Normal bone marrow cells: Development and cytology

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CHAPTER 2 Normal bone marrow cells

Development and cytology

The bone marrow (BM), the major site of hemopoiesis, is comprised of hemopoietic cells and stromal cells. This chapter will describe the morphological appearances, cytochemical characteristics and antigen expression profile of BM cells throughout their development. The emphasis will be to provide a broad understanding of normal BM development as a necessary baseline for further understanding of blood and BM pathology. Only brief mention will be made of cell ultrastructure. Although this method has provided significant insight into cell development, it now has little practical role in diagnostic hematology; for a detailed review of the ultrastructural appearances of BM cells the reader is referred to the previous edition of this book.

Hemopoietic cells

Hemopoiesis is the process by which mature blood elements of all lineages are derived from a common pluripotent stem cell. There are two types of hemopoietic systems:

Embryonic hemopoiesis

Hemopoiesis commences from a transient population of primitive HSC generated in the yolk sac. This commences on the 14th to 19th day of embryogenesis and persists there until the end of the 12th week of gestation. The yolk sac primarily produces nucleated erythroid cells that are megaloblastic and contain embryonic hemoglobins, Gower I (ζ2ε2), Gower II (α2ε2) and Portland I (ζ2γ2) (Fig. 2.1). Yolk sac erythropoiesis is referred to as ‘primitive’. In the 6th and 7th weeks of gestation, the blood islands within the yolk sac also contain a few megakaryocytes. Hemopoietic activity then occurs in a region of the para-aortic splanchopleural mesoderm. This region contains the dorsal aorta, gonadal ridge and mesonephros and is known as the aorta-gonad-mesonephros or AGM region.1,2 Hemopoiesis in the AGM region develops from ‘definitive’ HSC that eventually populate the adult BM. Some adult-type HSC also develop in the yolk sac and the placenta.

HSC derived from the AGM migrate to the liver and erythropoietic foci are detectable in the 6th week of gestation. The liver remains the main site of erythropoiesis from the 3rd to the 6th month when erythroblasts account for about 50% of the nucleated cells of the liver. The erythroblasts are mainly extravascular (Fig. 2.2), located near and within Kupffer cells (emperipolesis) and continue their maturation inside sinusoids.3 Erythroblasts are initially megaloblastic but subsequently become macronormoblastic. Fetal hepatic erythropoiesis is associated with the synthesis of fetal hemoglobin (HbF; α2γ2) and results in the production of nucleated, macrocytic red cells. The liver continues to produce red cells in decreasing numbers after the 6th month of gestation until the end of the 1st postnatal week.

Small foci of erythropoietic cells are present in the vascular connective tissue of some BM cavities from 2.5 to 4 months’ gestation. From the 6th month the BM is the major site of hemopoiesis and the myeloid/erythroid (M/E) ratio is about 1 : 4. Erythropoiesis in fetal BM occurs extravascularly, is macronormoblastic and results in the production of macrocytic red cells containing HbF and HbA (α2β2). The mean cell volume (MCV) in the cord blood of full-term newborns is 90–118 fl. Erythropoieisis in fetal BM appears to be regulated by erythropoietin produced extrarenally, probably in the liver. From the 6th month there is also proliferation of HSC in the fetal liver which generate erythroid, myeloid and some lymphoid cells. Small foci of erythroblasts, a few granulocytopoietic cells and occasional megakaryocytes also occur in many other embryonic and fetal tissues and organs (including lymph nodes, spleen and kidneys); however, their contribution to overall hemopoietic activity is small.

