Many anticancer therapies such as antibody-based therapies, cellular therapeutics (e.g.,
genetically modified cells, regulators of cytokine signaling, and signal transduction), and
other biologically tailored interventions strongly influence the immune system and require
tools for research, diagnosis, and monitoring. In flow cytometry, in vitro diagnostic (IVD)
test kits that have been compiled and validated by the manufacturer are not available for
all requirements. Laboratories are therefore usually dependent onmodifying commercially
available assays or, most often, developing them to meet clinical needs. However, both
variants must then undergo full validation to fulfill the IVD regulatory requirements. Flow
cytometric immunophenotyping is a multiparametric analysis of parameters, some of
which have to be repeatedly adjusted; that must be considered when developing specific
antibody panels. Careful adjustments of general rules are required to meet legal and
regulatory requirements in the analysis of these assays. Here, we describe the relevant
regulatory framework for flow cytometry-based assays and describe methods for the
introduction of new antibody combinations into routine work including development
of performance specifications, validation, and statistical methodology for design and
analysis of the experiments. The aim is to increase reliability, efficiency, and auditability
after the introduction of in-house-developed flow cytometry assays