WO2022001824A1 - 一种检测小细胞肺癌患者外周血循环肿瘤细胞pd-l1基因突变的试剂盒及检测方法 - Google Patents
一种检测小细胞肺癌患者外周血循环肿瘤细胞pd-l1基因突变的试剂盒及检测方法 Download PDFInfo
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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Definitions
- the invention provides a kit and a detection method for detecting PD-L1 gene mutation in peripheral blood circulating tumor cells of patients with small cell lung cancer, belonging to the technical field of molecular biology.
- Lung cancer is one of the main malignant tumors that cause the death of cancer patients. In my country, the incidence and mortality of lung cancer are the first. Small cell lung cancer (SCLC) accounts for about 15% to 20% of the incidence of lung cancer. Compared with non-small cell lung cancer, it has the characteristics of faster tumor doubling time, rapid growth and early metastasis.
- SCLC Small cell lung cancer
- Circulating tumor cells are tumor cells that are shed from solid tumors and enter the peripheral blood circulation. Since their discovery in 1989, various methods have been used to detect circulating tumor cells in peripheral blood. Recent studies have shown that its detection has important clinical significance for evaluating the prognosis of tumor patients, especially those with advanced tumors, and selecting appropriate individualized therapy. Because CTC detection has the characteristics of minimally invasive and real-time detection, it is called "liquid biopsy" of tumors.
- Shandong First Medical University, Shandong Pharmaceutical Research Institute and Shandong Kaige Intelligent Machine Co., Ltd. are cooperating on the development and production of key technologies, detection equipment, and kits for the detection and identification of circulating tumor cells.
- Shandong Qixin Biotechnology Co., Ltd., Shandong Yuxiao Biotechnology Co., Ltd., Jinan Xingen Biotechnology Co., Ltd., Shandong Discovery Biotechnology Co., Ltd. and other units have promoted and applied. This project is a major scientific and technological innovation project in Shandongzhou.
- the research institute is the core, implements the registrant system, relies on the core diagnostic technology of circulating tumor cell detection and identification, and further registers the identification and diagnosis kits, including PD1, PD-L1, ER, PR, Her-2, GPC-3, VEGF, P53 , Vimentin, TKI-EGFR, RAS, CK, ALK-D5F3, CD20, ALK/EML4, Beta-catenin, E-Cadherin, EP-CAM, HPV, IDH-1, PSA, PSMA, VEGF, GFAP, cytokeratin , AE1/AE3, estrogen receptor, progesterone receptor, BCA-225, CA 125, CEA, EMA, ERCC1, HPV, Ki-67, P53, TOP2A, etc.
- the efficacy of PD-1 or PD-L1 immunotherapy is mostly related to the immunohistochemical expression level of PD-L1 in tumor tissue, suggesting that PD-L1 expression level may be a biomarker to predict the efficacy of PD-1 immunotherapy; there are also studies It indicated that the high expression of PD-L1 in small cell lung cancer tissues was positively correlated with tumor invasiveness. Therefore, detection of PD-L1 expression in circulating tumor cells (CTCs) is of great value for the prognosis of small cell lung cancer and the evaluation of the efficacy of immunotherapy.
- CTCs circulating tumor cells
- the present invention provides a non-diagnostic non-diagnostic PD-L1 gene mutation in peripheral blood circulating tumor cells of patients with small cell lung cancer.
- Objective Detection method To separate and obtain CTCs in peripheral blood of patients with advanced or recurrent small cell lung cancer whose tissue specimens cannot be obtained by membrane filtration device, and further use immunohistochemistry to detect the expression of PD-L1 in CTCs.
- the invention provides a kit for detecting PD-L1 gene mutation in peripheral blood circulating tumor cells of patients with small cell lung cancer, including 45 mL of diluent, 1 mL of decolorizing solution, 0.5 mL of staining solution A, 1 mL of staining solution B, PD-L1 (human) 100 ⁇ L of primary antibody, 100 ⁇ L of goat anti-human IgG/HRP, 100 ⁇ L of 0.1% Triton X-100, 100 ⁇ L of 0.3% H 2 O 2 , 0.5 mL of reagent A, 1 mL of reagent B, 60 mL of 6 ⁇ PBS buffer; The pH is 7.4.
