BLOOD
Blood flows throughout the body in the vascular system, and
consists of plasma and three cellular components:
• red cells, which transport oxygen from the lungs to the tissues
white cells, which protect against infection
• platelets, which interact with blood vessels and clotting factors to maintain vascular integrity.
SITES OF HAEMATOPOIE5IS
Haematopoiesis is the process of formation of blood cells. In the embryo this occurs initially in the yolk sac, followed by the liver and spleen; by 5 months in utero, haematopoiesis is established in the bone marrow. At birth, haematopoietic (red) marrow is found in the medullary cavity of all bones, but with age this is progressively replaced by fat (yellow marrow) so that by adulthood, haematopoiesis is restricted to the vertebrae, pelvis, sternum, ribs, clavicles, skull,
upper humeri and proximal femora. Bone marrow usually accounts for 5% of an adult s weight but red marrow can expand in response to increased demands for blood cells.
Bone marrow occupies the intertrabecular spaces in trabecular bone and contains a range of immature haemato¬poietic precursor cells and a storage pool of mature cells for release at times of increased demand. Haematopoietic cells are set in and interact closely with a connective tissue stroma made of reticular cells, macrophages, fat cells, blood vessels and nerve fibres. This stroma provides the suitable microenvironment for blood cell growth and development. Normal marrow has a characteristic organisation . Nests of red cell precursors cluster around a central macrophage which provides iron and phagocytoses extruded nuclei. Megakaryocytes are large cells which produce and release platelets into vascular sinuses. White cell precursors are clustered next to the bone trabeculae; maturing cells migrate into the marrow spaces towards the vascular sinuses. Plasma cells normally represent 5% or less of the marrow population and are scattered throughout the internabecular spac
STEM CELLS
Haematopoiesis is an active process that must maintain normal numbers of circulating blood cells and be able to respond rapidly to increased demands such as bleeding or infection. All blood cells are derived from a pluripotent stem cell which has the ability to self-renew (make more stem cells) and to differentiate to form any of the blood elements. These comprise only0.01% of the total marrow cells and produce a hierarchy of lineage-committed stem cells. As primitive progenitor cells cannot be distinguished morpho¬logically, they are named according to the types of cell (or colony) they form during cell culture experiments. CFU-GMcolony-forming unit-granulocyte, monocyte) i
stem cell that produces granulocytic and monocytic lines. CFU-E produces erythroid cells and CFU-Meg produces megakaryocytes and ultimately platelets (The proliferation and differentiation of stem cells and their progeny are under the control of a range of growth factors produced by several cells, including stromal cells and lymphocytes. These growth factors bind to specific receptors on the cell surface and promote not only proliferation and differentiation but also survival and function of mature cells. Growth factors are often synergistic with other growth factors. Some, such as granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-3 (II,-3) and stem cell factor (SCF), act on a wide number of cell types at both early and late time points. Others, such as erythropoietin (Epo), granulocyte-colony stimulating factor (G-CSF) and thrombopoietin (Tpo), are lineage-specific. Many of these growth factors are now synthesised by recombinant DNA technology and used as treatments.
Recent evidence suggests that the bone marrow contains stem cells which can differentiate into cells from tissues other than the blood, such as nerve, skeletal muscle, cardiac muscle, liver and blood vessel endothelium. This is termed stem-cell plasticity and may have exciting clinical applications in the future.
Red cells
Red cell precursors formed from the erythroid progenitor cells are called erythroblasts or normoblasts These nucleated cells divide and acquire haemoglobin
which turns the cytoplasm pink; the nucleus then condenses and is extruded from the cell. The first non-nucleated red cell is a reticulocyte which still contains ribosomal material in the cytoplasm. Under normal staining conditions reticulocytes are large cells with a faint blue tinge, termed polychromasia. Reticulocytes lose their ribosomal material and mature over 3 days, during which time they are released into the circulation. Increased numbers of circulating reticulocytes (reticulocytosis) reflect increased erythro¬poiesis. Red cell production is controlled by erythropoietin, a polypeptide hormone produced by renal tubular cells in response to hypoxia. Erythropoietin stimulates committed erythroid stem cells to proliferate and decreases maturation time. Patients with renal failure are anaemic due to failure of erythropoietin production, and exogenous recombinant hormone can be used to treat this.
