价值.     

肺腺癌患者血清CEA和蛋白质谱联合检测的临床意义

目的:探讨在肺腺癌患者中联合应用CEA检测和SELDI蛋白芯片技术诊断的价值。方法:应用免疫化学发光技术检测69例肺腺癌患者及71例健康对照者血清标志CEA;同时应用表面增强激光解吸电离飞行时间质谱技术(SELDI-TOF)和CM10芯片检测该血清,经BiomarkerWizard软件分析,结合临床病理资料分析两种手段的联合诊断价值。结果:免疫化学发光法检测CEA的敏感度为62.319%(43/69),特异度为92.958%(66/71);SELDI-TOF-MS技术检测的敏感度为88.406%(61/69),特异度为68.182%(15/22);两者联合检测的敏感度为82.609%(57/69),特异度为92.958%(66/71)。结论:免疫化学发光法检测CEA和SELDI-TOF-MS技术联合检测蛋白标志比单用免疫化学发光法检测CEA能提高对肺腺癌诊断的敏感度。但与单独使用SELDI-TOF-MS技术相比,两者联合检测的敏感度无明显提高。     

肿瘤标志对煤矿工人尘肺并发肺癌的鉴别诊断价值

目的:探讨肿瘤标志在煤矿工人尘肺并发肺癌中的诊断价值。方法:应用电化学发光技术,检测98例尘肺并发肺癌患者、151例尘肺患者和252位健康煤矿工人血清细胞角蛋白21-1(Cyfra21-1)、神经元特异性烯醇化酶(NSE)、癌胚抗原(CEA)以及糖类抗原125(CA125)水平。结果:尘肺并发肺癌患者血清Cyfra21-1、NSE、CEA和CA125表达水平均显著高于患尘肺或健康煤矿工人,P<0.001。其中腺癌的CEA及CA125水平、鳞癌的Cyfra21-1表达水平以及小细胞癌的NSE表达水平比其他2种类型均高,且差异有统计学意义,P<0.001。以上单项肿瘤标志鉴别诊断尘肺和尘肺并发肺癌的准确性分别达到81.1%(202/249)、71.1%(177/249)、71.5%(178/249)和74.7%(186/249),联合检测准确性达87.6%(218/249)。结论:Cyfra21-1、NSE、CEA和CA125联合检测可用于煤矿工人尘肺并发肺癌的鉴别诊断,而且对判断肺癌的病理类型有辅助价值。     

血清肿瘤标志物联合检测在肺癌诊断中的价值

 目的　探讨血清5种肿瘤标志物联合检测在肺癌诊断中的价值。方法　采用电化学发光免疫法检测168例肺癌患者、46例肺良性疾病患者及健康对照组癌胚抗原（CEA）、糖类抗原125 （CA125）、糖类抗原15-3 （CA15-3）、细胞角蛋白19 片段（CYFRA21-1）和神经元特异性烯醇化酶（NSE）水平,并计算上述指标联合检测在肺癌诊断中的敏感度、特异度、准确度和约登指数（YI）。结果　肺癌组5种肿瘤标志物水平均明显高于肺良性疾病组和健康对照组（P＜0.01）。这5种血清肿瘤标志物在肺癌的不同病理类型、临床分期之间均有不同程度的差异。单项检测各种肿瘤标志物诊断肺癌的敏感度、准确度和YI）以CYFRA21-1最高,分别为58.9 %、65.9 %和0.37 %。联合检测较单项检测敏感性、准确性和YI明显提高,5项联合检测敏感度95.8 %、特异度79.2 %、准确度87.9 %、YI为0.75。结论　单项检测对肺癌的诊断价值有限,5种血清肿瘤标志物联合检测可提高肺癌的敏感度和准确度。     

