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.

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.

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.

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.

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.

Immunophenotyping is an important part of the integrated haematopathologic diagnostics of bone marrow (BM) samples. Integrated diagnosis should include clinical information, peripheral blood (PB) and BM smear cytology, flow cytometry (FCM) of BM aspirate, BM trephine biopsy (BMB) morphology, BMB immunohistochemistry (IHC) and cytogenetic/molecular genetic data if appropriate. Flow cytometry and IHC provide complementary information [1]. Immunophenotyping by FCM has the advantage of measuring high numbers of cells and the possibility to evaluate co-expression of several markers in various cell populations in a multicolour setting. Immunohistochemistry provides a possibility of in situ interpretation of morphology and immunophenotype simultaneously. Double IHC stains are possible but not widely used as of yet.