- the diluent is composed of 1mmol/L EDTA+0.1%BSA+0.1%trehalose+0.2% polyoxyethylene polyoxypropylene ether block copolymer.
- the decolorizing solution is composed of 95% alcohol and 100% xylene in a volume ratio of 1:1.
- the staining solution A is DAB staining solution
- the staining solution B is hematoxylin staining solution.
- the reagent A is a 0.6% hydroxypropyl methylcellulose aqueous solution
- the reagent B is composed of ethanol and 1,2-propanediol in a volume ratio of 3:1.
- the present invention also provides a method for detecting PD-L1 gene mutation in peripheral blood circulating tumor cells of patients with small cell lung cancer using the above-mentioned kit for non-diagnostic purposes, which is characterized by comprising the following steps:
- the specific method of the present invention detecting the PD-L1 expression of CTC is as follows:
- the membrane filtration device for separating tumor cells used in the present invention includes a filter, a blood sample container, a waste liquid tank and an iron stand, the iron stand is provided with a base, a stand and a bracket, and the blood sample container is arranged on the upper part of the iron stand through the stand, Below the blood sample container is a filter, and the filter is connected to a waste liquid tank through an infusion set, and the waste liquid tank is arranged on the base.
- the filter includes an upper filter port, a filter membrane, a filter-carrying membrane platform and a filter lower port, the filter membrane is placed on the filter-carrying membrane platform; the upper filter port is connected to the blood sample container, and the lower port of the filter is connected to a waste liquid tank through an infusion device.
- the filter membrane is made of hydrophobic material, and is evenly covered with filter holes with a diameter of 8 microns.
- the detection method provided by the present invention can detect the PD-L1 expression in patients with advanced or recurrent small cell lung cancer without obtaining tissue samples by needle biopsy.
- the technique is minimally invasive and enables real-time detection.
- the method provided by the present invention can avoid false positive results caused by edge effects that may occur in the dyeing process, has good stability, reduces cell loss, and improves detection accuracy.
- FIG. 1 is a schematic structural diagram of a membrane filtration device of the present invention
- FIG. 2 is a schematic cross-sectional view of the structure of the filter of the membrane filtration device of the present invention
- Fig. 3 is the structural representation of the filter membrane of the membrane filtration device of the present invention.
- Figure 4 is an image of circulating tumor cells obtained from peripheral blood of lung cancer patients
- the filter device is composed of a filter 3, a filter membrane 7, a blood sample container 2, a waste liquid tank 5, and an iron stand 1;
- the diameter of tumor cells is generally greater than 15 microns, and the diameter of blood cells (including red blood cells and white blood cells) is generally less than 8 microns. Therefore, when peripheral blood containing CTCs is filtered, the blood cells can be filtered because the diameter is smaller than the filter hole 10, while the diameter of CTCs is larger than The filter pores 10 are trapped on the filter membrane 7 .
- the filter was dried and observed under a microscope to determine the presence of CTCs.
- the detection accuracy after solid-sealing can reach 100%.
- the accuracy rate of single ethanol is 85%, while the accuracy rate of single 1,2-propanediol is only 70%, which can avoid false positive results caused by edge effects that may occur during the staining process. loss and improve the detection accuracy.
- Figure 4 is an image of circulating tumor cells isolated from peripheral blood of patients with small cell lung cancer.
- the nuclei are large, the cell diameter (long end) is greater than 15 ⁇ m, the shape of the nucleus is irregular, and the ratio of nuclear to cytoplasm is high.