Hemopoietic stem cells and progenitor cells

The uncommitted HSC has the capacity for self-renewal and is pluripotent, that is, it has the ability to differentiate into lineage-committed progenitors. It is not recognizable morphologically but can be identified by antigen expression profile. The differentiation of HSC generates multipotent myeloid progenitors and lymphoid progenitors (Fig. 2.3).4 Evidence for the presence of pluripotent HSC has come from a number of pieces of evidence including:

All pluripotent HSC express CD34 antigen and are usually AC133+, CD59+; Thy1low; CD38; C-kit−/low; CD33; lineage. By flow cytometry (FCM) CD34+ HSC constitute most cells of the CD45dim/SSClow (blast) region and are a heterogeneous cell population. A small fraction of pluripotent HSC with long-term repopulating cell activity have been associated with the CD34+/CD38 phenotype.5,6 These cells are very rare in normal BM (usually <0.1%), but may increase in regenerating BM and in myelodysplastic syndromes.7,8 CD34+/CD45dim cells include a major fraction of progenitor cells that are already committed to specific hematopoietic lineages (erythroid, neutrophil, monocytic, dendritic cell (DC), basophil, mast cell (MC), eosinophil and megakaryocytic) and variable numbers of CD34+ B-cell precursors (BCP).9

The multipotent (or pluripotent) HSC undergo gradual restriction in their hemopoietic potential as they eventually give rise to unipotent progenitor cells10,11 (Fig. 2.4). The progeny of HSC therefore become progressively restricted to one cell lineage and they lose the capacity to self-renew. The earliest branching between myeloid and lymphoid development is to committed progenitor cells of myeloid or lymphoid types, as follows:11,12

The complex mechanisms involved in the regulation of hemopoietic stem cells and progenitor cells are described in Chapter 4. Growth-promoting cytokines, such as granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF), other cytokines and transcription factors are key regulators of hemopoiesis and may also enhance some functions of the mature cells.

Erythropoiesis

The CMP undergoes further differentiation to generate megakaryocyte-erythroid progenitors (MEP) or granulocyte-macrophage progenitors (GMP).4,13 The bipotent MEP has potential to form erythroid or megakaryocytic cells, both of which require GATA-1 (globin transcription factor 1) for their terminal differentiation. The most immature lineage-specific erythroid progenitor cells are the erythroid burst-forming units (BFU-E) and the most mature are the erythroid colony-forming units (CFU-E). The CFU-E develop into proerythroblasts, the earliest morphologically recognizable BM red cell precursors. Proerythroblasts progress through several morphologically defined cytologic classes. These are, in order of increasing maturity, the basophilic erythroblasts, early and late polychromatic erythroblasts and reticulocytes. Cell division occurs in the proerythroblasts, basophilic erythroblasts and early polychromatic erythroblasts but not in more mature cells. There are, on average, four cell divisions in the morphologically recognizable precursor pool so that one proerythroblast may give rise to 24 (16) red cells. In normal adults, the time taken for a proerythroblast to mature into BM reticulocytes and for reticulocytes to enter the circulation is about 7 days; of this, about 2.5 days is spent in the marrow reticulocyte pool. The time taken for blood reticulocytes to mature into erythrocytes is 1–2 days. In normal individuals erythrocytes circulate for approximately 120 days before they are removed and broken down by the mononuclear phagocyte system.

Within the BM, erythroblasts are present in erythroid islands composed of one or more central macrophages surrounded by one or two layers of erythroblasts (Fig. 2.5); fine processes of macrophage cytoplasm are found between the erythroid progenitors. Surface receptors on the macrophages are involved in macrophage–erythroblast interactions. One such receptor, erythroblast macrophage protein (Emp), mediates attachment of erythroid cells to macrophages in the erythroid island. Emp is required for normal erythroid differentiation and nuclear extrusion.14 A receptor on erythroblasts, intercellular adhesion molecule-4, binds to alpha (V) integrins on macrophages and is important for the formation of erythroblastic islands.15 Interactions between the macrophage and erythroblasts may modulate erythropoiesis by affecting gene expression and apoptosis and are required for iron delivery for the production of hemoglobin.16,17

Erythroid progenitor cells that do not develop successfully into erythrocytes undergo apoptosis and are phagocytosed by BM macrophages. The loss of potential erythrocytes, or ‘ineffective’ erythropoiesis, is small in normal BM but substantial in certain anemias.