White cells
Granulocytes (neutrophils, eosinophils, basophils) and monocytes are formed from the CFU-GM progenitor cell. The first recognisable granulocyte in the marrow is the myeloblast, a large cell with a small amount of basophilic cytoplasm and a primitive nucleus. As the cells divide and mature, the nucleus segments and the cytoplasm acquires specific neutrophilic, eosinophilic or basophilic granules This takes about 14 days.
A large storage pool of mature neutrophils exists in the bone marrow. Every day some 1014 neutrophils enter the circulation, where cells may be freely circulating or attached
to endothelium in the marginating pool. These two pools are equal in size; factors such as exercise or catecholamines increase the cells flowing in the blood, so increasing the white cell count. Neutrophils spend 6-10 hours in the circulation before being removed, principally by the spleen. Alternatively, they pass into the tissues and either are consumed in the inflammatory process or undergo apoptotic cell death and phagocytosis by macrophages. Myelocytes or metamyelocytes are normally only found in the marrow but may appear in the circulation in infection or toxic states. The appearance of more primitive myeloid precursors in the blood is often associated with the presence of nucleated red cells and is termed a leucoerythroblastic picture; this indicates a serious disturbance of marrow function. Monocytes are large cells derived from monoblasts. These cells circulate for a few hours and then migrate into the tissues where they can mature into macrophages which can proliferate for years. The cytokines G-CSF, GM-CSF and M-CSF are involved in the production of myeloid cells and can be used clinically, e.g. to hasten recovery of blood neutrophil counts after chemotherapy.
Lymphocytes are also derived from the pluripotent haematopoietic stem cell. There are two main types: T cells (80% of circulating lymphoid cells) and B cells. Lymphoid cells which migrate to the thymus develop into T cells, whereas B cells develop in the bone marrow.
Platelets
Platelets are derived from megakaryocytes. Megakaryocytic stem cells (CFU-Meg) divide to form a megakaryoblast; megakaryocytes are formed by endomitotic reduplication where the nucleus divides but not the cell. Thus mature megakaryocytes are large cells with several nuclei and cytoplasm containing platelet granules. Up to 3000 platelets
then fragment off from each megakaryocyte into the circulation in the marrow sinusoids. The formation and maturation of megakaryocytes are under the influence of Tpo, a recombinant form of which is in clinical use. Platelets circulate for 8-14 days before they are destroyed in the reticulo-endothelial system. Some 30% of peripheral platelets are normally pooled in the spleen and do not circulate.
MAJOR FUNCTIONS OF BLOOD CELLS
RED CELLS
The mature red cell is an 8 ~tm biconcave delivers oxygen to the tissues from the lungs, dioxide in the reverse direction. It has no nucleus and no mitochondria; the normal red cell lifespan is about 120 days and in this time it will travel approximately 300 miles around the circulation. Red cells have to pass through the smallest capillaries in the circulation and their membrane structure is adapted to be deformable. The membrane has a lipid bilayer to which a `skeleton of filamentous proteins is attached via special linkage proteins .Inherited abnormalities of any of these proteins result in loss of membrane as cells pass through the spleen, and the formation of abnormally shaped cells called spherocytes or elliptocytes .Red cells are exposed to osmotic stress in the pulmonary and renal circulation; to maintain normal homeostasis, the membrane contains ion pumps which control intracellular levels of sodium, potassium, chloride and bicarbonate. The energy for these functions is provided by the metabolic pathways of the cytosol; 90% of glucose metabolism occurs via anaerobic glycolysis
White cells
Granulocytes (neutrophils, eosinophils, basophils) and monocytes are formed from the CFU-GM progenitor cell. The first recognisable granulocyte in the marrow is the myeloblast, a large cell with a small amount of basophilic cytoplasm and a primitive nucleus. As the cells divide and mature, the nucleus segments and the cytoplasm acquires specific neutrophilic, eosinophilic or basophilic granules This takes about 14 days.
A large storage pool of mature neutrophils exists in the bone marrow. Every day some 101`r neutrophils enter the circulation, where cells may be freely circulating or attached
(To convert kPa to mmHg, multiply by 7.5.)
produces adenosine triphosphate (ATP), and 10% via the pentose phosphate pathway which produces nicotinamide adenine dinucleotide phosphate (reduced) (NADPH). Membrane proteins inserted into the lipid bilayer also form the antigens recognised by blood grouping. The ABO and Rhesus systems are the most commonly recognised (pp. 1020-1022) but over 400 blood group antigens have been described.
