Normalization of mass cytometry data with bead standards

Mass cytometry uses atomic mass spectrometry combined with isotopically pure reporter elements to currently measure as many as 40 parameters per single cell. As with any quantitative technology, there is a fundamental need for quality assurance and normalization protocols. In the case of mass cytometry, the signal variation over time due to changes in instrument performance combined with intervals between scheduled maintenance must be accounted for and then normalized. Here, samples were mixed with polystyrene beads embedded with metal lanthanides, allowing monitoring of mass cytometry instrument performance over multiple days of data acquisition. The protocol described here includes simultaneous measurements of beads and cells on the mass cytometer, subsequent extraction of the bead‐based signature, and the application of an algorithm enabling correction of both short‐ and long‐term signal fluctuations. The variation in the intensity of the beads that remains after normalization may also be used to determine data quality. Application of the algorithm to a one‐month longitudinal analysis of a human peripheral blood sample reduced the range of median signal fluctuation from 4.9‐fold to 1.3‐fold. © 2013 International Society for Advancement of Cytometry