Blood consists of

- white cells
- red cells
- platelets
- plasma, in which the above elements are suspended.

Plasma is the liquid constituent element of blood, which includes of soluble fibrinogen in it. Serum is what remains after the formation of the fibrin clot.

The formation of blood cells (haemopoiesis)

The haemopoietic system includes the bone marrow, liver, spleen, lymph nodes and thymus. The turnover of cells is enormous; red cells survive 120 days, platelets around 7 days but granulocytes only 7 hours. The production of as many as 1013 new myeloid cells (all blood cells except for lymphocytes) per day in the normal healthy state obviously requires to be tightly regulated according to the needs of the body.

Blood islands are formed in the yolk sac in the third week of gestation and produce primitive blood cells which migrate to the liver and spleen. These organs are the chief sites of haemopoiesis from 6 weeks to 7 months, when the bone marrow becomes the main source of blood cells. The bone marrow is the only source of blood cells during normal childhood and adult life.

At birth, haemopoiesis is present in the marrow of nearly every bone. As the child grows the active red marrow is gradually replaced by fat (yellow marrow) so that haemopoiesis in the adult becomes confined to the central skeleton and the proximal ends (trabecular area) of the long bones. Only if the demand for blood cells increases and persists do the areas of red marrow extend. Pathological processes interfering with normal haemopoiesis may result in resumption of haemopoietic activity in the liver and spleen, which is referred to as extramedullary haemopoiesis.

All blood cells are derived from pluripotent stem cells. These stem cells are supported by stromal cells (see below) which also influence haemopoiesis. The stem cell has two properties – the first is self-renewal, i.e. the production of more stem cells, and the second is its proliferation and differentiation into progenitor cells, committed to one specific cell line.

There are two major ancestral cell lines derived from the pluripotential stem cell: lymphocytic and myeloid (non-lymphocytic) cells. The former gives rise to T and B cells. The myeloid stem cell gives rise to the progenitor CFU-GEMM (colony-forming unit, granulocyte-erythrocyte-monocyte-megakaryocyte). The progenitor cells such as CFU-GEMM cannot be recognized in bone marrow biopsies but are recognized by their ability to form colonies when haemopoietic cells are immobilized in a soft gel matrix. The CFU-GEMM can go on to form CFU-GM, CFU-Eo, and CFU-Meg, each of which can produce a particular cell type (for example, neutrophils, eosinophils and platelets) under appropriate growth conditions. Haemopoiesis is under the control of growth factors and inhibitors, and the microenvironment of the bone marrow also plays a role in its regulation.

Haemopoietic growth factors are glycoproteins which regulate the differentiation and proliferation of haemopoietic progenitor cells and the function of mature blood cells. They act on receptors expressed on haemopoietic cells at various stages of development to maintain the haemopoietic progenitor cells and to stimulate increased production of one or more cell lines in response to stresses such as blood loss and infection.

The pluripotential stem cells are under the influence of a number of haemopoietic growth factors including interleukin-3 (IL-3), IL-6, -7, -11, β-catenin and stem cell factor (SCF, Steel factor or C-kit ligand). Colony-stimulating factors (CSFs, the prefix indicating the cell type, as well as interleukins and erythropoietin (EPO) regulate the lineage committed progenitor cells. Thrombopoietin (TPO, which, like erythropoietin, is produced in the kidneys and the liver) along with IL-6 and IL-11 control platelet production. In addition to these factors stimulating haemopoiesis, other factors inhibit the process and include tumour necrosis factor (TNF) and transforming growth factor-β (TGF-β). Many of the growth factors are produced by activated T cells, monocytes and bone marrow stromal cells such as fibroblasts, endothelial cells and macrophages; these cells are also involved in inflammatory responses.

Many growth factors have been produced by recombinant DNA techniques and are being used clinically. Examples include G-CSF, which is used to accelerate haemopoietic recovery after chemotherapy and haemopoietic cell transplantation, and erythropoietin, which is used to treat anaemia in patients with chronic renal failure. Thrombopoietin is undergoing clinical trials in patients with idiopathic thrombocytopenic purpura.

Automated cell counters are used to measure the level of haemoglobin (Hb) and the number and size of red cells, white cells and platelets (Table 8.1). Other indices can be derived from these values. The mean corpuscular volume (MCV) of red cells is the most useful of the indices and is used to classify anaemia.

The white cell count (WCC, or WBC, white blood count) gives the total number of circulating leucocytes, and many automated cell counters produce differential counts as well.

