Blood, haematopoiesis and bone marrow

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Blood, haematopoiesis and bone marrow



Introduction


Blood is a suspension of cells in fluid. It is circulated around the body by the heart and, as a result of this circulation, blood serves as the transport vehicle for gases, nutrients, waste products, cells and hormones.


The fluid is known as plasma and a typical sample is composed of 90% water, 8% protein, 1% inorganic salts, 0.5% lipids, 0.1% glucose and other minor components. The proteins are numerous and diverse, including albumin, blood coagulation factors, anti-proteases, transport proteins and antibodies (immunoglobulins). Collectively, these proteins exert a water-binding effect known as colloidal osmotic pressure which helps regulate the distribution of fluid between the plasma and the extracellular space, serving to keep the fluid in the circulation.


Plasma components, including hormones, lipids, salts, water molecules and small proteins, are constantly exchanged with the extracellular fluid of body tissues in accordance with the blood’s transport functions. Proteins and plasma are not demonstrated by light microscopy except as a background colour.



Blood Cell Types


The cellular components of the blood are:



• Red blood cells (erythrocytes) are specialised cells containing the red pigment haemoglobin. They provide most of the oxygen transport from the lungs and much of the return carbon dioxide carriage. They are immotile and serve their function only as the result of being passively circulated around the vascular system. The fraction of blood (by volume) occupied by erythrocytes is called the haematocrit and is in the range of 0.35 to 0.50 in adults.


• White blood cells (leucocytes) constitute an important part of the defence and immune systems of the body but undertake these functions mainly in the tissues; leucocytes in circulating blood are in transit or are simply waiting in reserve.


• Platelets (thrombocytes) are specialised cells which bind to and coat damaged vessel walls, plug small defects in blood vessel walls and help activate the blood-clotting cascade. They are essential for haemostasis, the system that controls bleeding.


In adults these cells are formed in the bone marrow, a process known as haemopoiesis or haematopoiesis.



Methods Used to Study Blood and Bone Marrow Cells


Blood cell numbers are now usually counted by sophisticated laboratory analysers. Blood cell morphology is examined by microscopy using cytological methods. A standard method is to place a drop of blood on a glass slide and spread (smear) it into a very thin layer one cell thick. This smear is then air dried, which results in the cells spreading like fried eggs, making the cells appear larger and giving a clear view into the thinned cytoplasm. These smears are alcohol fixed and stained with Romanowsky-type stains which use polychromatic dyes containing multiple molecular variants to give subtle complexity in the staining. These are the best stains for morphology of blood and bone marrow; common examples are Giemsa and Wright. Distinctive staining characteristics are easily identified and reflect the affinity of the various cellular organelles for different stain components:



Examination of haematopoietic bone marrow in adults involves sampling from the axial skeleton, usually the iliac crest in the pelvis, with an aspiration sample and often a bone core. Aspiration samples, but not usually bone cores, can also be obtained from the sternum. Aspirated tissue fragments are smeared and stained like blood. Larger tissue pieces and cores of bone are examined as histology preparations, often stained with haematoxylin and eosin (H&E).



Clinical use of blood tests


Laboratory analysis of many components of blood, including cells, salts, various proteins, hormones, etc., are a convenient way and are often the only way of examining many body functions. ‘Blood tests’ have a large role in diagnosis and management of disease. For example, increased enzymes in the blood, leaking from damaged heart muscle cells, can diagnose a myocardial infarct (heart attack).



White Cell Series


Five types of leucocytes are normally present in human blood and are classified into two groups:




Granulocytes


Granulocyte types are named for the staining characteristics of their prominent type-specific cytoplasmic granules: neutrophil (lilac), eosinophil (red) and basophil (blue). Granulocytes have a single nucleus segmented into multiple lobes, assuming variable shapes that led to the use of the term polymorphonuclear leucocytes or polymorphs, mainly because early microscopists mistook the multi-lobed nuclei for multiple nuclei. The term polymorph is sometimes used as a synonym for granulocyte. To confound matters further, polymorph is often used specifically for neutrophils as they are the commonest granulocyte. Granulocytes are also referred to as myeloid cells due to their origin from bone marrow, but they are not the only white blood cells to be formed in bone marrow.


Granulocytes are important components of the innate (non-learned) defences against infection (see Ch. 11); however, this role usually takes place in the tissues, not in blood. All leucocytes carry surface proteins which can bind to receptors on the endothelial cells of blood vessels (see Ch. 8) and, via this binding, granulocytes actively adhere to vessel walls and then migrate into the tissues using pseudopodial movement.


