Abstract 5669: Performance characterization of the Cellometer Ascend automated cell counter following ISO cell counting standards

Abstract In modern therapeutics, cell counting and viability are extremely common, yet vital measurements in research, process development, and manufacturing for novel discoveries and development. Many challenges are associated with these frequent readouts, specifically due to the absence of reference materials, cell sample heterogeneity, stability of living cells, complexity of cell sample preparation, and differences in chemical staining methods. The International Organization for Standardization (ISO) has published two documents to help support cell counting procedures: ISO 20391 - 1:2018 Biotechnology - Cell Counting - Part I: General Guidance on Cell Counting Methods; and ISO 20391 - 2:2019 Biotechnology - Cell Counting - Part II: Experimental Design and Statistical Analysis to Quantify Counting Method Performance. The Cellometer Ascend, Revvity’s next generation automated cell counter, has been designed to provide reliable and consistent cell counting and viability measurements as an all-in-one standalone instrument with a user-friendly streamlined workflow allowing for cell counting at a larger concentration range. In this work, we characterized the cell counting performance of the Cellometer Ascend, following ISO cell counting standards. Linearity tests using Jurkat cells showed that Cellometer Ascend reliably covered a concentration range from 2.6 × 104 cells/mL to 4 × 107 cells/mL, with a precision below 10%. A truncated version of the ISO cell counting Part II protocol was developed internally to evaluate the performance and compare quality metrics for four distinct cell counting methods. These methods were compared simultaneously to minimize the variation from cell sample stability and results revealed comparable mean cell counts, precision, coefficient of determination (R2), and proportionality indices (PI). We further conducted multiple cell counting comparisons using the Bland-Altman comparative analysis, and showed that the Cellometer Ascend was in line with the other three instruments with a less than 6% difference observed in all cases. In addition, we investigated instrument-to-instrument consistency using two different concentrations of fluorescent beads on five Cellometer Ascend instruments. Results from the 5 × 106 beads/mL and 2.5 × 105 beads/mL concentrations showed coefficient of variation (CV) of 0.8% and 2.2%, respectively. In conclusion, the Cellometer Ascend can be used to provide high-quality cell counts and viability readouts according to ISO Cell Counting standards, is comparable to other cell counting methodologies, and could therefore be the benchtop solution for multiple cell counting needs. Citation Format: Carolina Franco Nitta, Yongyang Huang, Daniel Browe, Allen Lin, Sopaul Hem, Leo L. Chan, Bo Lin, Dmitry Kuksin. Performance characterization of the Cellometer Ascend automated cell counter following ISO cell counting standards [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5669.