- the detected circulating tumor cells were confirmed by immunohistochemistry to confirm the expression of PD-L1 and compared with the results of PD-L1 in the gross specimens of small cell lung cancer to observe the differences, mainly for patients whose gross specimens were negative for PD-L1 expression but positive for circulating tumor cells. , guide the targeted therapy of small cell lung cancer, and provide new ideas for the targeted therapy of small cell lung cancer.
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Abstract
一种检测小细胞肺癌患者外周血循环肿瘤细胞PD-L1基因突变的试剂盒及检测方法,属于分子生物学技术领域。试剂盒包括稀释液、脱色液、染色液A、染色液B、PD-L1(人)一抗、山羊抗人IgG/HRP、0.1%Triton X-100、0.3%H 2O 2、试剂A、试剂B、6×PBS缓冲液;提供的检测方法,不用穿刺活检获取组织标本即可检测到晚期或复发小细胞肺癌患者PD-L1表达情况。技术属于微创,能够实时检测。能够避免染色过程中可能产生的边缘效应导致的假阳性结果,稳定性好,降低细胞的损失,提高检测的准确性。
Description
本发明提供了一种检测小细胞肺癌患者外周血循环肿瘤细胞PD-L1基因突变的试剂盒及检测方法,属于分子生物学技术领域。
肺癌是导致癌症患者死亡的主要恶性肿瘤之一,在我国,肺癌的发病率和死亡率均居第一位。小细胞肺癌(small cell lung cancer,SCLC)约占肺癌发生率的15%~20%,相较非小细胞肺癌,具有更快的肿瘤倍增时间,快速生长以及易于早期转移的特点。
循环肿瘤细胞(Circulating tumor cell,CTC)是从实体肿瘤脱落进入外周血液循环的肿瘤细胞,自1989年被发现以来,目前已有多种方法用于外周血循环肿瘤细胞的检测。近期研究表明,其检测对于评估肿瘤患者尤其是晚期肿瘤患者的预后以及选择合适的个体化治疗具有重要的临床意义。因CTC检测具有微创、实时检测等特点,被称为肿瘤的“液态活检”。
目前,山东省第一医科大学、山东省药物研究院联合山东凯歌智能机器有限公司就循环肿瘤细胞检测鉴定关键技术、检测设备、试剂盒开发与生产进行合作,山东祺欣生物科技有限公司、山东喻晓生物科技有限公司、济南杏恩生物科技有限公司、山东发现生物技术有限公司等单位进行推广应用,本项目为山东省重大科技创新工程项目,以山东第一医科大学济南校区的山东省药物研究院为核心,落实注册人制度,依托循环肿瘤细胞检测鉴定核心诊断技术,进一步注册鉴定诊断试剂盒,以包括PD1、PD-L1、ER、PR、Her-2、GPC-3、VEGF、P53、Vimentin、TKI-EGFR、RAS、CK、ALK-D5F3、CD20、ALK/EML4、Beta-catenin、E-Cadherin、EP-CAM、HPV、IDH-1、PSA、PSMA、VEGF、GFAP、细胞角蛋白、AE1/AE3、雌激素受体、孕激素受体、BCA-225、CA 125、CEA、EMA、ERCC1、HPV、Ki-67、P53、TOP2A等作为CTCs表达的示踪剂,注册超灵敏、超快速、高覆盖、低成本、准确特异的鉴定诊断试剂盒,通过与在济南注册的山东凯歌智能机器有限公司、山东祺欣生物科技有限公司、山东喻晓生物科技有限公司、济南杏恩生物科技有限公司、山东发现生物技术有限公司合作进行研发与推广。
