Physiological haematopoiesis and bone marrow

Haematopoiesis is the process underlying the production, differentiation and maturation of blood cell lineages starting from a common precursor within the bone marrow, the pluripotent stem cell. During intrauterine life, at the beginning of pregnancy, the first precursors are localized within the yolk sac; subsequently haematopoiesis takes place within the liver and spleen to then find its definitive site, at the end of pregnancy, within the bone marrow. In physiological conditions the haematopoietic tissue accounts for 4-6% of the body weight. The bone marrow is localized within the cavities of long and axial bones; it consists of islands of haematopoietic and adipose tissue supported by vascular sinuses intermixed with bone trabeculae.

Pluripotent stem cells selectively differentiate into committed myeloid and lymphoid stem cells. Myeloid-committed stem cells are functionally defined as “colony-forming units” in view of their ability, shown experimentally, to form mixed haematopoietic colonies within the spleen of laboratory animals. These consist of CFU-GEMM (colony-forming unit-granulocyte, erythroid, monocyte, megakaryocyte), CFU-GM (colony-forming unit-granulocyte, monocyte), CFU-G (colony-forming unit-granulocyte), GFU-M (colony-forming unit-monocyte), CFU-Meg (colony-forming unit- megakaryocyte), BFU (burst-forming unit-erythroid macrocolonies), CFU-E (colony-forming unit-erythroid), CFU-Eos (colony-forming unit-eosinophil) and CFU-Baso (colony-forming unit-basophil). Specific cytokines and growth factors may selectively stimulate the differentiation of the different cell lineages (Fig. 1).

Erythropoiesis (Fig. 2) identifies the process by which mature erythrocytes are formed starting from an erythroid blast (erythroblast). A sequence of mitotic events determines the progressive reduction of cellular volume, and hence of DNA content, until reaching the stage of metarubricyte, at which point mitosis is interrupted in view of cytoplasmic saturation on account of haemoglobin. Subsequent stages consist in the expulsion of the nucleus, the production of reticulocytes (red blood cells with a cytoplasmic residue of RNA) and the formation of mature red blood cells. Four (4) mitotic divisions take place in a period of 3-4 days to produce around 16 metarubricytes, which are no longer capable of dividing.

Erythropoiesis is regulated by erythroblastic islands; these are made up by a macrophage surrounded by erythroid precursors which draw growth factors from it (Fig. 3). Erythropoietin (EPO) may concomitantly stimulate erythropoiesis and inhibit apoptosis within the entire process. The mechanism which allows the synthesis of EPO by the kidney (and in minimal part by the liver) is hypoxia; following a hypoxic event there is an immediate release of EPO, with its peak activity reached within 24 hours.

The earliest cell of the erythroid series is the rubriblast. At light microscopy rubriblasts are large cells (20-25µm), they have an increased nuclear:cytoplasmic (N:C) ratio, a round nucleus with finely granular chromatin, presence of 1-2 nucleoli and an intensely basophilic cytoplasm (Fig. 4). These are followed by the prorubricyte, rubricyte, metarubricyte and the polychromatophil. Prorubricytes (Fig. 5), although similar to the previous cell, are smaller in size (16-18µm), nucleoli in the nucleus are absent and the chromatin is in clumps. Rubricytes (Fig. 6) are medium-sized (8-12µm), nucleoli in the nucleus are absent, chromatin is in clumps and the cytoplasm may vary from moderately basophilic (basophilic rubricyte) to mildly basophilic (polychromatophilic rubricyte). Metarubricytes (Fig. 7) are the most mature erythroid cells provided with a nucleus but with no mitotic activity; the nucleus appears piknotic (with dark and clumped chromatin) and the cytoplasm is weakly basophilic. With polychromatophils (Fig. 8) the nucleus is definitively lost and the cytoplasm is weakly basophilic in view of the persistence of limited residual nuclear material (ribosomes). With the subsequent loss of ribosomes and the cessation of protein synthesis the mature erythrocyte is formed (Fig. 9).

Table 1. Morphology of erythroid precursors.

Granulopoiesis regulates the production of the different granulocytes (neutrophils, eosinophils and basophils) starting from a common precursor and under the influence of specific cytokines involved in the process of proliferation, differentiation and cell maturation.

Neutrophil granulocytes in particular, together with their specific precursors, are the prevalent nucleated cell lineage within the bonemarrow as well as in the peripheral blood.

Two compartments are found within the bone marrow:

• mitotic (or proliferation) compartment;
• maturation compartment.

The mitotic compartment consists of approximately from 10% to 30% of cells capable of cell division (myeloblasts, promyelocytes and myelocytes). The maturation comportment and the storage subcompartment consist of approximately from 70% to 90% of granulocytes (myelocytes, metamyelocytes, band neutrophils and segmented neutrophils). The time necessary for a neutrophil to complete its maturation cycle is on average of seven days; such period may be shorter depending on peripheral requirements. Once in the periphery neutrophils enter respectively into the circulating neutrophil pool (CNP) and the marginal neutrophil pool (MNP) in a 1:1 ratio in the dog and 1:3 in the cat (Fig. 10).