Haemoglobin
Haemoglobin is a protein specially adapted for gas transport to and from the lungs. It is composed of four globin chains, each containing an iron-containing porphyrin pigment termed haem. Globin chains are a combination of two alpha and two non-alpha chains; haemoglobin A (aa/(3(3) represents over 90% of adult haemoglobin, whereas haemoglobin F (aalyy) is the predominant type in the fetus. Each haem molecule contains a ferrous ion (Fe Z+) to which oxygen reversibly binds; the final oxygen to bind does so with 20 times the affinity of the first. When oxygen is bound, the beta chains `swing closer together; they move apart as oxygen is lost. In the `open deoxygenated state, 2,3 diphosphoglycerate (DPG), a product of red cell metabolism, binds to the haemoglobin molecule and lowers its oxygen affinity. These complex interactions produce the sigmoid shape of the oxygen dissociation curve (Fig. 24.5). The position of this curve depends upon the concentrations of 2,3 DPG, H+ ions and COZ; increased levels shift the curve to the right and cause oxygen to be released more readily. Tissue hypoxia increases all three and favours increased availability of oxygen from the red cell. Haemo¬globin F is unable to bind 2,3 DPG and has a left-shifted oxygen dissociation curve; this increased affinity, together with the low pH of fetal blood, ensures fetal oxygenation. Amino acid mutations affecting the haem-binding pockets of globin chains or the `hinge interactions between globin chains result in haemoglobinopathies or unstable haemo¬globins. Alpha globin chains are produced by two genes on chromosome 16 and beta globin chains by a single gene on chromosome 11; imbalance in the production of globin chains produces the thalassaemias (p. 1038).
Destruction
Red cells at the end of their lifespan are phagocytosed by the reticulo-endothelial system. Amino acids from globin chains are recycled and iron is removed from haem for reuse in haemoglobin synthesis. The remnant haem structure is degraded to bilirubin and conjugated to glucuronic acid before being excreted into bile. In the small bowel, bilirubin is converted to stercobilin; most of this is excreted, but a small amount is reabsorbed and excreted by the kidney as urobilinogen. Increased red cell destruction due to haemo¬lysis or ineffective haematopoiesis will result in jaundice and increased urinary urobilinogen. Free intravascular haemoglobin is toxic and is normally bound by hapto¬globins, which are plasma proteins produced by the liver.
WHITE CELLS
White cells or leucocytes in the blood consist of granulo¬cytes (neutrophils, eosinophils and basophils), monocytes and lymphocytes (
Neutrophils, the most common white blood cells in the blood of adults, are 10-14 pm in diameter with a multi¬lobular nucleus containing 2-5 segments and granules in their cytoplasm. Their main function is to recognise, ingest and destroy foreign particles and microorganisms. Two main types of granule are recognised: primary or azurophil granules, and the more numerous secondary or specific granules. Primary granules contain myeloperoxidase and other proteins important for killing ingested microbes. Secondary granules contain a number of membrane proteins including adhesion molecules, and components of the NADPH oxidase with which neutrophils produce superoxide anions for microbial killing. Granules fuse with the plasma mem¬brane upon degranulation and their contents are released extracellularly. Granule staining becomes more intense in response to infection and is termed `toxic granulation .
Eosinophils
Eosinophils represent 1-6% of the circulating white cells. They are a similar size to neutrophils but have a bi-lobedl
Causes of neutrophilia
Infection Bacterial Fungal Trauma Surgery Burns Infarction Myocardial infarct Pulmonary embolus Sickle-cell crisis Inflammation Gout Rheumatoid arthritis Ulcerative colitis Crohn s disease
Malignancy Solid tumours Hodgkin lymphoma Myeloproliferative disease Polycythaemia
Chronic myeloid leukaemia Physiological
Exercise Pregnancy
l
Causes of eosinophilia
Allergy
Hay fever Asthma Eczema Infection Parasitic Drug hypersensitivity
e.g. Gold, sulphonamides
Causes of basophilia
Myeloproliferative disease Polycythaemia
Chronic myeloid leukaemia Inflammation
Acute hypersensitivity Ulcerative colitis Crohn s disease
Iron deficiency
Causes of monocytosis
Infection
Bacterial, eg tuberculosis Inflammation
Connective tissue disease Ulcerative colitis
Crohn s disease Malignancy
Solid tumours
Skin disease
Connective tissue disease Polyarteritis nodosa Malignancy
Solid tumours Lymphomas
Lymphocyte
Causes of lymphopenia
Infection Viral
Bacterial, e.g. Bordetella pertussis Lymphoproliferative disease Chronic lymphatic leukaemia Lymphoma
Post-splenectomy
Inflammation
Connective tissue disease Lymphoma
Renal failure Sarcoidosis Drugs
Corticosteroids Cytotoxics Congenital
Severe combined immunodeficiency
nucleus and prominent orange granules on Romanowsky staining. Eosinophils are phagocytic and their granules contain a peroxidase capable of generating reactive oxygen species and proteins involved in the intracellular killing of protozoa and helminths (p. 296). They are also involved in allergic reactions (e.g. atopic asthma, p. 670; see also p. 83).