血清肿瘤标志及SF联合检测对肺癌早期诊断价值的探讨

为了探讨血清肿瘤相关抗原CEA、CA153、CA125、SCC、CY FRA21-1、NSE及SF水平与肺癌的关系,以及联合测定7项指标对肺癌早期诊断的应用价值,采用电化学发光法和化学发光微粒子免疫分析法(CMIA)分别对133例肺癌患者血清CEA、CA153、CA125、SCC、CY FRA21-1、NSE及SF水平进行检测,并与51例肺部良性疾病(BLD)患者、45名健康体检者比较.肺癌患者血清CEA、CA153、CA125、SCC、CY FRA21-1、NSE及SF水平均明显高于BLD与正常对照组(P均<0.01);CYFRA21-1敏感度在肺鳞癌组为83.78%,明显高于其他类型的肺癌(P<0.01);CEA敏感度在肺腺癌组为79.73%,明显高于其他类型的肺癌(P<0.01);NSE敏感度在小细胞肺癌组为77.27%,明显高于其他类型的肺癌(P<0.01).7项联合检测肺癌敏感度为94.10%,可明显提高肺癌诊断的敏感度.初步研究结果提示,联合检测肺癌患者血清CEA、CA153、CA125、SCC、CY FRA21-1、NSE及SF对肺癌的早期诊断、分型具有重要的临床意义.     

CA-125、CYFRA21-1、NSE在肺癌临床诊断中的应用及评价

目的:探讨糖类抗原-125（CA-125）、细胞角蛋白19片段(CYFRA21-1)、神经特异性烯醇化酶(NSE)及其联合检测在肺癌临床诊断中的价值。方法:应用电化学发光法检测145例肺癌患者、80例肺良性病变患者及60例健康体检者血清中三种标志物的水平并进行统计学分析。结果:NSE、CYFRA21-1、CA-125三项标志物水平均高于肺良性病组及对照组,CA-125+CYFRA21-1联合对腺癌的敏感性较高,NSE+CYFRA21-1或者NSE+CA-125+CYFRA21-1联合对鳞癌的敏感性高达80%以上,差异具有统计学意义。结论:CA-125、CYFRA21-1、NSE对肺癌的诊断有一定的临床价值,特别是联合应用意义更大。     

Cyfra21-1和CEA及CRP联合检测对食管癌诊断价值的探讨

目的:探讨外周血肿瘤相关细胞角蛋白19片段抗原21-1(Cyfra21-1)、癌胚抗原(CEA)及C反应蛋白(CRP)联合检测对食管癌的诊断价值。方法:电化学发光免疫分析法测定278例食管癌患者与100名健康体检者外周血清的Cyfra21-1、CEA和CRP的水平,t和χ2检验进行数据统计分析。结果:食管癌组血清Cyfra21-1、CEA和CRP的水平及阳性率分别为(6.4±4.8)ng/mL与41.7%、(6.1±2.2)ng/mL与30.2%及(57.8±63.6)mg/L与53.2%,均显著高于健康对照组的(4.5±1.5)ng/mL与14.0%、(5.8±0.9)ng/mL与8.0%及(10.2±1.9)mg/L与22.0%,P<0.05。随着临床分期的递增,血清Cyfra21-1、CEA和CRP的水平和阳性率也不同程度上升。食管癌患者血清Cyfra21-1、CEA和CRP联合检测的敏感性和特异性明显高于单项检测,P<0.05。结论:联合检测食管癌血清中Cyfra21-1、CEA和CRP水平变化,对食管癌早期诊断和鉴别具有重要的临床价值。     

血清CEA和CYFRA21-1检测与NSCLC临床诊断的相关性研究

目的: 研究血清CEA和CYFRA21-1与NSCLC临床诊断的相关性.方法: 采用电化学发光法对86例NSCLC患者和33例正常人的血清样本进行检测和分析.结果: NSCLC患者的CEA和CYFRA21-1浓度和阳性率高于正常人,二者有显著性差异(P<0.01).肺腺癌的CEA浓度高于肺鳞癌和大细胞肺癌(P<0.01),肺鳞癌的CYFRA21-1浓度高于肺腺癌和大细胞肺癌(P<0.01),CEA和CYFRA21-1联合检测对阳性率均有所提高.CEA和CYFRA21-1浓度随肿瘤分期的增加而升高.结论: 血清CEA和CYFRA21-1与NSCLC临床诊断具有明显相关性,可作为NSCLC患者临床诊断和肿瘤分期的判断指标之一.     

