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.

Fig. 2.2 Histologic appearances of normal fetal liver during the middle trimester of pregnancy. About 40% of the area of the section consists of erythropoietic cells which are present singly or in clusters between the plates of liver cells. Hematoxylin–eosin stain.

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.

Postnatal changes in the distribution of hemopoietic marrow

At birth all BM cavities contain red marrow consisting mainly of hemopoietic cells. By 1 year virtually all the hemopoietic cells in the terminal phalanges are replaced by fat cells. After the first 4 years there is an increase in fat cells amongst the hemopoietic cells of other marrow cavities. Between 10 and 14 years, the hemopoietic cells in the middle of the shafts of the long bones become virtually completely replaced by fat cells. Subsequently, these zones of non-hemopoietic yellow marrow spread proximally and distally. Distal spread is the more rapid and by about the 25th year the only regions of the long bones that contain red, hemopoietic marrow are the proximal shafts of the femora and humeri. Other sites of hemopoiesis in an adult are the skull, ribs, clavicles, scapulae, sternum, vertebrae and pelvis.

General characteristics of hemopoiesis

The formation of blood cells of all types involves two processes:

1. cell proliferation, which amplifies the number of mature cells produced from a cell that has become committed to any particular cell lineage

2. cell differentiation, or the progressive development of biochemical, functional and structural characteristics specific for a given cell type.

During intrauterine life and in the growing child, there is a progressive increase in the total number of hemopoietic and blood cells. In normal adults, the total number of hemopoietic and blood cells remains relatively constant. There is steady-state cell renewal with a relatively constant rate of loss of mature cells (erythrocytes, granulocytes, monocytes and platelets) balanced precisely by the production of new cells.

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:

Fig. 2.3 Schematic diagram showing the differentiation of hemopoietic stem cells to mature blood cells. B-cell P, B-cell precursor; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulocyte-macrophage progenitor; HSC, hemopoietic stem cell; MEP, megakaryocyte-erythroid progenitor; T-cell P, T-cell precursor; TSP, thymus-seeding progenitor.

1. Cytogenetic studies of chronic myeloid leukemia, which showed that the Philadelphia (Ph) chromosome is present in both myeloid (granulocytic, erythroid and megakaryocytic) and lymphoid cells.

2. The demonstration that in a case of sideroblastic anemia with glucose-6-phosphate-dehydrogenase (G6PD) mosaicism, a single G6PD isoenzyme was present in myeloid cells as well as T- and B-lymphocytes.

3. Studies of xenogeneic transplant models in immuno-incompetent animals.

All pluripotent HSC express CD34 antigen and are usually AC133⁺, CD59⁺; Thy1^low; CD38⁻; C-kit^−/low; CD33⁻; lineage⁻. By flow cytometry (FCM) CD34⁺ HSC constitute most cells of the CD45^dim/SSC^low (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⁺/CD45^dim 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 cells ^10,¹¹ (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

Fig. 2.4 Normal adult hemopoiesis in a bone marrow aspirate showing a range of normal erythroid and granulocytic progenitors at different stages of differentiation. May–Grünwald–Giemsa stain. × 400.

1. Common myeloid progenitor (CMP) which gives rise to cells of all myeloid lineages (i.e. granulocytic, erythroid, megakaryocytic). The CMP subsequently generates granulocyte-macrophage progenitors (GMP) and megakaryocyte-erythroid progenitors (MEP).

2. Common lymphoid progenitor (CLP), which gives rise to T- and B-lymphocytes and natural killer (NK) cells.

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

Fig. 2.5 (A, B) Erythroid islands in normal bone marrow. (A) Erythroid islands consisting of early and late polychromatic erythroblasts surrounding macrophages. May–Grünwald–Giemsa stain. × 400. (B) Electron micrograph of an erythroid island showing a macrophage (central) surrounded by a layer of erythroblasts. Uranyl acetate and lead citrate.

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):

Fig. 2.6 (A, B) Erythroblasts in a normal bone marrow aspirate showing all stages of erythropoiesis from pronormoblast to late polychromatic normoblasts. May–Grünwald–Giemsa stain. × 400. (A) Two pronormoblasts and many late polychromatic normoblasts. (B) Early and late polychromatic normoblasts.

• Pronormoblasts: large cells (diameter 12–20 µm) with a large rounded nucleus surrounded by a small amount of deeply basophilic cytoplasm; the intensity of cytoplasmic basophilia is greater than of myeloblasts. The cytoplasm may have a pale-staining area adjacent to the nucleus (corresponding to the Golgi apparatus). The nuclear chromatin has a finely granular or reticular appearance and there are prominent nucleoli. With maturation erythroid cells decrease in size and there is a progressive increase in cytoplasm relative to that of the nucleus.