Basophils
These cells are less common than eosinophils, representing less than 1 % of circulating white cells. They contain dense black granules which obscure the nucleus. Mast cells resemble basophils but are only found in the tissues. Basophils bind IgE antibody on their surface, and exposure to specific antigen results in degranulation with release of
Basophil
Monocyte
Causes of neutropenia
Infection
Viral
Bacterial, e.g. Salmonella Protozoal, e.g. malaria Drugs
Autoimmune
Connective tissue disease Alcohol
Bone marrow infiltration Leukaemia Myleodysplasia Congenital
Kostmann s syndrome
FUNCTIONAL ANATOMY, PHYSIOLOGY AND INVESTIGATIONS
loss from a damaged vessel by securing haemostasis, and also prevent the cessation of flow due to thrombosis. Haemostasis depends upon interactions between the vessel wall, platelets and clotting factors. There are two phases of haemostasis: primary and secondary. In the initial primary phase, the damaged vessel constricts and platelets aggregate at the site of damage to form a plug to arrest haemorrhage within a few minutes. This is followed by activation of the coagulation system with secondary deposition of a fibrin mesh to secure the platelet plug. These two phases are interlinked; damaged endothelium and the subendothelial matrix activate platelets, which then provide the optimal surface for the binding of the plasma clotting factors and the generation of insoluble fibrin.
PLATELETS
Under normal conditions platelets are discoid, with a diameter of 2-4 ltm (Fig. 24.7). The surface membrane invaginates to form a tubular network, the canalicular system, which provides a conduit for the discharge of granule content. Three types of granule are present in the cytoplasm: alpha, delta and lysosomes. Their contents are shown in Figure 24.7.
When platelets are activated by ADP, thrombin or collagen they contract to become spherical and extend long pseudopodia which adhere to the subendothelium and other platelets. Upon activation, platelet granules discharge their contents, which encourages further platelet aggregation and fibrin formation. At the same time, arachidonic acid is released from the platelet membrane and converted by cyclo-oxygenase to endoperoxides and the powerful platelet aggregating agent, thromboxane A2. Aspirin and non¬steroidal anti-inflammatory drugs (NSAIDs) inhibit platelet cyclo-oxygenase and impair platelet function. Platelet¬binding to the subendothelium is dependent on high molecular weight von Willebrand factor released from endothelial cells and platelets, which bridges platelet
histamine, leukotrienes and heparin. These cells are involved in hypersensitivity reactions (p. ).
Monocytes
Monocytes are the largest of the white cells, with a diameter of 12-20 gm and an irregular nucleus in abundant pale blue cytoplasm containing occasional cytoplasmic vacuoles. These cells migrate into the tissue where they become macrophages, Kupffer cells or antigen-presenting dendritic cells. The former phagocytose debris, apoptotic cells and microorganisms. They produce a variety of cytokines when activated, such as interleukin-1 (IL-1), tumour necrosis factor-a (TNF-a) and GM-CSF.
Lymphocytes
In children aged up to 7 years, lymphocytes are the most abundant white cell in the blood. They are heterogeneous, the smallest cells being the size of red cells and the largest being the size of neutrophils. Small lymphocytes are circular with scanty cytoplasm but the larger cells are more irregular with abundant blue cytoplasm. The majority of lymphocytes in the circulation are T cells (80%), which can be recognised by their expression of the CD antigens CD1, 2, 3, 4, 5, 7 and 8. The T cells mediate cellular immunity and two major types are recognised: CD4 positive helper cells and CD8 positive suppressor cells. The B cells mediate humoral immunity and can be recognised by their expression of immunoglobulin light chains (kappa or lambda in a ratio of 2:1). Lymphocyte subpopulations can be defined with specific functions and their lifespan can vary from several days to many years.