The formation of blood cells (haemopoiesis)

The haemopoietic system includes the bone marrow, liver, spleen, lymph nodes and thymus. There is huge turnover of cells with the red cells surviving 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 requires tight regulation according to the needs of the body.

Blood islands are formed in the yolk sac in the 3rd 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. However, in childhood and adult life, the bone marrow is the only source of blood cells in a normal person.

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 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 (Fig. 8.1). 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 CFU-GEMM can go on to form CFU-GM, CFU-Eo, and CFU-Meg, each of which can produce a particular cell type (i.e. neutrophils, eosinophils and platelets) under appropriate growth conditions. 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. Haemopoiesis is under the control of growth factors and inhibitors, and the microenvironment of the bone marrow also plays a role in its regulation.

Figure 8.1 Role of growth factors in normal haemopoiesis. Some of the multiple growth factors acting on stem cells and early progenitor cells are shown. baso, basophil; BFU, burst-forming unit; CFU, colony-forming unit; CSF, colony-stimulating factor; E, erythroid; Eo, eosinophil; EPO, erythropoietin; G, granulocyte; GEMM, mixed granulocyte, erythroid, monocyte, megakaryocyte; GM, granulocyte, monocyte; IL, interleukin; M, monocyte; Meg, megakaryocyte; SCF, stem cell (Steel) factor or C-kit ligand; TNF, tumour necrosis factor; TPO, thrombopoietin.

Haemopoietic growth factors

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 the cytokine-receptor superfamily 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 (Fig. 8.1).

These haemopoietic growth factors including erythropoietin, interleukin 3 (IL-3), IL-6, -7, -11, -12, β-catenin, stem cell factor (SCF, Steel factor or C-kit ligand) and Fms-tyrosine kinase 3 (Flt3) act via their specific receptor on cell surfaces to stimulate the cytoplasmic janus kinase (JAK) (see p. 25). This major signal transducer activates tyrosine kinase causing gene activation in the cell nucleus. Colony-stimulating factors (CSFs, the prefix indicating the cell type, see Fig. 8.1), 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) controls platelet production, along with IL-6 and IL-11. 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. Bone marrow stem cells can differentiate into other organ cell types, e.g. heart, liver, nerves, bone and this is called stem cell plasticity.

Peripheral blood

Automated cell counters are used to measure the haemoglobin concentration (Hb) and the number and size of red cells, white cells and platelets (Table 8.1). Other indices can be derived from these values. A carefully evaluated blood film is still an essential adjunct to the above, as definitive abnormalities of cells can be seen.

Table 8.1 Normal values for peripheral blood

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.

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 (HbA) has a molecular weight of 68 000 and consists of two α and two β globin polypeptide chains (α₂β₂). HbA comprises about 97% of the Hb in adults. Two other haemoglobin types, HbA₂ (α₂δ₂) and HbF (α₂γ₂), are found in adults in small amounts (1.5–3.2% and <1%, respectively) (see p. 390).

Haemoglobin synthesis occurs in the mitochondria of the developing red cell (Fig. 8.2). 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 and with the addition of iron haem is formed. Haem is then inserted into the globin chains to form a haemoglobin molecule. The structure of Hb is shown in Figure 8.3.

Figure 8.2 Haemoglobin synthesis. Transferrin attaches to a surface receptor on developing red cells. Iron is released and transported to the mitochondria, where it combines with protoporphyrin to form haem. Protoporphyrin itself is manufactured from glycine and succinyl-CoA. Haem combines with α and β chains (formed on ribosomes) to make haemoglobin.

Figure 8.3 Model of the haemoglobin molecule showing α (red) and β (blue) chains. 2,3-BPG (bisphosphoglycerate) binds in the centre of the molecule and stabilizes the deoxygenated form by cross-linking the β chains (also see Fig. 8.4). M, methyl; P, propionic acid; V, vinyl.

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 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 (Fig. 8.4). 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). 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.

Figure 8.4 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.

A summary of normal red cell production and destruction is given in Figure 8.5.

Figure 8.5 Red cell production and breakdown (see p. 307).

Iron

Absorption

Factors influencing iron and haem iron absorption (Fig. 8.8) are shown in Table 8.2.