Regulation of erythropoiesis

A key factor determining the rate of red cell production is the glycoprotein hormone erythropoietin (EPO) which, in the adult, is produced mainly by the peritubular cells of the kidney. EPO receptors are expressed on erythroid progenitor cells and the binding of EPO to its receptor results in the activation of JAK2 tyrosine kinase, which causes tyrosine phosphorylation in a number of proteins and triggers the activation of several signal transduction pathways involved in proliferation and in the prevention of apoptosis.18 An important effect of EPO is, therefore, to maintain the viability and proliferation of erythroid progenitor cells, by preventing apoptosis. In synergy with stem cell factor (SCF), GM-CSF, interleukin (IL)-3 and insulin-like growth factor-1 (IGF-1), EPO stimulates the rate of differentiation of CFU-E to pronormoblasts. EPO also stimulates terminal differentiation and decreases the time taken for the maturation of a pronormoblast to a marrow reticulocyte and its release into the circulation. The plasma level of EPO is inversely related to the capacity of the blood to deliver oxygen to the kidneys and other tissues. Reduction of the oxygen supply to the kidney results in enhanced EPO gene expression via a hypoxia-regulated transcription factor, HIF (hypoxia-inducible factor).19 Thus, in most anemic states there is an EPO increased level in the plasma, which in turn causes an enhancement of the rate of erythropoiesis.

Erythropoiesis is also influenced by the secretions of various endocrine glands. For example, hypofunction of the thyroid, testes, adrenal glands or anterior lobe of the pituitary gland result in mild to moderate anemia. Erythropoiesis is stimulated by thyroxine (stimulates terminal differentiation of erythroid progenitor cells), androgens (proliferation and expansion of erythroid progenitors) and growth hormone partly by an effect on the kidneys resulting in increased EPO production.20 Corticosteriods enhance erythropoiesis by stimulating EPO production and by a direct effect on progenitor cells. There is some evidence that estrogens may inhibit erythropoiesis;21 the sex difference in the hemoglobin levels of adults appears to be largely due to the higher androgen levels in males.

Light microscope cytology

The term erythroblast is used to define erythroid progenitors. When these appear normal the term normoblast is applied. In Romanowsky-stained normal BM smears morphologically identifiable erythroid cells have the following features (Figs 2.5 and 2.6):

Ultrastructure

Electron microscope studies show that all erythroblasts contain characteristic surface invaginations which develop into small intracytoplasmic vesicles (rhopheocytotic vesicles) (Fig. 2.5B). Morphological changes can be seen with maturation from pronormoblasts to mature late polychromatic normoblasts. The major changes are an increase in the amount of heterochromatin, decrease in the number of ribosomes, increase in the electron-density of the cytoplasm due to the accumulation of hemoglobin, a decrease in the number and size of mitochondria and aggregation of ferritin molecules into siderosomes. A Golgi apparatus persists in polychromatic normoblasts.

Megakaryopoiesis

Megakaryopoiesis is the process of development of megakaryocytes and platelets within the marrow. Humans generate 1011 platelets per day, and production can be increased 20-fold when in demand.24 Megakaryocytes are derived following a cascade of differentiation from the megakaryocyte-erythroid progenitor (MEP). The bipotent MEP commits to megakaryopoiesis under the influence of thrombopoietin (TPO), the primary regulator of platelet production, IL-6 and IL-11 to generate megakaryocyte colony-forming units (CFU-MK). CFU-MK are a diploid cell population, in which DNA synthesis and nuclear division (karyokinesis) is followed by cell division (cytokinesis). CFU-MK undergo further maturation to megakaryoblasts, the earliest morphologically recognizable member of the megakaryocyte series.

