Objectives: Given that method validation is mandatory for compliance with the International Organization for Standardization (ISO) 15,189 standard requirements, we evaluated the analytical performance of the AQUIOS CL system (Beckman Coulter) and compared it with two bead-based flow cytometry (FCM) protocols (BD FACSCAnto TM- II and Beckman Coulter DxFLEX). There are no comparative literature data on standardized protocols for counting lymphocyte subsets on the new-generation cytometer DxFLEX., Methods: We evaluated the AQUIOS CL's performance with regard to accuracy, linearity and stability by using dedicated control cell samples and patient samples. We also compared the lymphocyte counts measured on the AQUIOS CL (n=69 samples) with those measured on the BD FACSCAnto TM- II and DxFLEX FCM systems. For 61 samples, FCM results were compared with those measured on the XN-3000 Sysmex hematology analyzer., Results: AQUIOS CL showed acceptable performance - even outside the manufacturer's quantification ranges- and strong correlations with bead-based FCM methods. The FCM techniques and the XN-3000 gave similar absolute lymphocyte counts, although values in samples with intense lymphocytosis (B cell lymphoma/leukemia) were underestimated., Conclusions: The AQUIOS CL flow cytometer is a time-saving, single-platform system with good performance, especially when the manufacturer's instructions for use are followed. However, AQUIOS CL's possible limitations and pitfalls impose validation of a bead-based FCM method for immunophenotyping verification or as a back-up system. Although the DxFLEX flow cytometer is more time-consuming to use, it can provide standardized lymphocyte subset counts in case of aberrant results on AQUIOS CL or in the event of equipment failure., (© 2024 Walter de Gruyter GmbH, Berlin/Boston.)