Flow cytometric immunophenotyping: principles and pitfalls

Within the last decade flow cytometry (FCM) has become an integral part of basic immunological research. Elaboration of this technology has been intensively stimulated by a rapidly growing sophistication in monoclonal antibody technology and vice versa. At present numerous applications are established that allow an increasingly detailed insight into the immune system, however, automation still must be considered the "cinderella of the arts". Thus, transition of this powerful approach from a basic to a routine clinical procedure is much more difficult than expected. Sufficient usage of flow cytometers still requires some knowledge of physics and its technical applications. Moreover, several problems arise from the complexity of the biological systems investigated. Here we give a brief introduction to immunofluorescence and FCM followed by a discussion of six exemplary pitfalls that we hope will emphasise the general importance of methodological considerations.