肺癌患者血清中CK19-2G2和Cyfra21-1的测定及其对肺癌的诊断价值

目的 测定肺癌患者血清中细胞角蛋白19(CK19)降解片段CK19-2G2的浓度,并评价其对肺癌的诊断价值.方法 采用CK19-2G2和Cyfr21-1的检测试剂盒,分别检测104例肺癌患者、7l例肺部炎症患者和105例健康体检者的血清标本,采用Pearson相关分析判断二者的相关性.运用受试者工作特性曲线(ROC)分析CKl9-2G2和Cyfra21-1的检测效能,并确定CK19-2G2诊断肺癌的临界值.比较CK19-2G2和Cyfra21-1诊断肺癌的灵敏度和准确率.结果 肺癌组、肺部炎症组和健康对照组血清CK19-2G2的中位浓度分别为2.87、1.02和0.01 mU/ml,肺癌组显著高于肺部炎症组和健康对照组(均P<0.01).血清CK19-2G2诊断肺癌的灵敏度(72.1%)和准确率(82.9%)均高于Cyfra21-1(分别为41.35%和73.93%,均P<0.05).CK19-2G2与Cyfra21-1的血清浓度呈正相关(r=0.543).在不同病理类型的肺癌中,肺鳞癌患者的CK19-2G2血清浓度(M=8.35)显著高于肺腺癌患者(M=1.81)和小细胞肺癌患者(M=1.65),差异均有统计学意义(P<0.05).结论 CK19-2G2是一种较好的非小细胞肺癌肿瘤标志物,对肺癌的检测性能优于Cyfra21-1,对肺鳞癌的检测可能有更高的特异性.     

肺鳞癌患者血清CYFRA21-1和SCC检测及ROC曲线和截断点的理论分析

目的:探讨血清细胞角蛋白19片段(CYFRA21-1)抗原和鳞癌抗原(SCC)对肺鳞癌的临床意义.方法:将研究对象分组,试验组为102例肺鳞癌患者,对照组为53例良性肺病患者.分别采用电化学发光法和酶联免疫法检测血清CYFRA21-1及SCC,测定值用t检验,率比较用x2检验.接受者工作特征(ROC)曲线及截断点选择理论评估CYFRA21-1及SCC的临床价值.结果:肺鳞癌组与良性肺病组CYFRA21-1和SCC水平差异有统计学意义;CYFRA21-1以3.3 ng/mL、SCC以1.5 ng/mL为诊断临界点,CYFRA21-1诊断肺鳞癌敏感性为57.84％,特异性为92.45％,准确性为69.68％,ROC曲线下面积为0.788;SCC诊断肺鳞癌敏感性为33.33％,特异性为92.45％,准确性为53.55％,ROC曲线下面积为0.691;CYFRA21-1诊断肺鳞癌最佳截断点为2.485或2.365 ng/mL,对应敏感性和特异性分别为65.7％、92.5％和67.6％、90.6％;SCC诊断肺鳞癌最佳截断点为0.55 ng/mL,对应敏感性和特异性分别为72.5％和62.3％.随肺鳞癌分期增高,CYFRA21-1和SCC浓度增高差异有统计学意义,CYFRA21-1敏感性增高差异有统计学意义,SCC敏感性无明显变化.结论:CYFRA21-1对肺鳞癌的诊断具有较高的临床价值,而SCC对肺鳞癌的诊断价值较低.     

用Cyfra21—1检测血中细胞角质素片断CKS—19在肺癌诊断中的意义

用酶联免疫方法，试剂Cyfra21－1，测定42例健康人，23便非肿瘤性肺部疾病病人，68例肺癌病人血清中的细胞角质素片断CKS－19。健康人为1．45±0．86ng／ml，非肿瘤性肺疾病病人为1．64±1．40ng／ml。肺癌中鳞癌16例，腺癌33例，小细胞肺癌19例，Cyfra21－1阳性率各为75．0％，66．7／，31．6％。比较Cyfra21－1和CEA，NSE三种肺癌标志物的ROC曲线，Cyfra21－1的曲线最接近左上角，证明Cyfra21－1是敏感性，特异性较好的肺癌标志物。     