Figure 8.8 (a) Regulation of the absorption of intestinal iron. The iron-absorbing cells of the duodenal epithelium originate in the intestinal crypts and migrate toward the tip of the villus as they differentiate (maturation axis). Absorption of intestinal iron is regulated by at least three independent mechanisms, although the protein hepcidin is key. First, iron absorption is influenced by recent dietary iron intake (dietary regulator). After a large dietary bolus, absorptive cells are resistant to iron uptake for several days. Second, iron absorption can be modulated considerably in response to body iron stores (stores regulator). Third, a signal communicates the state of bone marrow erythropoiesis to the intestine (erythroid regulator). (b) Duodenal crypt cells sense body iron status through the binding of transferrin to the HFE/B₂M/TfR1 gene complex. Cytosolic enzymes change the oxidative state of iron from ferric (Fe³⁺) to ferrous (Fe²+). A decrease in crypt cell iron concentration upregulates the divalent metal transporter (DMT1). This increases as crypt cells migrate up the villus and become mature absorptive cells. (c) Apical cell. Dietary iron is reduced from the ferric to the ferrous state by the brush border ferrireductase. DMT1 facilitates iron absorption from the intestinal lumen. The export proteins, e.g. ferroportin 1 and hephaestin, transfer iron from the enterocyte into the circulation depending on the hepcidin level. A second pathway absorbs intact haem iron into the circulation via BRCP and FLVCR. BCRP, breast cancer resistant protein; B2M, β₂-microglobulin; FLVCR, feline leukaemia virus subgroup C; HCP1, haem carrier protein-1; HFE, hereditary haemochromatosis gene; TfR1, transferrin receptor.

Table 8.2 Factors influencing iron absorption

Haem iron is absorbed better than non-haem iron Ferrous iron is absorbed better than ferric iron Gastric acidity helps to keep iron in the ferrous state and soluble in the upper gut Formation of insoluble complexes with phytate or phosphate decreases iron absorption Iron absorption is increased with low iron stores and increased erythropoietic activity, e.g. bleeding, haemolysis, high altitude There is a decreased absorption in iron overload, except in hereditary haemochromatosis, where it is increased

Dietary haem iron is more rapidly absorbed than non-haem iron derived from vegetables and grain. Most haem is absorbed in the proximal intestine, with absorptive capacity decreasing distally. The intestinal haem transporter HCP1 (haem carrier protein 1) has been identified and found to be highly expressed in the duodenum. It is upregulated by hypoxia and iron deficiency. Some haem iron may be reabsorbed intact into circulation via the cell by two exporter proteins – BCRP (breast cancer resistant protein) and FLVCR (feline leukaemia virus subgroup C) (Fig. 8.8).

Non-haem iron absorption occurs primarily in the duodenum. Non-haem iron is dissolved in the low pH of the stomach and reduced from the ferric to the ferrous form by a brush border ferrireductase. Cells in duodenal crypts are able to sense the body’s iron requirements and retain this information as they mature into cells capable of absorbing iron at the tips of the villi. A protein, divalent metal transporter 1 (DMT1) or natural resistance-associated macrophage protein (NRAMP2), transports iron (and other metals) across the apical (luminal) surface of the mucosal cells in the small intestine.

Once inside the mucosal cell, iron may be transferred across the cell to reach the plasma, or be stored as ferritin; the body’s iron status at the time the absorptive cell developed from the crypt cell is probably the crucial deciding factor. Iron stored as ferritin will be lost into the gut lumen when the mucosal cells are shed; this regulates iron balance. The mechanism of transport of iron across the basolateral surface of mucosal cells involves a transporter protein, ferroportin 1 (FPN 1) through its iron-responsive element (IRE). This transporter protein requires an accessory, multicopper protein, hephaestin (Fig. 8.8).

The body iron content is closely regulated by the control of iron absorption but there is no physiological mechanism for eliminating excess iron from the body. The key molecule regulating iron absorption is hepcidin, a 25 amino acid peptide synthesized in the liver. Hepcidin acts by regulating the activity of the iron exporting protein ferroportin by binding to ferroportin causing its internalization and degradation, thereby decreasing iron efflux from iron exporting tissues into plasma. Therefore, high levels of hepcidin (occurring in inflammation states) via inflammatory cytokines, e.g. IL-6 will destroy ferroportin and limit iron absorption, and low levels of hepcidin (e.g. in anaemia, low iron stores, hypoxia) will encourage iron absorption. For example, in patients with haemochromatosis, mutations in the genes HFE, HJV and TfR2 will interrupt hepcidin synthesis. Therefore, in the intestinal cells, a deficiency of hepcidin would lead to less ferroportin being bound and thus more iron will be released into the plasma.

A longstanding mystery is why anaemias characterized by ineffective erythropoiesis such as thalassaemia are associated with excessive and inappropriate iron absorption. Preliminary evidence again suggests that the increased iron absorption in β-thalassaemia is mediated by downregulation of hepcidin and upregulation of ferroportin.

FURTHER READING

Andrews NC. Closing the iron gate. N Engl J Med 2012; 366:376−377.

Fleming RE, Ponka P. Iron overload in human disease. N Engl J Med 2012; 366:1548−1550.

