Normal Blood and Bone Marrow Populations
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4 CHAPTER 4 Normal Blood and Bone Marrow Populations It is essential to have a sound understanding of the nature of acute leukaemia (AL) blast cells, even if they occur at low and immunophenotypic characteristics of the normal cell frequencies. The sequential expression of antigens during populations encountered in bone marrow and peripheral normal marrow cell maturation and differentiation from a blood. It is also important to have some understanding of common stem cell population is well described in the lymphoid cellular interactions in the normal lymph node literature but needs to be fully understood. and thymus. This process is necessary if we are to under- Normal haematopoietic stem cells (HSC) are CD34+ stand the origin of the myeloid and lymphoid neoplasms (conversely not all leukaemic blasts are CD34+, a com- encountered in routine diagnostic immunophenotyping mon source of confusion) [1]. In normal steady state and helps us understand why certain neoplasms carry a marrow the total CD34+ fraction comprises HSC (early particular repertoire of antigens. It not only helps in progenitor cells; that is, uncommitted cells that can pro- the diagnosis of neoplastic disorders but equally gives duce multiple lineages), myelomonocytic (also CD123+), support to the recognition of reactive phenomena. erythroid (CD71+) and B-cell (CD19+) precursor cells [2]. The populations of cells which inhabit the bone Other antigens expressed by uncommitted HSC include marrow are constantly changing throughout the life of a HLA-DRdim and CD133, with the latter broadly correlat- normal individual. When this environment is challenged ing with CD34 expression. As the HSC population by infection, drugs, intercurrent medical disease and, of differentiates, they gain CD38 expression, a so-called course, primary haematological disease, the populations ‘activation marker’ in conjunction with the early lineage change or can respond and react in a variety of ways. We commitment antigens detailed above. need to be aware of these reactions such that we do not By definition, true HSC lack lineage commitment anti- over or under interpret the findings. This chapter is set gens (i.e. markers of lymphoid or myeloid differentiation), a out as a framework or foundation of normal myeloid and situation often abbreviated to lin–. Furthermore, many lymphoid ontogeny. Our understanding of this process is primitive haematopoietic stem cells are quiescent (out of still incomplete but we can only safely recognize abnor- cell cycle), with absence of both activation markers and mal situations once we are fully familiar with the normal markers of cell division, such as Ki67. These cells comprise resting blood and bone marrow cellular environment. only a very small fraction of total marrow CD34+ cells, how- ever, as this antigen continues to be expressed into the early- committed myeloid and lymphoid progenitor compartments Normal stem and precursor cell before it is lost during further maturation. Despite this populations caveat, CD34 is a very useful surrogate marker for the nor- mal HSC compartment, and it is routinely used for assess- Haematopoietic progenitor cells ment of stem cell harvests in the transplantation setting, for Recognition of the immunophenotypic patterns of matu- example, as well as in stem cell research. CD34+lin+ cells are ration in normal bone marrow facilitates the identification therefore committed progenitors and represent the first Practical Flow Cytometry in Haematology Diagnosis, First Edition. Mike Leach, Mark Drummond and Allyson Doig. © 2013 John Wiley & Sons, Ltd. Published 2013 by John Wiley & Sons, Ltd. 31 32 CHAPTER 4 identifiable stage through which cells mature sequentially further complication is that aberrant or asynchronous down a particular differentiation pathway. maturation can be observed in normal situations, for exam- Normal precursor cell populations in the marrow may ple expression of CD56 on granulocyte and monocyte occasionally cause confusion in the diagnosis and monitor- precursor cells in regenerating marrows both with and ing of acute leukaemias. This is largely because they share without G-CSF administration. Furthermore, unusually many antigens of immaturity in common with leukaemic synchronous maturation may be observed in this situation blast cells as well as morphological similarities. One of the (e.g. accumulation of promyelocytes as the regenerating most obvious differences is that AL blasts are usually much cells expand and pass through maturational stages together) more numerous than normal precursors; for the most part, requiring meticulous attention to flow, morphology, genet- progenitor cells in normal steady-state marrow samples are ics as well