随着分子生物学的发展,PD-1/PD-L1为免疫靶点的免疫疗法为小细胞肺癌治疗带来了新的曙光。研究发现,免疫抑制与免疫逃逸和肿瘤细胞PD-L1的过表达密切相关,肿瘤 细胞可通过其表面的PD-L1与免疫细胞T细胞表面的PD-1结合,传导抑制性信号,使得T细胞不能识别肿瘤细胞和向肿瘤细胞发出攻击信号,导致了肿瘤细胞免疫逃逸。基于此理论提出假设,自原发灶脱落进入循环系统的循环肿瘤细胞(CTC)出现凋亡、免疫清除或存活、转移结局与PD-L1表达密切相关。PD-1或PD-L1免疫制剂的疗效多与肿瘤组织中PD-L1的免疫组化表达水平有关,提示PD-L1表达水平可能是预测PD-1免疫治疗疗效的生物标志物;还有研究表明小细胞肺癌组织中PD-L1的高表达与肿瘤侵袭性呈正相关。因此,检测循环肿瘤细胞(CTC)PD-L1表达情况对小细胞肺癌预后及免疫治疗疗效评估具有重要价值。
发明内容
为了克服晚期或复发小细胞肺癌患者无法实时或反复穿刺获取组织标本、进而不能评估患者PD-L1状态的不足,本发明提供了一种小细胞肺癌患者外周血循环肿瘤细胞PD-L1基因突变非诊断目的的检测方法:利用膜过滤装置分离获得无法获取组织标本的晚期或复发小细胞肺癌患者外周血中的CTC,进一步运用免疫组化技术检测CTC的PD-L1表达情况。
本发明采用的技术方案如下:
本发明提供了一种检测小细胞肺癌患者外周血循环肿瘤细胞PD-L1基因突变的试剂盒,包括稀释液45mL、脱色液1mL、染色液A 0.5mL、染色液B 1mL、PD-L1(人)一抗100μL、山羊抗人IgG/HRP 100μL、0.1%Triton X-100 100μL、0.3%H
2O
2 100μL、试剂A 0.5mL、试剂B 1mL、6×PBS缓冲液60mL;所述PBS缓冲液的pH值为7.4。
进一步的,所述稀释液是由1mmol/L EDTA+0.1%BSA+0.1%海藻糖+0.2%聚氧乙烯聚氧丙烯醚嵌段共聚物组成。
进一步的,所述脱色液是由95%酒精与100%二甲苯按容积比1:1组成。
进一步的,所述染色液A为DAB染色液;所述染色液B为苏木素染色液。
进一步的,所述试剂A为0.6%的羟丙基甲基纤维素水溶液;所述试剂B为乙醇和1,2-丙二醇按照体积比3:1组成。
本发明还提供了一种利用上述试剂盒非诊断目的检测小细胞肺癌患者外周血循环肿瘤细胞PD-L1基因突变的方法,其特征在于,包括以下步骤:
(1)利用膜过滤装置分离获取无法获得组织标本的晚期或复发小细胞肺癌患者外周血中的CTC:采集无法获取组织标本的晚期或复发小细胞肺癌患者外周血:肘正中静脉外周血5ml;
(2)外周血样预处理:将采集的外周血样采用稀释液进行10倍稀释,稀释后加多聚甲醛 固定外周血样10分钟,固定终浓度为0.25%;
(3)利用膜过滤分离肿瘤细胞装置过滤外周血样,分离获得外周血CTC:将预处理的外周血样加入到膜过滤分离肿瘤细胞装置的血样容器中,使其依靠重力自然过滤;
(4)过滤结束后,从膜过滤分离肿瘤细胞装置中取下滤器,将循环肿瘤细胞染色液A液0.5ml加入到滤器中,染色3min,PBS缓冲液冲洗干净;滤液过滤完全后加入染色液B液1ml,染色2min,纯水1ml冲洗2次,取下滤膜,放置在载玻片上,干燥后在显微镜下观察,确定是否存在CTC;