肺癌相关肿瘤标记物的诊断价值探讨

目的 :探讨血清癌胚抗原 (CEA)、细胞角质蛋白 (CYFRA2 1 1)和神经烯醇化酶 (NSE)对肺癌诊断的价值。方法 :采取血清标本 98例 ,其中腺癌 2 9例、鳞癌 2 4例、小细胞肺癌 11例 ,肺部良性病变 34例 ,采用放射免疫法检测。结果 :CEA、Cyfra2 1 1和NSE在肺癌组的敏感性分别为 4 3 8%、37 5 %和 4 3 8% ,特异性为 82 4 %、91 2 %和 85 3%。其中CEA对腺癌的敏感性为 5 8 6 % ,CYFRA2 1 1对鳞癌的敏感性为 5 8 3% ,NSE对小细胞肺癌的敏感性为 72 7% ,均明显高于肺部良性病变对照组(P <0 0 1)。NSE在非小细胞肺癌中的敏感性为 37 7% ,亦高于肺部良性病变对照组 (P <0 0 5 )。Ⅲ期、Ⅳ期肺癌CEA、Cy fra2 1 1及NSE的平均水平显著高于Ⅱ期肺癌 (P <0 0 1)。将 3项联合检验 ,对肺癌诊断的敏感性上升到 6 7 2 % ,高于 3项单检时的敏感性 (P <0 0 1) ,且特异性无明显下降。结论 :CEA、Cyfra2 1 1、NSE分别对腺癌、鳞癌及小细胞癌的诊断有一定的意义。肿瘤标记物的水平与病程密切相关 ,较高水平常出现于晚期病例。将CEA、Cyfra2 1 1、NSE 3项联检可提高肺癌的诊断率 ,有一定的临床价值     

Evaluation of the soluble fragments of cytokeratin 19 (CK19) in non-small cell lung cancer (NSCLC)

This study compared the diagnostic efficacy of serum CK19 determination (Cyfra 21-1) with other tumour markers, such as CEA, SCC, NSE, TPA, in patients with resected non-small lung cancer. Tumour marker levels were tested in 90 patients with benign lung disease and at diagnosis in 72 patients with proven NSCLC, 39 squamous cell carcinoma and 33 adenocarcinoma. At presentation baseline levels of all tumor markers were significantly higher (p<0.05) in lung cancer patients than in control subjects, except for NSE. A significant increase (p<0.05) in serum concentrations was observed from stage I to stage IIIb only for Cyfra 21-1 (stage I/II, median=2.7 ng/ml; stage IIIb, median=6.3 ng/ml) and TPA (stage I/II, median=89.8 IU/ml; stage IIIb, median=170.7 IU/ml). Receiver operating characteristic (ROC) analysis was performed to evaluate the best threshold values and the global accuracy of each marker. The highest global sensitivity for NSCLC was reached by TPA (70.8%), whereas that of Cyfra 21-1 was 50%. According to tumour histology, significant difference (p<0.05) in serum levels were found only for CEA (adenocarcinomas, median=5.6 ng/ml; squamous cell carcinoma, median=3.2 ng/ml) and SCC (adenocarcinomas, median=1.0 ng/ml; squamous cell carcinoma, median=1.5 ng/ml). As regards squamous cell carcinoma histotype, the highest sensitivity was obtained by TPA (74.4% at a specificity of 62.2%) and for adenocarcinomas by CEA (78.8% at a specificity of 85.6%). Tumour marker levels were also determined during the follow-up of 10 patients. The best sensitivity in detecting relapses was shown by CEA (90%), followed by TPA (70%), SCC (50%), Cyfra 21-1 (40%) and NSE (10%), even though the CEA test displayed a high percentage of false positive results (98.1%) in patients with no evidence of disease (NED).     