Table 8-1. Normal values for peripheral blood

	Male	Female
Hb (g/dL)	13.5-17.5	11.5-16
PCV (haematocrit; L/L)	0.4-0.54	0.37-0.47
RCC (1012/L)	4.5-6.0	3.9-5.0
MCV (fL)		80-96
MCH (pg)		27-32
MCHC (g/dL)		32-36
WBC (109/L)		4.0-11.0
Platelets (109/L)		150-400
ESR (mm/h)		< 20
Reticulocytes	0.5-2.5% (50-100 ×109/L)

Normally, less than 2% of the red cells are reticulocytes (p. 422). The reticulocyte count gives a guide to the erythroid activity in the bone marrow. An increased count is seen with increased marrow maturity, e.g. following haemorrhage or haemolysis, and during the response to treatment with a specific haematinic. A low count in the presence of anaemia indicates an inappropriate response by the bone marrow and may be seen in bone marrow failure (from whatever cause) or where there is a deficiency of a haematinic.

A carefully evaluated blood film is still an essential adjunct to the above, as definitive abnormalities of cells can be seen.

Erythrocyte sedimentation rate (ESR)

This is the rate of fall of red cells in a column of blood and is a measure of the acute-phase response. The pathological process may be immunological, infective, ischaemic, malignant or traumatic. A raised ESR reflects an increase in the plasma concentration of large proteins, such as fibrinogen and immunoglobulins. These proteins cause rouleaux formation, when cells clump together like a stack of coins, and therefore fall more rapidly. The ESR increases with age, and is higher in females than in males. It is low in polycythaemia vera, owing to the high red cell concentration, and increased in patients with severe anaemia.

Plasma viscosity

Plasma viscosity measurement is used instead of the ESR in many laboratories. As with the ESR, the level is dependent on the concentration of large molecules such as fibrinogen and immunoglobulins. There is no difference between levels found in males and females, and viscosity increases only slightly in the elderly. It is not affected by the level of Hb and the result may be obtained within 15 minutes.

C-reactive protein

C-reactive protein is a pentraxin, one of the proteins produced in the acute-phase response. It is synthesized exclusively in the liver and rises within 6 hours of an acute event. It rises with fever (possibly triggered by IL-1, IL-6 and TNF-α and other cytokines) and in inflammatory conditions and after trauma. It follows the clinical state of the patient much more rapidly than does the ESR and is unaffected by the level of Hb, but it is less helpful than the ESR or plasma viscosity in monitoring chronic inflammatory diseases. Its measurement is easy and quick to perform using an immunoassay that can be automated. High-sensitivity assays have shown that increased levels predict future cardiovascular disease.

Erythropoiesis

Red cell precursors pass through several stages in the bone marrow. The earliest morphologically recognizable cells are pronormoblasts. Smaller normoblasts result from cell divisions, and precursors at each stage progressively contain less RNA and more Hb in the cytoplasm. The nucleus becomes more condensed and is eventually lost from the late normoblast in the bone marrow, when the cell becomes a reticulocyte.

Reticulocytes contain residual ribosomal RNA and are still able to synthesize Hb. They remain in the marrow for about 1-2 days and are released into the circulation, where they lose their RNA and become mature red cells (erythrocytes) after another 1-2 days. Mature red cells are non-nucleated biconcave discs.

Nucleated red cells (normoblasts) are not normally present in peripheral blood, but are present if there is extramedullary haemopoiesis and in some marrow disorders (see leucoeryothroblastic anaemia, p. 464).

About 10% of erythroblasts die in the bone marrow even during normal erythropoiesis. Such ineffective erythropoiesis is substantially increased in some anaemias such as thalassaemia major and megaloblastic anaemia.

Erythropoietin is a hormone which controls erythropoiesis. The gene for erythropoietin is on chromosome 7 and codes for a heavily glycosylated polypeptide of 165 amino acids. Erythropoietin has a molecular weight of 30400 and is produced in the peritubular cells in the kidneys (90%) and in the liver (10%). Its production is regulated mainly by tissue oxygen tension. Production is increased if there is hypoxia from whatever cause – for example, anaemia or cardiac or pulmonary disease. The erythropoietin gene is one of a number of genes that is regulated by the hypoxic sensor pathway. The 3′-flanking region of the erythropoietin gene has a hypoxic response element which is necessary for the induction of transcription of the gene in hypoxic cells. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor which binds to the hypoxia response element and acts as a master regulator of several genes that are responsive to hypoxia. Erythropoietin stimulates an increase in the proportion of bone marrow precursor cells committed to erythropoiesis, and CFU-E are stimulated to proliferate and differentiate. Increased ‘inappropriate’ production of erythropoietin is also seen in patients with renal disease and neoplasms in other sites which result in polycythaemia (see Table 8.16).