(5)运用免疫组化技术检测CTC的PD-L1表达情况。
本发明检测CTC的PD-L1表达的具体方法如下:
(1)脱色:将带有CTC的滤膜从载玻片上取下,置于脱色液中浸泡4-6小时,脱去CTC染色液;
(2)滴加100μl 0.1%Triton X-100,室温孵育15min,DI水洗2min×3次;
(3)滴加100μl 0.3%H
2O
2,室温孵育10min,PBS洗2min×3次;(4)滴加100μlPD-L1(人)一抗,室温孵育2h或4℃过夜,PBS洗2min×3次;
(5)滴加100μl山羊抗人IgG/HRP,18~26℃温度下孵育20min,PBS洗2min×3次;
(6)滴加100μl DAB显色液,18~26℃孵育并随时在显微镜下观察显色情况,观察时间为3~10min;
(7)显色完成后,弃掉DAB显色液,流水冲洗5min,苏木素染色5min;
(8)盐酸酒精分化8秒,自来水返蓝5min;
(9)将返蓝后的CTC采用75%乙醇(1min),95%乙醇(1min),100%乙醇(1min)梯度乙醇脱水,然后加入0.5mL试剂A,振荡均匀后,加入1mL试剂B,摇动混合均匀后,离心沉淀,将沉淀物采用中性树脂封固;
(10)光学显微镜下镜检。
本发明所使用的膜过滤分离肿瘤细胞装置,包括滤器、血样容器、废液缸和铁架台,所述铁架台设有底座、立架和支架,所述血样容器通过支架设置于铁架台上部,血样容器的下方为滤器,滤器通过输液器联通至废液缸,废液缸设置于底座上。
所述滤器包括滤器上口、滤膜、载滤膜平台和滤器下口,滤膜置于载滤膜平台上;滤器上口接血样容器,滤器下口通过输液器接废液缸。
所述滤膜为疏水材料制成,其上均匀布满口径为8微米的滤孔。
本发明的有益效果是:
(1)本发明提供的检测方法,不用穿刺活检获取组织标本即可检测到晚期或复发小细胞肺癌患者PD-L1表达情况。该技术属于微创,并能够实时检测。
(2)本发明提供的方法,能够避免染色过程中可能产生的边缘效应导致的假阳性结果,稳定性好,降低细胞的损失,提高检测的准确性。
图1为本发明的膜过滤装置结构示意图;
图2为本发明膜过滤装置的滤器的结构示意剖视图;
图3为本发明膜过滤装置的滤器滤膜的结构示意图;
图4为肺癌患者外周血分离获取的循环肿瘤细胞影像图;
图中:1铁架台、2血样容器、3滤器、4输液器、5废液缸、6滤器上口、7滤膜、8载滤膜平台、9滤器下口、10滤孔、11底座、12立架、13支架。
下面结合附图和实施例对本发明阐述如下。
本发明所使用的试剂盒具体规格如表1所示:
表1
组分 | 含量 |
6×PBS缓冲液 | 60mL |
稀释液 | 45mL |
脱色液 | 1mL |
染色液A | 0.5mL |
染色液B | 1mL |
PD-L1(人)一抗 | 100μL |
山羊抗人IgG/HRP | 100μL |
0.1%Triton X-100 | 100μL |
0.3%H 2O: | 100μL |
试剂A | 0.5mL |
试剂B | 1mL |
运用此技术方法分离获取并鉴定9例小细胞肺癌患者(同时检测9例正常人样本做阴性对照)外周血循环肿瘤细胞的实施例。
实施例1
一、利用膜过滤装置分离获取无法获得组织标本的晚期或复发小细胞肺癌患者外周血中的CTC,确定CTC是否存在:
自肘正中静脉采集空腹8-12小时的空腹血5ml,用45ml稀释液(成分:1mmol/L EDTA+0.1%BSA+0.1%海藻糖+0.2%聚氧乙烯聚氧丙烯醚嵌段共聚物)稀释外周血,然后加入3ml的4%多聚甲醛固定稀释后的血样10分钟;
在固定的间期,组装膜过滤装置:如附图1、图2、图3所示,该过滤装置由滤器3、滤膜7、血样容器2、废液缸5、铁架台1构成;