• Basophilic normoblast: the cytoplasm is even more blue-staining than that of the pronormoblast. Its nuclear chromatin has a coarsely granular appearance and there are no nucleoli.

• Early polychromatic normoblasts: have polychromatic cytoplasm due to hemoglobinization and a nucleus containing clumps of condensed chromatin.

• Late polychromatic normoblasts: are smaller (diameter 8–10 µm), have faintly polychromatic cytoplasm and a small eccentric nucleus (diameter less than 6.5 µm) and contain large clumps of condensed chromatin. The nucleus becomes pyknotic, is extruded and rapidly phagocytosed by adjacent macrophages. The morphology of the resulting marrow reticulocytes is similar to that of circulating reticulocytes, as described in Chapter 1.

Cytochemistry

Perls’ acid ferrocyanide stain identifies one small blue–black granule in up to 30% of polychromatic erythroblasts. These iron-containing siderotic granules are randomly distributed in the cytoplasm, and erythroblasts containing such granules are termed sideroblasts. Abnormalities of siderotic granulation include increased numbers of sideroblasts, increased granulation or abnormal granule localization. The term ring sideroblasts is used when there are five or more perinuclear siderotic granules. Erythroid cells at all stages of maturation frequently contain coarse acid phosphatase-positive paranuclear granules. Normal erythroblasts are periodic acid-Schiff (PAS)-, Sudan black- and myeloperoxidase (MPO)-negative. Occasional erythroblasts contain a few alpha-naphthol AS-D chloroacetate esterase-positive granules.

Antigen expression

Early erythroblasts have weak CD45 expression, are CD44 (strong), CD71, CD36, HLA-DR and CD117 positive. Glycophorin A (CD235a) is expressed at a low level at this stage. Maturation to the basophilic erythroblast is accompanied by a decrease in CD44, CD45 and acquisition of CD235a antigen. Transition to polychromatic erythroblast shows a further loss of CD45, HLA-DR and CD44, a mild decrease in CD36 expression and presence of hemoglobin.^22,²³

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):

Fig. 2.7 (A, B) Megakaryocytes in normal bone marrow. May–Grünwald–Giemsa stain. × 400. (A) Granulated megakaryocyte. (B) The bare nucleus of a senescent megakaryocyte.

1. megakaryoblasts (group I megakaryocytes)

2. promegakaryocytes (group II megakaryocytes)

3. granular megakaryocytes (group III megakaryocytes), which produce platelets

4. ‘bare’ nuclei.

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).

Antigen expression

Cells committed to the megakaryocyte lineage express platelet glycoproteins. The earliest is platelet glycoprotein IIIa (CD61; integrin αIIβ3) followed by glycoprotein IIb (CD41; integrin αIIb), glycoprotein Ib (CD42), glycoprotein V and factor VIII-related antigen. Thrombospondin receptor (CD36; glycoprotein IIIb) and platelet-endothelial cell adhesion moleculae (PECAM-1; CD31) are also expressed.

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.

Fig. 2.8 (A, B) Megakaryocytes showing emperipolesis. (A) Granulated megakaryocyte showing emperipolesis of a neutrophil granulocyte. May–Grünwald–Giemsa stain. × 400. (B) Electron micrograph of a megakaryocyte which is apparently showing an unusual degree of emperipolesis. Six cells (two eosinophil granulocytes, two neutrophil granulocytes and a monocyte) can be seen within the megakaryocyte profile. It is possible that at least some of these cells are not completely within the megakaryocyte but merely protruding into it. Uranyl acetate and lead citrate. × 6800.

Granulopoiesis and monocytopoiesis

Granulopoesis is the production of granulocytic cells (neutrophils, eosinophils and basophils, and cells of the monocyte–macrophage series) within the BM. Granulopoiesis commences with the differentiation of the HSC to the common myeloid progenitor (CMP). The CMP further develops into the bipotent granulocyte-macrophage progenitor (GMP). The GMP differentiates into cells that are irreversibly committed to mature into granulocytic cells (CFU-G) or macrophages (CFU-M). The granulocytic cells, including neutrophils, eosinophils and basophils, are all characterized by the presence of cytoplasmic granules (Table 2.1).

Table 2.1 The major granule proteins present in neutrophils, eosinophils, basophils and mast cells

Cell type	Primary granules	Specific granules
Neutrophil granulocytes	Myeloperoxidase Acid phosphatase Lysozyme Neutrophil elastase Defensins Bactericidal permeability-increasing protein Cathepsins α₁ antitrypsin Heparin-binding protein Sulphated mucosubstances Aryl sulphatase α-mannosidase	Secondary: Lysozyme Lactoferrin Transcobalamin I Collagenase Histaminase Vitamin B₁₂ binding protein Buy Membership for Pathology Category to continue reading. Learn more here