Abstract Background Cancer immunotherapy research, immune microenvironment exploration, and biomarker discovery are key application areas of immune checkpoint analysis. PD-1 and CD28 are crucial receptors expressed on the surface of T cells, playing vital roles in regulating T cell activation and immune response. Accurate detection of these immune checkpoints, such as PD-1 and CD28 on lymphocytes, is essential for understanding immune responses, particularly in clinical contexts such as cancer immunotherapy. Flow cytometry offers a precise approach to detect these markers in whole blood samples. Methods This study developed and optimized a flow cytometry-based detection method utilizing the CYTEK NL-CLC flow cytometer to quantitatively assess the expression of PD-1 and CD28 on lymphocytes. A detailed protocol was established and validated, focusing on key performance parameters. Results Our method showed high sensitivity and specificity, providing a powerful tool for immune monitoring and treatment decision-making. Validation results, including precision, dilution linearity, fluorescence stability, reference interval, and accuracy, all met acceptable criteria and have been reviewed and approved for clinical testing. Conclusions The CYTEK NL-CLC flow cytometer is positioned as a reliable and effective platform for immune checkpoint analysis in both clinical and research settings, supporting its integration into cancer immunotherapy workflows and personalized medicine strategies.
Chen et al. (Tue,) studied this question.