血清CEA、CA125及Cyfra21-1水平对中晚期非小细胞肺癌患者预后的影响

目的 探讨晚期非小细胞肺癌（NSCLC） 患者血清癌胚抗原（CEA） 、糖类抗原（CA125）、非小细胞肺癌相关抗原（Cyfra21-1）水平与无疾病进展生存期的相关性。方法 选取2012年6月至2014年5月于宜昌市第二人民医院确诊的非小细胞肺癌患者120例,对其临床资料进行回顾性分析,了解CEA、CA125、Cyfra21-1水平与无疾病进展生存期的相关性。结果 与鳞癌患者相比,血清CEA水平在肺腺癌患者中明显升高（P＜0.05）;血清CA125水平在Ⅳ期肺腺癌患者中明显升高（P＜0.05）;血清Cyfra21-1在患者疾病一般特征中差异未见统计学意义（P＞0.05）。血清CEA、CA125、Cyfra21-1水平升高的患者中位无疾病进展生存期分别为4.2、4.5、4.3月,与正常组相比差异均有统计学意义（P＜0.05）。结论 晚期非小细胞肺癌患者CEA、CA125、Cyfra21-1升高与无疾病进展生存期存在明显的相关性,临床医师可通过检测患者血清CEA、CA125、Cyfra21-1水平判断预后。     

五项血清肿瘤标志物联合检测在肺癌诊断中的应用

目的：探讨癌胚抗原（CEA）、神经元特异性烯醇化酶（NSE）、鳞状上皮抗原（SCC）、细胞角蛋白19片段抗原（Cyfra21—1）和糖链抗原125（CA125）5项肿瘤标志物联合检测在肺癌诊断中的价值。方法：采用电化学发光法及酶化学发光法测定81例肺癌、32例良性肺病患者和30例健康人的血清CEA、NSE、SCC、cyfra21—1和CA125水平。结果：肺癌组血清CEA、NSE、SCC、Cyfra21—1和CA125的阳性检出率（分别为49．38％、55．56％、23．46％、62．96％、39．51％）明显高于良性肺病组和健康对照组。肺癌组5项肿瘤标志物阳性率随肿瘤临床分期而升高。其中CEA在肺腺癌中明显升高（P〈0．05），NSE以小细胞癌升高明显（P〈0．05），SCC在肺鳞癌中明显升高（P〈0．01），Cyfra21—1以非小细胞肺癌升高明显（P〈0．01）。5项肿瘤标志物联合检测比单项检测的阳性率和准确性更高。结论：外周血CEA、NSE、SCC、Cyfra21—1和CA125联合检测可提高肺癌的阳性检出率，CEA、NSE、SCC和Cyfra21—1对肺癌病理分型有重要的临床参考价值。     

Follow-up with serum Cyfra 21-1 in patients with squamous cell carcinomas of the head and neck

OBJECTIVE: Finding tumor markers for disease progression, and especially development of distant metastases, is desirable for patients with squamous cell carcinoma of the head and neck (SCCHN). Elevated serum levels of Cyfra 21-1 (cytokeratin fraction 21-1) have been frequently associated with disease progression in patients with lung cancer. In SCCHN, Cyfra 21-1 has not been established as a routine tumor marker yet, probably due to difficulties in finding the appropriate cut-off for the serum level. The aim of this study was to investigate whether assessment of changes in serum Cyfra 21-1 over time can predict distant metastases in patients with SCCHN, without attempting to establish an arbitrary cut-off for abnormal levels. METHODS: Cyfra 21-1 serum levels of 25 patients with SCCHN and distant metastases were evaluated by means of an ELISA test kit. RESULTS: There was a wide range of Cyfra 21-1 serum levels at the time of primary diagnosis, without correlation with tumor size, lymph node status, time to recurrence, or presence of distant metastases. All patients had a clear increase of Cyfra 21-1 levels which preceded the appearance of distant metastases clinically. CONCLUSIONS: Due to the wide range of Cyfra 21-1 levels at the time of primary tumor diagnosis, Cyfra-21-1 is neither a suitable screening marker for SCCHN, nor for diagnosis of distant metastases at the time of initial diagnosis of the tumor, but is of evident prognostic value for follow-up, especially for early detection of distant metastases.     

Monitoring of therapy in head and neck patients during the radiotherapy by measurement of Cyfra 21-1.

