Objective: To investigate the optimal experimental conditions (including antigen retrieval time, antibody titers and antibody incubation time) for reliable detection of programmed death-ligand 1 (PD-L1) expression using PD-L1 (22C3) antibody concentrate, and to establish a laboratory developed test for PD-L1 detection. Methods: Using Dako PD-L1 IHC 22C3 pharmDX staining procedure and scoring guidelines as the standard reference (group A), the PD-L1 expression in 25 tissue specimens (including 15 lung cancer tissues, 5 tonsil tissues and 5 placenta tissues) was detected with Flex+/HRP detection kit (EnVision) under 8 different experimental conditions (groups B1 to B8). The staining results were then compared to those in group A. Results: In group B1, 3 tissue samples showed the percentages of PD-L1 positive tumor cells were similar to those in group A, while the percentages of PD-L1 positive tumor cells were lower than those in group A in the other samples. In group B7, two case showed a positive rate higher than that in group A that was also above the positive cut-off value, and the rest of the samples had a percentage of PD-L1 positive tumor cells slightly higher than that in group A, but still below the positive cut-off value. The staining results of group B8 were the closest to those of group A compared with the other groups. Although the percentages of PD-L1 positive tumor cells in the B2 to B6 groups were decreased in various degrees as compared with group A, they were still concordant with group A's classification (positive vs. negative) and would not change the choice of clinical treatments. Conclusions: The experimental conditions are associated with detection rate of PD-L1 expression using 22C3 antibody. In the present study, the most-suitable alterative conditions in the PD-L1 detection using 22C3 antibody concentrate are those applied in the group B8 (including antigen retrieval in Dako PT Link tank at 97 ℃, pH 6.0 for 40 min and incubation with 22C3 antibodies (1∶100 dilution) at room temperature for 60 min, incubation with EnVision Flex+Linker at room temperature for 30 min, incubation with EnVision/HRP at room temperature for 30 min and DAB staining for 5 min), which could provide reliable results at minimum costs.
目的： 摸索程序性死亡配体1（PD-L1，22C3）浓缩抗体最佳处理条件（抗原修复时间、抗体稀释度、抗体孵育时间），稳定PD-L1检测质量，建立PD-L1实验室内部检测体系。 方法： 以PD-L1（22C3）标准检测试剂盒的检测条件、染色结果作为参照，评分标准参照Dako公司 PD-L1 IHC 22C3 pharmDX 标准检测试剂盒的判读标准，采用Dako公司 PD-L1（22C3）浓缩抗体及EnVision Flex+/HRP检测试剂盒与PD-L1（22C3）标准检测试剂盒，检测15例肺癌、5例扁桃体及5例胎盘组织在8种不同处理条件下（B1~B8）的免疫组织化学染色结果，并比对其与PD-L1（22C3）标准检测试剂盒（A组）检测结果的一致性。 结果： 8组不同的处理条件，B1组除弥漫强阳性表达及阴性病例（共3例）阳性肿瘤细胞的百分比与A组接近外，其余病例阳性肿瘤细胞百分比略低于A组；B7组有2例肿瘤细胞的阳性百分比明显高于A组，超出阈值范围，其余病例阳性肿瘤细胞的百分比略高于A组，但在阈值范围内；B8组结果与A组结果最接近；B2~B6组阳性肿瘤细胞的百分比均有不同程度的降低，但在阈值范围。 结论： PD-L1（22C3）检测条件与检测结果密切相关，为达到理想的检测效果同时节约成本，以B8组的检测条件最为合适［用Dako公司 pH6.0 抗原修复液在Dako PT Link 97 ℃修复 40 min；22C3（1∶100）室温孵育60 min；EnVision Flex+Linker室温孵育30 min；EnVision/HRP室温孵育30 min；二氨基联苯胺显色5 min］。.
Keywords: Immunohistochemistry; Laboratory techniques and procedures; PD-L1; Staining and labeling.