快速微生物检测方法在肿瘤浸润淋巴细胞(TIL)产品中的应用  

Application of Rapid Microbiological Assay in Tumor-Infiltrating Lymphocyte(TIL)Products

作　　者：王涛 周足力[2] 初明[3,4] 王星星 董兆惠 王铁山 陈萌 张秀军 WANG Tao;ZHOU Zuli;CHU Ming;WANG Xingxing;DONG Zhaohui;WANG Tieshan;CHEN Meng;ZHANG Xiujun(The Fifth Medical Center of the PLA General Hospital,Beijing 100039,China;People′s Hospital of Peking University,Beijing 100044,China;School of Basic Medical Sciences of Peking University,Beijing 100191,China;Key Laboratory of Medical Immunology,National Health Commission,Beijing 100191,China;Aeonvital Institute of Clinical and Evolutionary Immunology,Beijing 102600,China;Beijing University of Chinese Medicine,Beijing 100029,China;China National Institute of Standardization,Beijing 100191,China)

机构地区：[1]解放军总医院第五医学中心,北京100039 [2]北京大学人民医院,北京100044 [3]北京大学基础医学院,北京100191 [4]国家卫生健康委员会医学免疫学重点实验室,北京100191 [5]北京循生免疫医学转化研究院,北京102600 [6]北京中医药大学,北京100029 [7]中国标准化研究院,北京100191

出　　处：《中国医药导刊》2024年第4期328-337,共10页Chinese Journal of Medicinal Guide

摘　　要：目的:采用呼吸信号法和定量聚合酶链反应(QPCR)技术对肿瘤浸润淋巴细胞(TIL)产品的无菌检测进行评估,旨在研究高效、可靠的实验方法,以快速完成免疫细胞产品微生物检测。方法:提取患者外周血或者肿瘤组织中免疫细胞,按照直接接种法、呼吸信号法以及QPCR法,对2020版《中华人民共和国药典》1101中规定的6种菌进行方法适用性验证。结果:结果显示,在<100 CFU·mL^(-1)条件下,TIL6种菌株在呼吸信号法适用性试验中,0.40~1.53 d检测出阳性信号,在QPCR方法检测适用性试验中,4 h内检出阳性信号。TIL中不存在干扰细菌和真菌检出的成分,两种方法专属性、耐用性良好。呼吸信号法检测限最优,QPCR法检测时间最短。结论:呼吸信号法的灵敏度和培养时间优于传统的检测方法,QPCR法将检测时间从传统无菌检测14 d缩短至4 h,两种方法都具有良好的特异性。因此在新鲜或者冻存等不同细胞使用条件下均可以作为快速检测方法的选择,在细胞治疗产品微生物检验中具有良好的应用前景。Objective:To evaluate sterility detection of tumor-infiltrating lymphocyte(TIL)products by means of respiratory signal-ing method and nucleic acid amplification(QPCR)technology method,to develop an efficient and reliable method for rapid microbial detection of immune cell products.Methods:Live immune cells with short shelf life and limit cell valume,cannot be sampled with traditional microbial detection methods,"This study evaluated sterility detection of tumor-infiltrating lymphocyte(TIL)products by means of respiratory signaling and nucleic acid amplification technology,aiming to develop an efficient and reliable method for rapid microbial detection of immune cell products.With the verification of the 6 strains specified in the 1101 section of 2020 edition of Chinese Pharmaco-poeia,we extract immune cells from the patient's peripheral blood or tumor tissue,andtest TIL product in direct inoculation method of Chinese Pharmacopoeia,respiratory signal method and QPCR method seperately.After in vitro activation,the tumor cells were infused back into the body to kill the tumor cells,at the same time,the immune system of the patients was further activated to achieve the goal of survival or cure with the tumor.Results:Under the condition of<100 CFU·mL^(-1),the respiratory test result showed that the positive signal was within 0.40~1.53 d.While the positive signal was detected within 4 h by QPCR method.There are no components interfering with the detection of bacteria and fungi in TIL products.The two methods have good specificity and durability with the respiratory signal method having the best detection limit and the QPCR method having the shortest detection time.Conclusion:The sensitivity and culture time of respiratory signal method are superior to the traditional direct inoculation method of Chinese Pharmacopoeia.QPCR reduces the detection time from 14 d to 4 hs.Both methods have good specificity.Therefore,they can be used as a rapid detection method under different product conditions such as fresh or frozen,and h

关 键 词：免疫细胞 肿瘤浸润淋巴细胞 定量聚合酶链反应 呼吸信号法 微生物检测 

分 类 号：R730[医药卫生—肿瘤] R37[医药卫生—临床医学]