PURPOSE: Cyfra 21-1, measuring serum fragments of cytokeratin 19, has been found to be related to tumour stage and tumour size in patients with cervical cancer. It could be a promising marker in squamous lung cancer. We evaluated this new marker with carcinoembryonic antigen, (CEA) and squamous cell carcinoma antigen (SCC-Ag) in the monitoring of 27 patients with head and neck cancer. PATIENTS AND METHODS: The retrospective study group consisted of 27 patients, 17 not suited for surgery and 10 after laser resection. Patients were clinically staged according to the TNM-classification. The mean age of the patients was 53 years (range 37-70 years). Serum levels of each marker were studied in relation to tumour stage and clinical status of the patients during radiotherapy and 6 weeks after the end of the treatment. The clinical performance of the various assays to separate those patients with complete remission from those patients with the presence of tumour was assessed. RESULTS: Pre-treatment serum Cyfra 21-1, CEA, and SCC-Ag levels were not related to stage of disease and were not found to be predictive of tumour response. The clinical performance of post-treatment serum SCC-Ag levels in predicting the presence of tumour was not better than the Cyfra 21-1 assays. CONCLUSION: We could not conclude from this study that Cyfra 21-1 marker is an additional parameter in identifying patients at risk of residual tumour after treatment, recurrent or progressive disease. An elevation of cyfra 21-1 marker was not detectable in 70% of the cases with macroscopic tumour. Therefore, Cyfra 21-1 is not a reliable parameter for the monitoring of patients with head and neck cancer during radiotherapy.     

食管癌患者血清CEA、SCC和Cyfra21-1含量检测及临床意义

目的：探讨血清肿瘤标志物癌胚抗原(CEA)、鳞状细胞癌相关性抗原(SCC)和角化素蛋白片段19(Cyfra21-1)在食管癌的诊断、治疗和预后判断及随访中的作用。方法：以电发光免疫测定法(ECLIA)和微粒酶联免疫测定法(MEIA)检测206例食管癌患者术前和其中71例术后血清中CEA、Cyfra21-1和SCC的水平。检测结果采用SPSS10．0统计软件进行t检验和X^2检验。结果：肿瘤体积愈大、病期愈晚、肿瘤浸润愈深,患者术前血清CEA、SCC和Cyfra21-1总体水平愈高,早期患者水平较低。三者中,CEA和Cyfra21-1的个体差异较大,Cyfra21-1相关性最好。术后检测血清的71例中,92．9％的患者三种血清标志物降至正常。全组患者CEA和Cyfra21-1的阳性率分别为29．1％和45．1％,两者联合检测阳性率为57．3％。165例手术切除者Ⅰ、Ⅱ、Ⅲ期的CEA阳性率分别为16．6％、26．8％和30．8％;Cyfra21-1分别为27．8％、37．5％和50．5％;两者联合检测阳性率分别为38．9％、50．0％和63．7％。结论血清CEA、SCC、Cyfra21-1联合检测可用于食管癌的辅助诊断以及对病期及预后的判断。三者中Cyfra21-1更有意义。     

Comparison of Cyfra 21-1 and SCC assays in head and neck tumours.

Patients with head and neck tumours (HNT) have a high risk of early locoregional relapse that is difficult to diagnose. This study evaluated the usefulness of the serum Cyfra 21-1 assay compared to squamous cell carcinoma antigen (SCC) assay for monitoring such patients. Three hundred and twelve HNT patients, including 204 newly diagnosed patients, were followed up for a median of 446 days with serial serum assays for SCC and Cyfra 21-1. Untreated patients showed SCC and Cyfra 21-1 serum levels correlated with each other: concentration was correlated to clinical stage, tumour size (as T1 + T2 vs. T3 + T4) and nodal status. Cyfra 21-1, but not SCC, was related to the presence of metastases and the primary tumour site, with a univariate prognostic value for disease-free survival (p = 0.015). Cox's regression analysis showed that only Cyfra 21-1 was associated with a risk of relapse (p = 0.027). The random coefficient growth curve model applied to serial SCC and Cyfra 21-1 measurements of 111 patients showed that only Cyfra 21-1 exhibited a significant difference between patients with and without relapses. We found Cyfra 21-1 to be more closely related to initial clinical data and disease evolution than SCC, and therefore propose the use of Cyfra 21-1 for monitoring head and neck cancers.