Haemoglobin synthesis

Haemoglobin performs the main functions of red cells – carrying O2 to the tissues and returning CO2 from the tissues to the lungs. Each normal adult Hb molecule (Hb A) has a molecular weight of 68 000 and consists of two α and two β globin polypeptide chains (α2β2) which have 141 and 146 amino acids respectively. HbA comprises about 97% of the Hb in adults. Two other types, Hb A2 (α2δ2) and Hb F (α2γ2), are found in adults in small amounts (1.5-3.2% and < 1%, respectively).

Haemoglobin synthesis occurs in the mitochondria of the developing red cell. The major rate-limiting step is the conversion of glycine and succinic acid to δ-aminolaevulinic acid (ALA) by ALA synthase. Vitamin B6 is a coenzyme for this reaction, which is inhibited by haem and stimulated by erythropoietin. Two molecules of δ-ALA condense to form a pyrrole ring (porphobilinogen). These rings are then grouped in fours to produce protoporphyrins. Finally, iron is inserted to form haem. Haem is then inserted into the globin chains to form Hb.

Haemoglobin function

The biconcave shape of red cells provides a large surface area for the uptake and release of oxygen and carbon dioxide. Haemoglobin becomes saturated with oxygen in the pulmonary capillaries where the partial pressure of oxygen is high and Hb has a high affinity for oxygen. Oxygen is released in the tissues where the partial pressure of oxygen is low and Hb has a low affinity for oxygen.

In adult haemoglobin (Hb A), a haem group is bound to each of the four globin chains; the haem group has a porphyrin ring with a ferrous atom which can reversibly bind one oxygen molecule. The haemoglobin molecule exists in two conformations, R and T. The T (taut) conformation of deoxyhaemoglobin is characterized by the globin units being held tightly together by electrostatic bonds. These bonds are broken when oxygen binds to haemoglobin, resulting in the R (relaxed) conformation in which the remaining oxygen-binding sites are more exposed and have a much higher affinity for oxygen than in the T conformation. The binding of one oxygen molecule to deoxyhaemoglobin increases the oxygen affinity of the remaining binding sites – this property is known as ‘cooperativity’ and is the reason for the sigmoid shape of the oxygen dissociation curve. Haemoglobin is, therefore, an example of an allosteric protein. The binding of oxygen can be influenced by secondary effectors – hydrogen ions, carbon dioxide and red-cell 2,3-bisphosphoglycerate (2,3-BPG, formerly called 2,3-diphosphoglycerate (2,3-DPG)). Hydrogen ions and carbon dioxide added to blood cause a reduction in the oxygen-binding affinity of haemoglobin (the Bohr effect). Oxygenation of haemoglobin reduces its affinity for carbon dioxide (the Haldane effect). These effects help the exchange of carbon dioxide and oxygen in the tissues.

Oxygenated and deoxygenated haemoglobin molecule. The haemoglobin molecule is predominantly stabilized by α-β chain bonds rather than α-α and β-β chain bonds. The structure of the molecule changes during O2 uptake and release. When O2 is released, the β chains rotate on the α1β2 and α2β1 contacts, allowing the entry of 2,3-BPG which causes a lower affinity of haemoglobin for O2 and improved delivery of O2 to the tissues.

Red cell metabolism produces 2,3-BPG from glycolysis. 2,3-BPG accumulates because it is sequestered by binding to deoxyhaemoglobin. The binding of 2,3-BPG stabilizes the T conformation and reduces its affinity for oxygen. The P50 is the partial pressure of oxygen at which the haemoglobin is 50% saturated with oxygen. P50 increases with 2,3-BPG concentrations, which increase when oxygen availability is reduced in conditions such as hypoxia or anaemia. P50 also rises with increasing body temperature, which may be beneficial during prolonged exercise. Haemoglobin regulates oxygen transport as shown in the oxyhaemoglobin dissociation curve. When the primary limitation to oxygen transport is in the periphery, e.g. heavy exercise, anaemia, the P50 is increased to enhance oxygen unloading. When the primary limitation is in the lungs, e.g. lung disease, high altitude exposure, the P50 is reduced to enhance oxygen loading.

