Good knowledge of immunophenotypic features of normal cells in various compartments is important when potentially pathological specimen are sent for examination in the flow cytometry platform. This chapter proposes a comprehensive description of these features, together with some functional and/or maturation characteristics of some cell types. Blood and bone marrow are considered, but also body fluids and, briefly, some tissues.

Malignant plasma cell proliferations are characterised by specific clinical, immunophenotypic and genetic features. Multiparameter flow cytometry (MFC) is an essential component of the diagnosis of these diseases. Clonal proliferations can be identified through their aberrant cell-surface immunophenotype or, more precisely, by demonstrating monotypy, i.e. selective expression of the same light chain in the cytoplasm of plasma-cells. This chapter reviews these immunophenotypic features, the technical points of caution to observe for proper use of MFC at diagnosis and during therapy to assess measurable residual disease.

Flow cytometry relies on the use of fluorochrome-conjugated antibodies, most of them identified and produced after the discovery of the technology allowing to generate large amounts of monoclonal antibodies. Hence, nearly all these reagents are named after the cluster of differentiation (CD) number that was given to newly discovered molecules they recognize, many of them having no other name. Although some CDs have become very popular and well known, others are less familiar. This chapter provides a guide to recover the characteristics of surface or cytoplasmic antigens explored with the CDs most frequently used in the field of haematological malignancies.

As indicated by the title of this chapter, some conditions may display features evocative of hematological malignancies and have to be recognized as not being tumoral. Here, these situations have been grouped as increased leucocyte types (leucocytosis) or as decreased cell counts (cytopenias), segregated in disease types. A third part considers abnormal immunophenotypes of lymphocytes and myeloid cells. Finally, the recurrent question of haemodilution of bone marrow aspirates, which decreases the otherwise helpful ability of flow cytometry to count large numbers of cells and thus perform accurate differentials, is discussed.

The term “acute leukemia” actually covers a large number of different diseases. This is mostly related to the lineage involved, yet, even in a single lineage, differences exist according to the differentiation stage where maturation blockade occurred or to the type of chromosomal/molecular anomalies associated with the disease. This chapter provides a guide of how immunophenotypic anomalies, typically identified very early in the diagnosis process, can orient further cytogenetic or molecular investigations, allowing for a faster integrated diagnosis and better focused patient management.

Although the fundamental idea of having cells focalised to be ’seen’ one by one by a detection system remains unchanged, flow cytometry technologies evolve. This chapter provides an overview of recent progress in this evolution. From a technical point of view, cameras can provide images of each of these cells together with their fluorescent properties, or the whole spectrum of emitted light can be collected. Markers coupled to heavy metals allow to detect each cell immunophenotype by mass spectrometry. On the analysis side, artificial intelligence and machine learning are developing for unsupervised analysis, saving time before a much better supervision of small populations.

This chapter is an introduction to flow cytometry aimed at newcomers in the field but also intended as a refresher for seasoned flow cytometrists confronted with unexpected data related to physical interferences, compensation problems, autofluorescence or aiming at harmonising instruments. It also provides counsel on panel building, sample handling and data display, fundamental points to consider in setting up new protocols.

Mixed-phenotype acute leukaemia is a generic classification item collecting leukaemias with two clones of different lineage or really abnormal cells expressing markers of several lineages. Their identification relies on both morphological and immunophenotypic features. From a cytogenetic/molecular point of view, their heterogeneity is amazing. Clinical management of such patients is getting progressively better stratified, allogeneic hematopoietic stem cell transplantation remaining the best option, with a possibly better approach for patients with Philadelphia chromosome. This is a typical example of the need for integrated diagnosis.