用10mlPBS润湿滤器3,然后将固定好的外周血样加入到膜过滤装置的血样容器2中,使其依靠重力自然过滤,CTC被截留在滤膜7上;
肿瘤细胞直径一般大于15微米,而血细胞(包括红细胞、白细胞)直径一般小于8微米,因此当含有CTC的外周血经过滤后,血细胞因直径小于滤孔10能够被滤过,而CTC因直径大于滤孔10被截留在滤膜7上。
过滤结束后,从过滤装置中取下滤器3,打开并移走滤器上口6,将将循环肿瘤细胞染色液A液0.5ml加入到滤器中,染色3min,PBS缓冲液冲洗干净;滤液过滤完全后加入B液,1ml,染色2min,纯水1ml,PBS缓冲液将滤器3冲洗干净,用眼科镊子取下滤膜7,细胞面朝上,放置在载玻片上;
将滤膜干燥后在显微镜下观察,确定是否存在CTC。
通过观察,8例健康志愿者均未查到CTC;除1例复发小细胞肺癌患者未检测到CTC外,其余均检测到CTC(表2),本次检测阳性率为77.8%,值得注意的是,当稀释液不添加0.1%海藻糖或者不添加0.2%聚氧乙烯聚氧丙烯醚嵌段共聚物时,单一的采用0.3%海藻糖或者0.3%聚氧乙烯聚氧丙烯醚嵌段共聚物,制备的血样稳定性差,部分血样还会形成分层,血液细胞容易发生聚集和粘连,影响最终的检测效果。
表2实施例CTC检测结果
二、运用免疫组化技术检测CTC的EGFR表达情况:
将载玻片上载有CTC的滤膜7从载玻片上取下,置于95%酒精与100%二甲苯按容积比1:1混匀的脱色液中浸泡4-6小时,脱去CTC染色液;滴加100μl 0.1%Triton X-100,室温孵育15min,DI水洗2min×3次;滴加100μl 0.3%H
2O
2,室温孵育10min,PBS洗2min×3次;滴加100μl PD-L1(人)一抗,室温孵育2h(或4℃过夜),PBS洗2min×3次;滴加100μl山羊抗人IgG/HRP,室温(18~26℃)孵育20min,PBS洗2min×3次;滴加100μl DAB显色液,室温(18~26℃)孵育并随时在显微镜下观察显色情况(一般为3~10min,时间不能超过10min);显色完成后,弃掉DAB显色液,流水冲洗5min,苏木素染色5min;盐酸酒精分化8秒,自来水返蓝5min;75%乙醇(1min),95%乙醇(1min),100%乙醇 (1min)梯度乙醇脱水,然后加入0.6%的羟丙基甲基纤维素水溶液,振荡均匀后,加入乙醇和1,2-丙二醇混合溶剂(V:V=3:1),摇动混合均匀后,离心沉淀,将沉淀物晾干,中性树脂封固;光学显微镜下镜检,细胞病理学专家阅片,根据细胞膜和细胞浆着色程度判定PD-L1表达情况。
当将试剂B采用单一的乙醇或者1,2-丙二醇时,采用中性树脂固封后,本发明乙醇和1,2-丙二醇混合溶剂,固封后的检测准确率能够达到100%,而采用单一的乙醇的准确率为85%,而采用单一的1,2-丙二醇的准确率则仅为70%,能够避免染色过程中可能产生的边缘效应导致的假阳性结果,稳定性好,降低细胞的损失,提高检测的准确性。
图4为小细胞肺癌患者外周血分离获取的循环肿瘤细胞影像图,其细胞核较大,细胞直径(长端)大于15μm,细胞核形状不规则;高核质比。
所检测的循环肿瘤细胞应用免疫组化证实PD-L1的表达并与小细胞肺癌大体标本PD-L1结果对比,观察其差异,主要针对大体标本PD-L1表达阴性而循环肿瘤细胞表达阳性的患者,指导小细胞肺癌的靶向治疗,为小细胞肺癌靶向治疗提供新的思路。
Claims (7)