Anaemia is present when there is a decrease in the level of haemoglobin in the blood below the reference level for the age and sex of the individual (Table 8.1). Alterations in the level of Hb may occur as a result of changes in the plasma volume. A reduction in the plasma volume will lead to a spuriously high Hb – this is seen with dehydration and in the clinical condition of apparent polycythaemia. A raised plasma volume produces a spurious anaemia, even when combined with a small increase in red cell volume as occurs in pregnancy. After a major bleed, anaemia may not be apparent for several days until the plasma volume returns to normal.

The various types of anaemia, classified in terms of the red cell indices, particularly the MCV. There are three major types:

• hypochromic microcytic with a low MCV
• normochromic normocytic with a normal MCV
• macrocytic with a high MCV.

Patients with anaemia may be asymptomatic. A slowly falling level of Hb allows for haemodynamic compensation and enhancement of the oxygen-carrying capacity of the blood. A rise in 2,3-BPG causes a shift of the oxygen dissociation curve to the right, so that oxygen is more readily given up to the tissues. Where blood loss is rapid, more severe symptoms will occur, particularly in elderly people.

Symptoms (all non-specific)

• Fatigue
• Headaches
• Faintness

(The above three symptoms are all very common in the general population.)

• Breathlessness
• Angina
• Intermittent claudication
• Palpitations.

Signs

• Pallor
• Tachycardia
• Systolic flow murmur
• Cardiac failure
• Rarely papilloedema and retinal haemorrhages after an acute bleed (can be accompanied by blindness).

Specific signs of the different types of anaemia will be discussed in the appropriate sections. Examples include:

• koilonychia – spoon-shaped nails seen in iron deficiency anaemia
• jaundice – found in haemolytic anaemia
• bone deformities – found in thalassaemia major
• leg ulcers – occur in association with sickle cell disease.

It must be emphasized that anaemia is not a diagnosis, and a cause must be found.

Peripheral blood

A low haemoglobin should always be considered in relation to:

• the white blood cell (WBC) count
• the platelet count
• the reticulocyte count (as this indicates marrow activity)
• the blood film, as abnormal red cell morphology may indicate the diagnosis.

Where two populations of red cells are seen, the blood film is said to be dimorphic. This may, for example, be seen in patients with ‘double deficiencies’ (e.g. combined iron and folate deficiency in coeliac disease, or following treatment of anaemic patients with the appropriate haematinic).

Bone marrow

Examination of the bone marrow is performed to further investigate abnormalities found in the peripheral blood (Practical box 8.1). Aspiration provides a film which can be examined by microscopy for the morphology of the developing haemopoietic cells. The trephine provides a core of bone which is processed as a histological specimen and allows an overall view of the bone marrow architecture, cellularity and presence/absence of abnormal infiltrates.

The following are assessed:

• cellularity of the marrow
• type of erythropoiesis (e.g. normoblastic or megaloblastic)
• cellularity of the various cell lines
• infiltration of the marrow
• iron stores.

Special tests may be performed: cytogenetic, immunological, cytochemical markers, biochemical analyses (e.g. deoxyuridine suppression test), microbiological culture.

Iron deficiency is the most common cause of anaemia in the world, affecting 30% of the world’s population equivalent to 500 million people. This is because of the body’s limited ability to absorb iron and the frequent loss of iron owing to haemorrhage. Although iron is abundant, most is in the insoluble ferric (Fe3+) form, which has poor bioavailability. Ferrous (Fe2+) is more readily absorbed. Free iron is toxic, and it is bound to various proteins for transport and storage.

Practical Box 8.1 Techniques for obtaining bone marrow

The technique should be explained to the patient and consent obtained Aspiration Site – usually iliac crest Give local anaesthetic injection Use special bone marrow needle (e.g. Salah) Aspirate marrow Make smear with glass slide Stain with: Romanowsky technique Perls’ reaction (acid ferrocyanide) for iron. Trephine Indications include: ‘Dry tap’ obtained with aspiration Better assessment of cellularity, e.g. aplastic anaemia Better assessment of presence of infiltration or fibrosis. Technique Site – usually posterior iliac crest Give local anaesthetic injection Use special needle (e.g. Jamshidi – longer and wider than for aspiration) Obtain core of bone Fix in formalin; decalcify – this takes a few days Stain with: Haematoxylin and eosin Reticulin stain.

The other causes of a microcytic hypochromic anaemia are anaemia of chronic disease, sideroblastic anaemia, and thalassaemia. In thalassaemia there is a defect in globin synthesis, in contrast to the other three causes of microcytic anaemia where the defect is in the synthesis of haem.

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