With the exception of eosinophils entering intestinal mucosa, granulocytes do not normally enter tissues in any number; they just circulate. They enter tissues in response to chemotactic signals and due to changes in the expression of endothelial cell surface receptors, induced by mediators of acute inflammation.


Neutrophils are highly phagocytic. They engulf and kill microorganisms and ingest cell debris and particulate matter. These cells release multiple important pro-inflammatory chemical signals and regulators contributing to the overall inflammatory process. Granulocytes have a short ‘one-shot’ functional life; having left the circulation they die in the tissues and do not re-enter the blood.



Lymphocytes and monocytes


Lymphocytes and monocytes have non-lobulated nuclei and were described as mononuclear leucocytes by early microscopists to distinguish them from the polymorphs.


Lymphocytes play a key role in all immune responses, facilitating and regulating inflammation. In contrast to other leucocytes, their activity is directed toward specific foreign agents (antigens), providing a learned and targeted response, both antibody- and cell-mediated. Lymphocytes routinely traffic through tissues, then through lymphatics and lymph nodes and finally re-enter the circulation, providing routine surveillance against foreign antigens. They have an indefinite lifespan and are capable of proliferation.


Monocytes are highly phagocytic cells, ingesting micro-organisms, cell debris and particulate matter. They routinely enter some tissues, can mature into macrophages (including becoming resident tissue macrophages) and can have extended tissue survival.


Monocytes and lymphoid cells produce, secrete and have receptors for a large number of inflammation-related chemical mediators.



White cell count


The white cells in blood are a cell population in waiting, a reserve pool. When stimulated by chemotaxins and aided by expression of leucocyte receptors on the endothelial cells (blood vessel lining cells) the blood leucocytes exit into tissue and become part of the inflammatory process. Numbers in blood might be expected to fall as a result, but a large number of mature granulocytes sit in waiting, attached to the lining of small vessels. These form a functional reserve, not included in the blood count, but rapidly mobilised by the chemotaxins and cytokine signals of inflammation.


The same chemical signals stimulate movement of mature and almost mature cells in bone marrow into the circulating blood, forming an additional functional reserve. These signals will also stimulate faster maturation of the existing immature granulocytes in the marrow, which forms yet another reserve, and stimulate increased cell production in marrow from stem cells; this is a slower process.


In most inflammation and infections, the granulocyte blood count will rise. Examples include:



Transient reduction in neutrophils in the blood (neutropenia) can occur due to cytokines produced in early viral infections. Continued reduction in numbers (cytopenia), however, implies that the demand is greater than the supply. This can arise from either increased utilisation or disrupted marrow production.


In very sudden and extremely severe infections, the blood count may fall as the reserve granulocytes are drained faster than increased production can replace them. In these circumstances, immature granulocyte precursors such as band forms, metamyelocytes and myelocytes (see Fig. 3.8) may be mobilised to enter the circulation. This is a phenomenon called left shift. A low neutrophil count (neutropenia) can be a blood count finding in overwhelming or severe sepsis, especially if there is left shift.


Malignant and pre-malignant diseases of white cell precursors can lead to circulating abnormal cells (leukaemia), sometimes in large numbers or, by disrupting production of normal cells, can produce cytopenias.



Haematopoiesis


Haematopoiesis is the process of production of mature blood cells from precursors. This is a major task as an adult produces 100 × 109, i.e. a hundred billion, granulocytes each day. This production is ultimately derived from the pluripotential stem cells and their more differentiated offspring, the haematopoietic stem cells. Stem cells are the cells which provide the lifelong reserve; they actively maintain their own population (self-renewing). They also proliferate and differentiate, contributing to the maintenance of the next level of more mature differentiated populations of stem cells or progenitors. As we move through the process, the emphasis for these cells progressively shifts from self-renewal to proliferation and differentiation.


These cells have been extensively studied in animal transplantation models and in laboratories, in short and long-term culture systems. Many of the identified precursors have been named colony forming units (CFU) or burst forming units (BFU) and the regulatory molecules have been named colony–stimulating factors (CSF), reflecting this laboratory background. Other regulatory molecules have been called interleukins, cytokines or growth factors, depending on their laboratory histories.