In both the cat and the dog the half-life of circulating neutrophils ranges between about 5.5 and 7.6 hours. The migration and chemotaxis to the inflammation site take place thanks to the interaction of the neutrophil with inflammatory mediators and with chemotactic substances which promote endothelial adhesion, migration to tissues, phagocytosis of microorganisms and the release of cytotoxic substances. Neutrophils of the marginal pool or which have migrated to tissues survive from 1 to 4 days; they can then be either phagocytized by tissue macrophages or eliminated transmucosally. Once recruited within the inflammatory site and once having contained the inflammatory event neutrophils die by apoptosis: the removal of intact neutrophils avoids the release of potentially toxic intracellular content which could be an additional cause of tissue damage.

Myeloblasts are the earliest cells of the granulocyte series (Fig. 11). These are large-sized cells, with an increased N:C ratio, a round to oval nucleus with finely reticulated chromatin, with 1-2 nucleoli and a moderately basophilic cytoplasm. Promyelocytes appear as large-sized cells (they may at times appear bigger than myeloblasts in view of their more abundant cytoplasm), with an increased N:C ratio, a round to oval nucleus with finely reticulated chromatin, absence of nucleoli and moderately basophilic cytoplasm with magenta-coloured granulations (Fig. 12). These are followed by myelocytes, of smaller size compared to the previous cell, with a round to oval nucleus and finely reticulated chromatin, absence of nucleoli and a slightly basophilic cytoplasm (Fig. 13). With metamyeolocytes the mitotic capacity is lost and a deep indentation appears on the nucleus (Fig. 14). Band neutrophils are non-lobulated and have a typical horseshoe shape (Fig. 15). Segmented neutrophils, the final stage of cell maturation, have a multilobulated nucleus with coarse chromatin and clear cytoplasm (Fig. 16).

Table 2. Morphology of myeloid precursors.

Eosinophil granulocytes (Fig. 17) and basophils (Fig. 18), which are in very low numbers within the normal bone marrow, share with neutrophils the same precursors until the stage of myelocyte, when cytoplasmic granulations specific for each cell lineage begin to appear. In eosinophilic differentiation a fundamental role is played in particular by interleukin 5 (IL-5); in basophilic differentiation this role is played by IL-3 and IL-4.

Monocytes (Fig. 19), like granulocytes, originate within the bone marrow from the CFU-GM precursor: the differentiation into monoblasts, promonocytes and monocytes takes place under the influence of specific growth factors, such as IL-3, GM-CSF (granulocyte-macrophage colony-stimulating factor) and M-CSF (macrophage colony-stimulating factor). Morphologically, monocytes appear as 14-20 μm diameter cells, with a convoluted and pleomorphic nucleus, lax chromatin and absence of nucleoli; the cytoplasm, usually abundant, varies from clear to weakly basophilic and may occasionally be vacuolated depending on the stage of activation. In spite of having an intermediate size between granulocytes and lymphocytes, the elevated cytoplasmic adhesiveness makes them look larger.

Although T and B lymphocytes (Fig. 20) originate from a pluripotent stem cell having a common lymphoid lineage orientation, the subsequent maturation stages differ in the two cell types.

The differentiation process of B cells may specifically be divided into two subsequent stages: the first, called “antigen-independent”, is a central or medullary differentiation process; the second, which is a secondary, peripheral or “antigen-dependent” differentiation process, takes place within secondary lymphoid organs. The duration of this latter phase varies depending on the antigenic stimulus that triggers the differentiation; in any case the final transformation stages result in the production of B cells which are highly specialized in the humoral (antibody) immune response, such as plasma cells and memory B cells. These latter, antigen-specific cells, take their name from the fact that they remain in a quiescent stage for years, while waiting for their reactivation following re-exposure to the antigen.

Differently from B cells, the precursors of T cells abandon the bone marrow early on and continue their development and maturation, both functionally and phenotypically, within the thymus. In the thymus each T cell acquires a specific immunological competence, becoming programmed to recognize a specific antigen sequence by means of a surface antigen (TCR, T-Cell receptor) which is complementary to the sequence itself; in addition there is the progressive differentiation into two distinct lymphocyte subpopulations: T-helper cells and T-cytotoxic cells. The last stage of development, also knows as the peripheral phase, takes place within secondary lymphoid organs [lymph nodes, spleen, MALT (mucosa-associated lymphoid tissue)] where the antigen-antibody interaction, mediated by TCR, allows the activation of T cells with consequent clonal proliferation and differentiation: many T cells with the same receptor are therefore replicated and directed to the exposure site where, via mechanisms involving cytokines and other phagocytic cell types, they can act to destroy the invader. Such process is known as the cell-mediated response.

Megacariocytopoiesis, which results in the production of platelets (Fig. 21), is a complex biological process: it originates from a pluripotent stem cell which, via a sequence of progenitors which are progressively directed to the production of elements morphologically recognizable as megakaryocyte precursors, allows the synthesis of megakaryoblasts, promegakaryocytes, megakaryiocytes and finally platelets.