as the clinical history to avoid erroneous inter- present at low levels. CD34+ cells are generally no more pretation. These situations are little mentioned in the than 1–2% of all marrow cells, while CD117+ cells, an anti- literature but should be recognized relatively easily by an gen that is expressed beyond CD34 in myeloid progenitor experienced cytometrist. cell populations, are generally no more than 2–4% [3–5]. CD45 (a pan-leucocyte antigen) is generally dimly Approximately 25% of CD34+ cells are CD117+ [6]. How- expressed on marrow precursor cells, including myelo- ever, regenerating marrows often express proportionally blasts, monoblasts, precursor B-cells and erythroblasts higher levels of progenitor cell antigens than marrows in [9]. It is, by comparison, bright on mature cells such as steady state, particularly in the case of normal B-cell pre- monocytes, neutrophils and lymphocytes, although it is cursors (haematogones) after chemotherapy or stem cell lost altogether by later erythroid precursors. CD45dim is transplantation for example [7, 8]. In general, for practical therefore a useful feature to allow gating on blast cells of purposes, the authors consider a CD34+ population of ≥ 3% many AL, and it is commonly used in conjunction with or a CD117 population ≥ 5% as significant and worthy of the low SSC that is also seen in precursor cells [10], full characterization, although this is dependent on the redrawn schematically in Figure 4.1. clinical scenario and assessing lower levels than this may be By incorporating CD13, an early marker of myeloid relevant in some circumstances (e.g. post induction therapy differentiation, the following progenitor populations can in AL or distinguishing MDS from aplastic anaemia in a therefore be easily defined in normal marrow: hypoplastic marrow). Particularly at low levels of blast 1 A CD34+CD117+CD45dimCD13+ pattern, which defines numbers, demonstration of an abnormal phenotype is of the normal myeloid/monocytic progenitor population. more significance than accurate quantification in most cir- 2 A CD34+CD117–CD45dimCD13– pattern, which defines cumstances, particularly when the potential pitfalls in blast the normal precursor B-cell population. These cells enumeration are taken into account (see below). One would in addition express B-cell antigens. 103 Bright 102 Dim Lymphocytes Monocytes CD45 Neutrophils 101 Neg Promyelocytes Meta/myelocytes Lymphoblasts Myeloblasts/monoblasts Nucleated erythroid cells Figure 4.1 Distribution of normal bone marrow populations according to CD45 expression and Side scatter side scatter properties. Normal Blood and Bone Marrow Populations 33 For the purposes of further discussion of normal normal counterparts. Knowledge of these differences maturation patterns we will define two major popu- is important in identifying such cells as leukaemic. lations: Variations in antigen intensity also occur during 1 Myeloid (neutrophils, eosinophils, basophils, mono- maturation; for example CD13 and CD33 expression on cytes, erythroid, megakaryocytes, mast cells and dendritic myeloblasts progressively increase in intensity up to and cells) including the promyelocyte stage. This CD33bright popula- 2 Lymphoid (B and T-cells) tion then loses intensity (but remains positive) as matura- It is worth drawing attention to two excellent resources tion to neutrophils takes place. This is in contrast to with regards precursor cells and normal marrow matura- monocyte maturation which is characterized by a contin- tion that are freely available. First, there exist useful ual increase in CD13 and CD33 intensity as cells mature. consensus descriptions of cytology in normal and abnor- Late antigens appearing during the myelocyte-neutrophil mal myelopoiesis which have recently been produced stages include CD15 (which first appears at a late promye- by the International Working Group on Morphology locyte stage) and CD11b [14]; CD16 and CD10 are of Myelodysplastic Syndrome (IWGM-MDS) [11, 12]. expressed from the metamyelocyte/band-form stage Second, is the most recent of several attempts to accu- onwards. Mature neutrophils therefore have a CD45+, rately document the maturation patterns of precursor CD13+, CD33+, CD11b+, CD15+, CD16+ phenotype. CD64 cells in normal marrow. The most comprehensive study to may be expressed on early myeloblasts, but disappears date has been performed on behalf of the European beyond this stage. It may be re-expressed by activated neu- Leukaemia Net (ELN) [13]. This study exhaustively docu- trophils, where bright CD64 expression can be indicative ments scatter plot patterns of antigen expression using of sepsis [15]. A graphic summary of myeloid maturation predefined standard antibody panels, and provides a nor- is shown in Figure 4.2. mal framework upon which abnormal populations