The stem cells, progenitors and the later differentiated forms all depend on a complex microenvironment for growth, proliferation and differentiation. This microenvironment is physical, with cell-cell attachments, signalling by these attachments and local secretion of growth factors. The analogy of seed (stem cell) and soil (microenvironment) has often been used.




BFU burst forming unit CFU colony forming unit E erythroid EMeg erythroid megakaryocyte Eo eosinophil G granulocyte GM granulocyte monocyte GMEo granulocyte monocyte eosinophil M monocyte Meg megakaryocyte



Major haematopoietic growth factors


Haematopoiesis is tightly controlled by growth factors and the microenvironment. Growth factors, with the possible exception of erythropoietin, are multifunctional. A single growth factor will influence several different stages of several lineages and promote proliferation and/or differentiation, maturation, egress from bone marrow and survival in tissue. The differences in effect are due to the developmental stage of the cell on which the growth factor is acting, combined with its differentiation, surface receptors and the multitude of other signals it is receiving (see Table 3.1).


The haematopoietic stem cell (HSC) is particularly influenced by stem cell factor (SCF), Flt-3 ligand and vascular endothelial growth factor (VEGF), while the supporting stromal cells of the microenvironment are responsive to interleukin-1 (IL-1) and tumour necrosis factor (TNF). These stem cells are not geographically constrained; they normally circulate in blood in small numbers. Granulocyte-CSF (G-CSF) will mobilise stem cells into the circulation in large numbers. These circulating stem cells then home to sources of stromal derived factor-1 (SDF-1), a product of the haematopoietic microenvironment.


Major generic drivers for haematopoiesis in general are interleukin 3 (IL-3), granulocyte-monocyte CSF (GM-CSF) and stem cell factor (SFC); these promote most lineages at most stages. Thrombopoietin, produced in the kidney and liver, is important for megakaryocyte and platelet production and also promotes the early stages of red cell production. Erythropoietin, a protein hormone produced mostly in the kidneys, drives the later part of red cell production (from CFU-E) but has little effect on the early red cell progenitors. Granulocyte CSF (G-CSF) and monocyte CSF (M-CSF) promote granulocyte and monocyte lineages while interleukin-5 (IL-5) helps drive eosinophil production.


These stem cells and progenitor cells are not recognisable by microscopy. Many resemble lymphocytes and are only identifiable by their expression of different combinations of cell surface molecules. The earliest committed cells recognised in marrow smears are the blasts e.g. myeloblasts, proerythroblasts, monoblasts, etc. These are quite late in the process, about to undertake final proliferation and differentiation. A BFU-E takes about 7 days to become very many CFU-E; each CFU-E takes about 7 days to become many recognisable proerythroblasts and each proerythroblast will form about 16 mature red cells, taking 6 to 7 days. Note that the adult requirement is for about 2.5 billion red cells/kg body weight each day. Many of these growth factors are now available and are used therapeutically.




Bone marrow transplantation


Bone marrow transplantation (BMT) is really haematopoietic stem cell (HSC) transplantation, with the aim of providing a new system of haematopoiesis and, via the lymphocyte pathway, a new immune system. The stem cells are collected either as anticoagulated aspirates of bone marrow from multiple sites or as white cell concentrates collected from peripheral blood after mobilisation of stem cells via injections of G-CSF.


The patient’s original haematopoietic system is destroyed by cytotoxic drugs and radiotherapy. The collected stem cell–containing material is then transfused and the stem cells home back into the haematopoietic microenvironment where they grow, re-establishing haematopoiesis and an immune system.


The donor source may be other people, called allogenic transplantation, or self, called autologous transplantation. When allogenic cells are used, HLA matching is important to minimise graft versus host disease (GVHD), a condition where mature lymphoid cells in the graft recognise the patient as foreign and attack.


BMT is used to treat severe genetic immunodeficiencies and haematopoietic disorders. Examples include aplastic anaemia, a condition of acquired failure of haematopoiesis, and leukaemia. Leukaemia is treated by BMT because the high-dose chemotherapy treatment kills the patient’s stem cells as well as the leukaemic cells. In a similar fashion, autologous transplantation is used to enable survival following very high doses of marrow-toxic drugs, thereby allowing higher-dose, more effective anti-cancer therapy.





Ap adipocyte B bone trabeculae E endothelial cell nucleus H hepatocyte Mk megakaryocyte S sinusoid


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Aug 22, 2016 | Posted by in HISTOLOGY | Comments Off on Blood, haematopoiesis and bone marrow

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