nTFHL is a heterogenous group of mature T-cell lymphomas with follicular helper T-cell (TFH) immunophenotype. It is designated when at least two of the following markers are expressed: CD10, PD1 (CD279), ICOS, BCL6, CXCL13, CXCR5, SAP, MAF, or CD200. It is characterized by frequent loss of surface CD3, almost exclusively CD4, frequently diminished CD7, and retained CD2 and CD5. TFH markers studied by flow cytometry include CD10, PD1, CD200, ICOS, and CXCR5, but they can be expressed in reactive T-cells. nTFHL has three morphologic subtypes: angioimmunoblastic, follicular, and not otherwise specified. nTFHL angioimmunoblastic type or angioimmunoblastic T-cell lymphoma (AITL) is the prototype. All three subtypes share similar immunophenotypic characteristics, indistinguishable by flow cytometry. In nFFHL, lymphoma cells are frequently detected in blood and bone marrow by flow cytometry, often without morphologic involvement by lymphoma. The nTFHL immunophenotype is detailed and differential diagnoses are reviewed. Lymphocyte-variant hypereosinophilic syndrome and CD10-positive reactive T-cells in healthy individuals are discussed as immunophenotypic mimickers of nTFHLs.

A number of miscellaneous nodal and extranodal mature NK/T-cell lymphomas that are not included in other chapters are discussed in this chapter. Entities include peripheral T-cell lymphoma not otherwise specified (PTCL, NOS), nodal EBV+ NK/T-cell lymphoma, primary cutaneous T-cell lymphomas other than mycosis fungoides/Sezary syndrome, intestinal T-cell lymphoma (enteropathy associated T-cell lymphoma and monomorphic epitheliotropic intestinal T-cell lymphoma), and extranodal NK/T-cell lymphoma. The discussion focuses on the immunophenotypic features of each entity and how they aid in differential diagnosis.

This chapter discusses the diverse immunophenotypic features of acute myeloid leukemia (AML) with mutated TP53. It also addresses immunophenotypic shifts that may occur over the course of the disease.

Hepatosplenic T-cell lymphoma (HSTCL) is an aggressive T-cell lymphoma with characteristic clinicopathologic features. This chapter highlights the key immunophenotype of HSTCL and underscores the distinct differences between HSTCL and its mimickers, such as γδ T-LGLL and aggressive NK-cell neoplasm.

Mixed-phenotype acute leukemia (MPAL) comprises acute leukemias with discrete admixed populations of myeloid and lymphoid blasts (bilineal or mixed lineage) or with coexpression of lymphoid and myeloid markers in a single blast population (biphenotypic or mixed phenotype). However, the distinctions between mixed-lineage and mixed-phenotype MPAL are not always clear-cut. This chapter focuses on explaining the diagnostic criteria for MPAL and lineage-defining markers, summarizing its biological and genetic features, and addressing common diagnostic pitfalls of these unusual leukemias as part of the differential diagnostic considerations.

This chapter provides the key immunophenotypic features of various subtypes of T-large granular lymphocytic leukemia (T-LGLL). The differential diagnosis of T-LGLL, including reactive T-LGL proliferation, T-cell clone of uncertain significance, and other T-cell leukemias/lymphomas, especially hepatosplenic T-cell lymphoma, is also discussed. Diagnostic pitfalls associated with T-LGLL are highlighted.

Flow cytometry plays an important role in the diagnosis of B-cell lymphomas/leukemias. The identification of aberrant B cells by flow cytometry supports a neoplastic or clonal process. In this chapter, we focus on mature B-cell lymphomas/leukemias and describe the common approaches used in flow cytometry for identification of abnormal B cells as well as challenges and pitfalls associated with these approaches. Normal B-cell differentiation will be discussed first, which is fundamental for understanding cell of origin and identifying abnormal B cells in different subtypes of lymphomas.

This chapter describes the normal maturation patterns of T and NK cells. Approaches and challenges in identifying their abnormal counterparts are discussed in detail.

This chapter focuses on the flow cytometry immunophenotypic evaluation of Burkitt lymphoma, diffuse large B-cell lymphoma or high-grade B-cell lymphoma with MYC and BCL2 rearrangements (with or without BCL6 rearrangement), high-grade B-cell lymphoma not otherwise specified, and diffuse large B-cell lymphoma. The differential diagnosis among these three entities and with other entities is also discussed, with a focus on flow cytometry analysis. The differential diagnosis between surface light chain negatve blastoid high-grade B-cell lymphoma and CD34-negative B-ALL sometimes can be very challenge. Features that may be helpful for such a differential diagnosis, mostly flow cytometric immunophenotypic features, are discussed in detail.

Neoplasms derived from plasma cells are collectively termed plasma cell neoplasms (PCNs), the most notable of which is multiple myeloma. Neoplastic plasma cells in PCNs have distinct immunophenotypes that differ from normal plasma cells and those associated with B-cell lymphoma. Thus flow cytometry can be used to identify aberrant plasma cells to facilitate diagnosis and to monitor treatment response, such as minimal residual (measurable) disease. In addition, flow cytometry is instrumental in differentiating plasma cell neoplasms from B-cell lymphomas with plasmacytic differentiation, such as lymphoplasmacytic lymphoma or marginal zone lymphoma. This distinction is of critical importance in terms of clinical treatment and prognosis. This chapter focuses on the applications of flow cytometry in characterizing the immunophenotypic features of plasma cell neoplasms and their key distinctions from B-cell lymphomas and other diagnostic challenges.

Mature B-cell lymphomas can exhibit considerable plasmacytic differention, the most typical examples of which are lymphoplasmcytic lymphoma (LPL) and marginal zone lymphoma (MZL). The degree of plasmacytic differentiation can sometimes blur the lines between a B-cell lymphoma and a plasma cell neoplasm by morphology or immunohistochemistry. However, characteristic immunophenotypic features by flow cytometry can facilitate this distinction, which is of particular importance for clinical management. This chapter focuses on the applications of flow cytometry in characterizing the immunophenotypic features of LPL and MZL and their key distinctions from each other, plasma cell neoplasms, and other diagnostic challenges.

Aggressive NK-cell leukemia (ANKL) is a systemic neoplasm of malignant NK cells driven by Epstein-Barr virus (EBV). It shares significant clinical and pathological overlap with extranodal NK/T-cell lymphoma. This chapter highlights the unique immunophenotypic characteristics of ANKL, providing insights into its distinction from related entities.

This chapter outlines the key immunophenotypic features of NK-large granular lymphocytic leukemia (NK-LGLL) and highlights the use of NK-cell receptor patterns, analyzed through flow cytometry, to establish NK-cell clonality. It also emphasizes the essential differences between NK-LGLL and its mimics, such as reactive NK-cell proliferation, NK-cell clones of uncertain significance, and aggressive NK-cell leukemia.

Sperm selection is a crucial step in any assisted reproduction treatment and will facilitate optimal results if the correct protocol is chosen as per the specifics of the case.

Although standard laboratory processes like classic swim-up or density gradients are suitable in a significant number of cases, special preparation methods are required for specific situations such as elevated sperm DNA fragmentation, infectious samples to avoid viral transmissions to partner or progeny, retrograde ejaculation, high viscosity semen, immotile spermatozoa, or testicular sperm.

The objective of this chapter is to supplement the information provided in the sixth edition of the WHO laboratory manual for the examination and processing of human semen by providing a clinical explanation of the various sperm preparation protocols and discussing the selection of the most appropriate technique for a given semen sample.

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.