Hentze MW, Muckenthaler MU, Galy B et al. Two to tango: regulation of mammalian iron absorption. Cell 2010; 142:24–38.

Iron deficiency

Iron deficiency anaemia develops when there is inadequate iron for haemoglobin synthesis. The causes are:

Most iron deficiency is due to blood loss, usually from the uterus or gastrointestinal tract. Premenopausal women are in a state of precarious iron balance owing to menstruation. A common cause of iron deficiency worldwide is blood loss from the gastrointestinal tract resulting from parasites such as hookworm infestation. The poor quality of the diet, predominantly containing vegetables, also contributes to the high prevalence of iron deficiency in developing countries. Even in developed countries, iron deficiency is not uncommon in infancy where iron intake is insufficient for the demands of growth. It is more prevalent in infants born prematurely or where the introduction of mixed feeding is delayed.

Clinical features

The symptoms of anaemia are described on page 375. The well known clinical features of iron deficiency listed below are generally only seen in cases of very longstanding iron deficiency:

Brittle nails

Spoon-shaped nails (koilonychia)

Atrophy of the papillae of the tongue

Angular stomatitis

Brittle hair

A syndrome of dysphagia and glossitis (Plummer–Vinson or Paterson–Brown–Kelly syndrome; see p. 243).

The diagnosis of iron deficiency anaemia relies on a clinical history which should include questions about dietary intake, self-medication with non-steroidal anti-inflammatory drugs (which may give rise to gastrointestinal bleeding), and the presence of blood in the faeces (which may be a sign of haemorrhoids or carcinoma of the lower bowel). In women, a careful enquiry about the duration of periods, the occurrence of clots and the number of sanitary towels or tampons (normal 3–5/day) used should be made.

Investigations

Blood count and film. A characteristic blood film is shown in Figure 8.9. The red cells are microcytic (MCV <80 fL) and hypochromic (MCH (mean corpuscular haemoglobin) <27 pg). There is poikilocytosis (variation in shape) and anisocytosis (variation in size). Target cells are seen.

Serum iron and iron-binding capacity. The serum iron falls and the total iron-binding capacity (TIBC) rises in iron deficiency compared with normal. Iron deficiency is regularly present when the transferrin saturation (i.e. serum iron divided by TIBC) falls below 19% (Table 8.3).

Serum ferritin. The level of serum ferritin reflects the amount of stored iron. The normal values for serum ferritin are 30–300 µg/L (11.6–144 nmol/L) in males and 15–200 µg/L (5.8–96 nmol/L) in females. In simple iron deficiency, a low serum ferritin confirms the diagnosis. However, ferritin is an acute-phase reactant, and levels increase in the presence of inflammatory or malignant diseases. Very high levels of ferritin may be observed in hepatitis and in a rare disease, haemophagocytic lymphohistiocytosis (p. 80).

Serum soluble transferrin receptors. The number of transferrin receptors increases in iron deficiency. The results of this immunoassay compare well with results from bone marrow aspiration at estimating iron stores. This assay can help to distinguish between iron deficiency and anaemia of chronic disease (Table 8.3), and may avoid the need for bone marrow examination even in complex cases. It may sometimes be helpful in the investigation of complicated causes of anaemia.

Other investigations. These will be indicated by the clinical history and examination. Investigations of the gastrointestinal tract are often required to determine the cause of the iron deficiency (see p. 257).

Figure 8.9 Hypochromic microcytic cells (arrow) on a blood film. Poikilocytosis (variation in size) and anisocytosis (variation in shape) are seen.

Table 8.3 Microcytic anaemia: the differential diagnosis

Differential diagnosis

The presence of anaemia with microcytosis and hypochromia does not necessarily indicate iron deficiency. The most common other causes are thalassaemia, sideroblastic anaemia and anaemia of chronic disease, and in these disorders the iron stores are normal or increased. The differential diagnosis of microcytic anaemia is shown in Table 8.3.

Treatment

The correct management of iron deficiency is to find and treat the underlying cause, and to give iron to correct the anaemia and replace iron stores. Patients with iron deficiency taking iron will increase their Hb level by approximately 10 g/L/week unless of course other factors such as bleeding are present.

Oral iron is all that is required in most cases. The best preparation is ferrous sulphate (200 mg three times daily, a total of 180 mg ferrous iron), which is absorbed best when the patient is fasting. If the patient has side-effects such as nausea, diarrhoea or constipation, taking the tablets with food or reducing the dose using a preparation with less iron such as ferrous gluconate (300 mg twice daily, only 70 mg ferrous iron) is all that is usually required to reduce the symptoms.

In developing countries, distribution of iron tablets and fortification of food are the main approaches for the alleviation of iron deficiency. However, iron supplementation programmes have been ineffective, probably mainly because of poor compliance.

