FACSCanto II and LSRFortessa flow cytometer instruments can be synchronized utilizing single-fluorochrome-conjugated surface-dyed beads for standardized immunophenotyping

Abstract

Background: Multiparameter flow cytometry is the preferred method to determine immunophenotypic features of cells present in a wide variety of sample types. Standardization is key to avoid inconsistencies and subjectivity of interpretations between clinical diagnostic laboratories. Among these standardization requirements, synchronization between different flow cytometer instruments is indispensable to obtain comparable results. This study aimed to investigate whether two widely used flow cytometers, the FACSCanto II and LSRFortessa, can be effectively synchronized utilizing calibration bead–based synchronization. Method: Two FACSCanto II and two LSRFortessa flow cytometers were synchronized with both multicolor hard-dyed and single-fluorochrome–conjugated surface-dyed beads according to the manufacturer's instructions. Cell staining was performed on five whole-blood samples obtained from healthy controls and were analyzed upon synchronization with the respective synchronization protocols. Results: Comparability criteria (defined as <15% deviation from the reference instrument) were met with both bead sets when synchronizing different FACSCanto II or LSRFortessa instruments. However, we observed that the criteria could not be met when synchronizing FACSCanto II with LSRFortessa instruments with multicolor hard-dyed beads. By utilizing single-fluorochrome–conjugated surface-dyed beads to determine and adjust PMT voltages, the accepted comparability criteria were successfully met. The protocol has been validated using five different eight-parameter stained samples. Conclusion: We show that FACSCanto II and LSRFortessa instruments can effectively be synchronized using single-fluorochrome–conjugated surface-dyed beads in case deviation criteria cannot be met using multicolor hard-dyed beads. Synchronization with single-fluorochrome–conjugated surface-dyed beads results in decreased deviations between instruments, allowing comparability criteria to become stricter.