Four types of megakaryocytic cells can be identified in Romanowsky-stained BM smears. These are, in increasing order of maturity (Fig. 2.7):

DNA synthesis occurs in 44% of megakaryoblasts, 18% of promegakaryocytes and in only 2% of granular megakaryocytes. DNA synthesis is not associated with cell division and therefore cycles of DNA synthesis result in the production of mononucleate polyploid cells. The DNA content of a megakaryoblast ranges from 4 n to 32 n (1 n = haploid DNA content) and that of the larger promegakaryocyte and granular megakaryocyte from 8 n to 64 n. These polyploid cells undergo massive cellular enlargement to enable large numbers of platelets to be formed; each megakaryocyte generates 1000–3000 platelets. The megakaryocyte cytoplasm contains a network of specialized membranes, the demarcation membrane system (DMS), dense bodies, secretory vesicles and other organelles. Long extensions from the DMS form branches and undergo evagination to form pro-platelet processes. Platelets, which form by the fragmentation of these cytoplasmic processes, have a diameter of 1–3 µm. During platelet release the granular megakaryocytes protrude cytoplasmic processes close to or directly into the marrow sinusoids, pieces of cytoplasm break away and fragment into platelets. The almost ‘bare’ nucleus that remains after the release of platelets is surrounded by a narrow rim of cytoplasm containing a few granules and other organelles. The time taken for a megakaryoblast to mature into a platelet-producing granular megakaryocyte is approximately 6 days. Although the majority of megakaryocytes are in the marrow, some enter the circulation via the sinusoids and become trapped in the lungs; some pulmonary megakaryocytes appear to produce platelets.

Light microscope cytology

Early megakaryoblasts are difficult to distinguish from BM myeloblasts (see later) but do have a distinct ultrastructural appearance and phenotype. Megakaryoblasts (20–30 µm diameter) have a single large oval, kidney-shaped or lobed nucleus with several nucleoli, a very high nucleus to cytoplasm ratio and deeply basophilic agranular cytoplasm. Promegakaryocytes are usually larger than megakaryoblasts and have a lower nucleus to cytoplasm ratio and less basophilic cytoplasm. They have overlapping nuclear lobes and the cytoplasm may contain azurophilic cytoplasmic granules. The granular megakaryocytes (Fig. 2.7) are up to 70 µm in diameter and possess abundant pale-staining cytoplasm and numerous azurophilic cytoplasmic granules. The nucleus has coarsely granular chromatin and multiple lobes which extend through much of the cell. Prior to the formation of platelets by the fragmentation of cytoplasmic processes, the nuclear lobes become fairly tightly packed together. Following completion of platelet formation, a ‘bare’ nucleus remains (Fig. 2.7B).

Ultrastructure

Extensive studies have been performed of megakaryocytes throughout differentiation; the reader is referred to the previous edition of this book and other references for details of the ultrastructural features.25,26 Ultrastructural cytochemical studies have demonstrated platelet peroxidase (PPO), distinct from myeloperoxidase (MPO), in the endoplasmic reticulum and perinuclear space of promegakaryoblasts, megakaryoblasts and megakaryocytes. PPO is also present in the dense bodies and dense tubular system of platelets.

Emperipolesis

Emperipolesis describes the presence and movement of one cell within the cytoplasm of another; the ‘engulfed’ cell can subsequently leave the ‘engulfing’ cell and appears morphologically unaltered by the interaction.27 Emperipolesis is most commonly seen within megakaryocytes and sometimes one megakaryocyte may contain several cells ‘inside’ it (Fig. 2.8). The engulfed cells may be neutrophils, eosinophils and their precursors, lymphocytes, erythroblasts or red cells. Megakaryocytic emperipolesis is of uncertain significance but may represent a transmegakaryocytic route for the entry of blood cells into the circulation; it has been suggested that some of the intramegakaryocytic cells may enter the circulation via the processes of megakaryocytic cytoplasm which protrude into adjacent marrow sinusoids. Emperipolesis has also been described in non-hemopoietic cells and malignant cells, including blast cells in the blast phase of chronic myelogenous leukemia.