Mature megakaryocytes (Fig. 22), estimated at around 0.1-0.5% of all nucleated cells, have a variable diameter ranging from 20µm to 50µm, are multinucleated and have an abundant, acidophilic cytoplasm rich in fine granulations. Megakaryocytes originate from a DNA replication not followed by mitosis, which results in the formation of large-sized, polyploid cells. When more mature, megakaryocytes localize themselves in proximity of the endothelial cells of medullary sinusoids and develop cytoplasmic protrusions known as “phylopods” which spill into capillaries and give origin, via their fragmentation, to numerous platelets.

The production of platelets is directly influenced by thrombopoietin (TPO), a glycoprotein synthesized in the liver and kidneys, the blood levels of which are inversely proportional to the megakaryiocyte and platelet mass. Both platelets and megakaryocytes expose high-affinity receptors for TPO and the binding promotes its degradation. In view of this, in the course of thrombocythemia the increased platelet mass binds to and metabolizes TPO, limiting its availability for megakaryocytes and thus limiting the consequent synthesis of platelets; on the contrary, in the presence of thrombocytopenia the peripheral consumption of TPO is limited, thus allowing the glycoprotein to act at medullary level and hence promote the synthesis of new platelets.

Following are some figures regarding the distribution of different cell lineages found in the normal bone marrow of dogs taken from a study published by Mischke et al. in 2002:

• Myeloid:Erythroid (M:E) ratio of 1.08 (±0.61 SD)
• Immature Erythroid:Mature Erythroid (I:Me) ratio of 0.27 (± 0.09 SD)
• Immature Myeloid:Mature Myeloid (I:M) ratio of 0.16 (± 0.05 SD)
• Megakariocytopoietic cells 0.23% (± 0.20 SD).

Other cell types, called accessory cells, are also present within the bone marrow in variable percentages. The following cells are included among accessory cells:

• Lymphocytes
• Plasma cells
• Macrophages
• Osteoclasts/osteoblasts.

Lymphocytes (Fig. 20) are physiologically estimated to represent around 6.39% (±3.75 SD) of all nucleated cells: these are small cells (7-10µm), characterized by a spherical nucleus and an elevated nuclear:cytoplasmic ratio; their activation, triggered by the encounter and recognition of the antigen, induces their blastic transformation which is characterized by a considerable volume increase and the appearance of a cytoplasm rich in mitochondria, ribosomes and endoplasmic reticulum.

Plasma cells (Fig. 23) account instead for around 2.98% (±1.65 SD) [Mischke et al., 2002] of all nucleated cells: these are medium-sized cells, with a moderate nuclear:cytoplasmic ratio, a round, eccentric nucleus, absence of nucleoli, chromatin aggregated in clumps and moderate, intensely basophilic cytoplasm; a clear area, the arcoplasm, which identifies the Golgi apparatus, is found in perinuclear position. The number of these two cell types varies greatly depending on the pathological process present. An increase of both may for example be present following a chronic inflammatory disorder, as an expression of a chronic antigenic stimulus. In the presence of cancer, more evident variations may be observed in the presence of acute and chronic lymphoproliferative disorders.

Osteoclasts (Fig. 24) are giant multinucleated cells, with well-separated nuclei (a characteristic which allows their differentiation from megakaryocytes) and abundant cytoplasm containing magenta-coloured granular material resulting from the removal and digestion of bone. Osteoblasts (Fig. 25), on the contrary, contain a single, round to oval, eccentric nucleus with reticulated chromatin. Within the nucleus 1-2 nucleoli may be present. Both osteoclasts and osteoblasts, which are rarely present in adult subjects, are a common finding during growth and developmental stages as an expression of active bone remodelling. Pathologic conditions characterized by the presence of disorders of calcium metabolism, such as for example paraneoplastic hypercalcaemia (ex. lymphoma and multiple myeloma) and chronic renal failure, may be associated with the finding of such elements.

Macrophages (Fig. 26) usually do not exceed 1% of all nucleated cells [0.22% (±0.24 SD)] (Mischke et al., 2002). These are large-sized cells, with a moderate nuclear:cytoplasmic ratio, a round, eccentric nucleus with finely compacted chromatin. Common findings in the cytoplasm are vacuoles and phagocytized material compatible with nuclear debris, haemosiderin and rarely erythrocytes and/or leukocytes. In chronic inflammatory conditions or in special neoplastic conditions their number may increase.

Suggested readings

1. Feldman B.F., Zinkl J.G., Jain N.C. – Schalm’s Veterinary haematology, 5° edition, ed Lippincott Wlliams & Wilkins, 2000
2. Harvey J.W. Atlas of veterinary hematology – Blood and bone marrow of domestic animals –  Saunders 2001
3. MischkeR, Busse L. – Reference values for the bone marrow aspirates in adult dogs. J Vet Med A Physiol Pathol Clin Med.Dec;49(10):499-502, 2002
4. Stockham S.L., Scott M.A. – Fundamentals of Veterinary Clinical Pathology – 1° edition, Blackwell 2002
5. Willard M.D., Tveden H. – Small Animal Clinical Diagnosis by laboratory Methods – 4° edition, Saunders 2004.