Oral iron should be given for long enough to correct the Hb level and to replenish the iron stores; this can take 6 months. The commonest causes of failure to respond to oral iron are:

Lack of compliance

Continuing haemorrhage

Incorrect diagnosis, e.g. thalassaemia trait.

These possibilities should be considered before parenteral iron is used. However, parenteral iron is required by occasional patients, e.g. intolerant to oral preparation, severe malabsorption, chronic disease (e.g. inflammatory bowel disease). Iron stores are replaced much faster with parenteral iron than with oral iron, but the haematological response is no quicker. Parenteral iron can be given by slow intravenous infusion of low-molecular-weight iron dextran (test dose required), iron sucrose, ferric carboxymaltose, iron isomaltoside 1000; oral iron should be discontinued.

Anaemia of chronic disease

One of the most common types of anaemia, particularly in hospital patients, is the anaemia of chronic disease, occurring in patients with chronic infections such as tuberculosis or chronic inflammatory disease such as Crohn’s disease, rheumatoid arthritis, systemic lupus erythematosus (SLE), polymyalgia rheumatica and malignant disease. There is decreased release of iron from the bone marrow to developing erythroblasts, an inadequate erythropoietin response to the anaemia, and decreased red cell survival.

The exact mechanisms responsible for these effects are not clear but it seems likely that high levels of hepcidin expression play a key role (see iron absorption).

The serum iron and the TIBC are low, and the serum ferritin is normal or raised because of the inflammatory process. The serum soluble transferrin receptor level is normal (Table 8.3). Stainable iron is present in the bone marrow, but iron is not seen in the developing erythroblasts. Patients do not respond to iron therapy, and treatment is, in general, that of the underlying disorder. Recombinant erythropoietin therapy is used in the anaemia of renal disease (see p. 623), and occasionally in inflammatory disease (rheumatoid arthritis, inflammatory bowel disease).

Sideroblastic anaemia

Sideroblastic anaemias are inherited or acquired disorders characterized by a refractory anaemia, a variable number of hypochromic cells in the peripheral blood, and excess iron and ring sideroblasts in the bone marrow. The presence of ring sideroblasts is the diagnostic feature of sideroblastic anaemia. There is accumulation of iron in the mitochondria of erythroblasts owing to disordered haem synthesis forming a ring of iron granules around the nucleus that can be seen with Perls’ reaction. The blood film is often dimorphic; ineffective haem synthesis is responsible for the microcytic hypochromic cells. Sideroblastic anaemias can be inherited as an X-linked disease transmitted by females. Acquired causes include myelodysplasia, myeloproliferative disorders, myeloid leukaemia, drugs (e.g. isoniazid), alcohol misuse and lead toxicity. It can also occur in other disorders such as rheumatoid arthritis, carcinomas, megaloblastic and haemolytic anaemias. A structural defect in δ-aminolaevulinic acid (ALA) synthase, the pyridoxine-dependent enzyme responsible for the first step in haem synthesis (Fig. 8.2), has been identified in one form of inherited sideroblastic anaemia. Primary acquired sideroblastic anaemia is one of the myelodysplastic syndromes (see p. 405) and this is the cause of the vast majority of cases of sideroblastic anaemia in adults. Lead toxicity is described in Chapter 17.

Vitamin B₁₂

Vitamin B₁₂ is synthesized by certain microorganisms, and humans are ultimately dependent on animal sources. It is found in meat, fish, eggs and milk, but not in plants. Vitamin B₁₂ is not usually destroyed by cooking. The average daily diet contains 5–30 µg of vitamin B₁₂, of which 2–3 µg is absorbed. The average adult stores some 2–3 mg, mainly in the liver, and it may take 2 years or more after absorptive failure before B₁₂ deficiency develops, as the daily losses are small (1–2 µg).

Structure and function

Cobalamins consist of a planar group with a central cobalt atom (corrin ring) and a nucleotide set at right-angles (Fig. 8.13). Vitamin B₁₂ was first crystallized as cyanocobalamin, but the main natural cobalamins have deoxyadenosyl-, methyl- and hydroxocobalamin groups attached to the cobalt atom.

Figure 8.13 Methylcobalamin structure. This is the main form of vitamin B₁₂ in the plasma.

The main function of B₁₂ is the methylation of homocysteine to methionine with the demethylation of methyl THF polyglutamate to THF. THF is a substrate for folate polyglutamate synthesis.

Deoxyadenosylcobalamin is a coenzyme for the conversion of methylmalonyl CoA to succinyl CoA. Measurement of methylmalonic acid in urine was used as a test for vitamin B₁₂ deficiency but it is no longer carried out routinely.