Case Study of a Cross‐Platform Evaluation of an Optimized Multicolor Flow Cytometry Immunophenotyping Assay for Human Peripheral Whole Blood

Flow cytometry is a commonly used single-cell analysis technique that can be applied for the immunophenotyping of human blood samples. Nowadays, increasing numbers of clinical trials include translational research programs utilizing flow-based immune monitoring assays to assess patients' longitudinal immune status. Over time, flow cytometers require regular calibration, quality controls, maintenance, and need to be replaced by new devices after certain years of operation. When flow-based assays are utilized in clinical studies, it is important to transfer and re-establish them thoroughly on new devices to ensure stable measurements. This project describes a detailed and optimized immunophenotyping assay and its transfer from the Gallios flow cytometer to the DxFlex (both from Beckman Coulter). According to the manufacturer, the DxFlex is intended as a successor to the Gallios and features similar lasers and filters. This enables all antibodies and assayed populations to be represented on the new device. However, after calculating the concentrations of cell populations in the blood, deviations were observed between the two devices. It is concluded that calculated absolute values cannot be compared between the two devices, even though the immunophenotyping assay can be successfully measured on the new DxFlex flow cytometer. Switching this specific IPT assay is not feasible in ongoing clinical trials. When transferring a complex assay, parallel comparative measurements are required to ensure reliable data acquisition, population detection, and result comparability.