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.

The ontogeny of native Langerhans cells (LCs) in the epidermis has been debated over the past decade, with most agreeing that such LCs are now best classified as specialized resident-tissue macrophages derived from the embryonic yolk sac that have a self-renewal capacity in the steady state, along with migratory dendritic cell (DC)–like properties (albeit with much slower migration as compared to conventional or classic DCs [cDCs]) (1). The diversity of the epidermal pool of resident and inflammatory macrophages and DCs may be greater than previously realized – with LCs, monocyte-derived LC-like cells, and inflammatory dendritic epidermal cells (IDECs) all acting as human antigen-presenting cells (1).

The appearance and phenotype of histiocytes can change with levels of maturation, activation, and the local tissue microenvironment, with only a few markers truly constitutive. Widely varying surface and cytoplasmic molecules are informative for these cells and are easily identified using flow cytometry, although many are not unique to these cell lines. Others are more useful for the identification of macrophages, dendritic cells, and their neoplastic counterparts in fixed tissues. Table 25.1 lists some antibodies informative for histiocytes by flow cytometry, only some of which are restricted to either macrophages or dendritic cells; many are common to both. Table 25.2 lists the common types of monocyte/macrophage and dendritic cells and the markers most useful in their identification in fixed tissues (1). Chapter 28 further highlights immunostain panels best adapted for neoplastic proliferations.

Immunodeficiency is a state of reduced ability to produce an adequate immune response due to an insufficiency or absence of antibodies, immune cells, or both. Immunodeficiency disorders can be primary (inherited/congenital) or acquired. Acquired immunodeficiencies can result from HIV infection, solid organ or hematopoietic stem cell transplantation, and other iatrogenic conditions, such as chemotherapy.

Immunodeficiency is associated with an increased risk of benign and malignant lymphoproliferative disorders (LPDs) [1]. As a result, a wide range of clinical manifestations and diseases can develop. Pathologists may face a challenge in making an accurate diagnosis and classification of lesion tissues from these patients because of the different nature of their immune systems in this context. Therefore, it is important that the pathologist be informed of the underlying condition when evaluating specimens from these patients [1].

Acute leukemias of ambiguous lineage (ALALs) are leukemias that show no clear evidence of differentiation along a single lineage [1]. ALALs are rare and comprise 2–3% of acute leukemia (0.35 cases per 1 million person-years) [1–5]. They occur in both children and adults, with a male predominance [1,2,4].

ALALs represent a heterogeneous group of diseases. Cases of ALAL mostly fall into two broad categories: acute undifferentiated leukemia (AUL) and mixed-phenotype acute leukemia (MPAL) [1–5]. The current classification is mainly based on immunophenotypes and genetic features outlined in World Health Organization (WHO) classification system (Table 17.1) [1,2,5,6].

Myelodysplastic syndromes (MDSs) and acute myeloid leukemias (AMLs), although most frequently occurring sporadically and in adult populations, have additionally been noted to occur in association with predisposing germline genetic abnormalities, with such cases commonly occurring in children and young adults. With the utilization of advanced diagnostic methodologies in clinical practice and the recognition of the significance of this set of conditions for clinical management, the spectrum of syndromic conditions has continued to expand in the medical literature.

Table 5.1 provides an overview of the inherited leukocyte disorders covered in this chapter.

Idiopathic (immune) thrombocytopenic purpura (ITP) is an acquired disorder of antibody-mediated platelet consumption [1]. It can be categorized based on the presence of underlying disease (primary versus secondary) or onset rapidity (acute versus chronic) [2]

Juvenile myelomonocytic leukemia (JMML) is a clonal myelomonocytic neoplasm of childhood with prominent granulocytic and monocytic proliferation, characteristically driven by RAS-pathway gene mutations. (See Table 10.1.)

The term histiocyte (tissue cell) has evolved and is now often used as a collective term for two related groups of immune regulatory cells, the monocyte-macrophages and the dendritic cell (DC)–accessory antigen-presenting cells (1). The histiocytic proliferations of childhood encompass benign and malignant accumulations of monocyte-macrophages and hematopoietic-derived DC with a clinical spectrum of indolent to aggressive lesions. Although distinguishing between their reactive and neoplastic states can be challenging at times, molecular-based testing can help refine the diagnosis of neoplastic accumulations (see also Chapter 28) (2, 3).

Enlarged lymph nodes are frequently encountered in children and often are transient. Persistently enlarged lymph nodes require biopsy and microscopic examination to classify the type of disease process. Lymphadenopathies are often characterized based on which lymph node compartment (paracortex, follicles, or medullary sinuses) is affected [1]. Thus, histologic evaluation of node architecture is paramount to determine the nature of the lymphadenopathy. In a large series of studies in children, approximately one-third showed evidence of infectious disease [2].

Lymphadenitis (lymphadenopathy due to infectious causes) is roughly characterized based on the type of microorganisms (bacteria, virus, fungus, etc.). Although many of these entities have well-described morphologic characteristics, others demonstrate a morphologic continuum with characteristics that overlap with one another, thus making the diagnosis based solely on morphologic grounds challenging. In the following discussion, the most common entities will be highlighted, recognizing that less common diseases do enter the differential of childhood lymphadenitis.