- 一种检测小细胞肺癌患者外周血循环肿瘤细胞PD-L1基因突变的试剂盒,其特征在于,包括稀释液45mL、脱色液1mL、染色液A 0.5mL、染色液B 1mL、PD-L1(人)一抗100μL、山羊抗人IgG/HRP 100μL、0.1%Triton X-100 100μL、0.3%H 2O 2 100μL、试剂A 0.5mL、试剂B 1mL、6×PBS缓冲液60mL;所述PBS缓冲液的pH值为7.4。
- 根据权利要求1所述的试剂盒,其特征在于,所述稀释液是由1mmol/L EDTA+0.1%BSA+0.1%海藻糖+0.2%聚氧乙烯聚氧丙烯醚嵌段共聚物组成。
- 根据权利要求1所述的试剂盒,其特征在于,所述脱色液是由95%酒精与100%二甲苯按容积比1:1组成。
- 根据权利要求1所述的试剂盒,其特征在于,所述染色液A为DAB染色液;所述染色液B为苏木素染色液。
- 根据权利要求1所述的试剂盒,其特征在于,所述试剂A为0.6%的羟丙基甲基纤维素水溶液;所述试剂B为乙醇和1,2-丙二醇按照体积比3:1组成。
- 一种利用权利要求1-5任一项所述的试剂盒非诊断目的检测小细胞肺癌患者外周血循环肿瘤细胞PD-L1基因突变的方法,其特征在于,包括以下步骤:(1)利用膜过滤装置分离获取无法获得组织标本的晚期或复发小细胞肺癌患者外周血中的CTC:采集无法获取组织标本的晚期或复发小细胞肺癌患者外周血:肘正中静脉外周血5ml;(2)外周血样预处理:将采集的外周血样采用稀释液进行10倍稀释,稀释后加多聚甲醛固定外周血样10分钟,固定终浓度为0.25%;(3)利用膜过滤分离肿瘤细胞装置过滤外周血样,分离获得外周血CTC:将预处理的外周血样加入到膜过滤分离肿瘤细胞装置的血样容器中,使其依靠重力自然过滤;(4)过滤结束后,从膜过滤分离肿瘤细胞装置中取下滤器,将循环肿瘤细胞染色液A液0.5ml加入到滤器中,染色3min,PBS缓冲液冲洗干净;滤液过滤完全后加入染色液B液1ml,染色2min,纯水1ml冲洗2次,取下滤膜,放置在载玻片上,干燥后在显微镜下观察,确定是否存在CTC;(5)运用免疫组化技术检测CTC的PD-L1表达情况。
- 根据权利要求6所述的检测方法,其特征在于,所述检测CTC的PD-L1表达的具体方法如下:(1)脱色:将带有CTC的滤膜从载玻片上取下,置于脱色液中浸泡4-6小时,脱去CTC染色液;(2)滴加100μl 0.1%Triton X-100,室温孵育15min,DI水洗2min×3次;(3)滴加100μl 0.3%H 2O 2,室温孵育10min,PBS洗2min×3次;(4)滴加100μl PD-L1(人)一抗,室温孵育2h或4℃过夜,PBS洗2min×3次;(5)滴加100μl山羊抗人IgG/HRP,18~26℃温度下孵育20min,PBS洗2min×3次;(6)滴加100μl DAB显色液,18~26℃孵育并随时在显微镜下观察显色情况,观察时间为3~10min;(7)显色完成后,弃掉DAB显色液,流水冲洗5min,苏木素染色5min;(8)盐酸酒精分化8秒,自来水返蓝5min;(9)将返蓝后的CTC采用75%乙醇(1min),95%乙醇(1min),100%乙醇(1min)梯度乙醇脱水,然后加入0.5mL试剂A,振荡均匀后,加入1mL试剂B,摇动混合均匀后,离心沉淀,将沉淀物采用中性树脂封固;(10)光学显微镜下镜检。
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CN111638341A (zh) * | 2020-07-01 | 2020-09-08 | 山东凯歌智能机器有限公司 | 一种检测小细胞肺癌患者外周血循环肿瘤细胞pd-l1基因突变的试剂盒及检测方法 |
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