Immunohistochemistry is a powerful diagnostic tool for practicing pathologists. Over the past few decades, the techniques used in immunohistochemistry have become exponentially more complex. By exploiting the specificity of antibody-antigen interactions, we can use commercially available labeled antibodies to determine the presence and dispersion of various macromolecules within tissue. Antigen retrieval techniques, tissue preservation and standardization have broadened the utility of immunohistochemistry from diagnostic ancillary test to screening for hereditary syndromes and serving as biomarkers in the era of personalized medicine. This chapter will describe the conceptual framework of immunohistochemistry, outline technical mechanisms, and explain its clinical relevance.

The diagnosis of cytopenic patients suspected of myelodysplastic syndrome (MDS) can be challenging, particularly when initial laboratory assessments are indecisive. In normal haematopoiesis, the expression of differentiation antigens is tightly regulated. Changes in expression patterns may therefore indicate dysplasia, the hallmark of MDS. Multiparameter flow cytometry (MFC) can identify aberrancies in differentiation antigen expression and maturation patterns not recognized by cytology. MFC performed according to recommendations defined by the International and European LeukemiaNet-associated Working Group focusing on standardisation of MFC in MDS (iMDSFlow) may reveal aberrancies in the myeloid progenitor cells, B-cell progenitors, maturing myelomonocytic cells and erythroid cells. Defined abnormalities can be counted in MFC scoring systems to provide a means to determine the extent of dysregulation of the maturation patterns, i.e. dysplasia according to MFC. Ideally, scores should enable a categorization of MFC results from bone marrow assessments in cytopenic patients as ’normal’, ’low probability of’ or ’high probability of’ MDS. Notably, MFC as a single technique is not sufficient for the diagnosis of MDS, and results should always be evaluated as part of an integrated diagnostic workup.

This chapter provides useful guidelines for the immunophenotypic identification of both indolent and aggressive B-cell lymphomas. An integrated diagnostics is necessary to provide the final classification, but flow cytometry allows for a quick orientation about the lymphoma subtype and may help in speeding targeted further assays and therapeutic decisions.

Hodgkin lymphoma, a nodal disease, is usually diagnosed using morphology and immunochemistry on lymph nodes biopsies. However, with the increased practice of fine-needle aspiration or core biopsy, multiparameter flow cytometry (MFC) can provide valuation information on cell suspensions from such samples. Here, the major markers and characteristics allowing, in MFC, to distinguish between the scarce Reed Sternberg cells and the inflammatory immunological infiltrate surrounding them are described. Guidelines and recent information are provided for readers willing to implement these investigations in their own settings.

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.

Measurable residual disease (MRD) is an established prognostic factor after induction chemotherapy in acute myeloid leukaemia patients. Over the past decades, molecular and flow cytometry-based assays have been optimized to provide highly specific and sensitive MRD assessment that is clinically validated. Flow cytometry is an accessible technique available in most clinical diagnostic laboratories worldwide and has the advantage of being applicable in approximately 90% of patients. Here, the essential aspects of flow cytometry-based MRD assessment are discussed, focusing on the identification of leukaemic cells using leukaemia associated immunophenotypes. Analysis, detection limits of the assay, reporting of results and current clinical applications are also reviewed. Additionally, limitations of the assay will be discussed, including the future perspective of flow cytometry-based MRD assessment.

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.

Acute lymphoblastic leukaemia (ALL) is the most common cancer in childhood but shows a very low frequency in adults. Even in the genomics era, multiparametric flow cytometry is still critical for ALL diagnosis and management. At diagnosis, it determines the proper therapeutic approach through blast characterization and lineage assignment. During treatment, it is an essential tool for response to therapy monitoring through minimal/measurable residual disease detection. Additionally, multiparametric flow cytometry is fundamental in the even more applied immunotherapy setting, recognizing any potential switch of blast immunophenotype.

Mature T- and natural killer (NK)-cell neoplasms comprise multiple distinct disease entities. Diagnosis and classification of these entities require the integration of morphology, immunophenotype and cyto- and molecular genetics and correlation with clinical presentation. Multiparameter flow cytometry (MFC) is an important tool to immunophenotype T and NK cells. Our knowledge of the constellation of immunophenotypic aberrancies associated with certain disease entities has increased by the simultaneous analysis of more markers and molecular genetic studies. Genotype-phenotype associations have been identified contributing to a better understanding of the disease biology and clinical behaviour. T- and NK-cell disease entities in which MFC plays a central role in the diagnosis and classification are reviewed in this chapter. T-cell clonality analysis by MFC has become an assay used in many diagnostic laboratories. The availability of the JOVI-1 antibody against the T-cell receptor β constant region 1 protein (TRBC1) has greatly facilitated the detection of clonal TCRαβ T cells with high specificity and sensitivity. Despite the major advances in the diagnostic flow cytometry assays for the detection of T- and NK-cell neoplasms, standardized protocols are needed to increase the accuracy of diagnosis and classification and facilitate the implementation of automated MFC data analysis.

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.