Chapter 8 Haematological disease
Introduction and general aspects
Haemopoietic growth factors
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.
Stem cell diseases
The clonal proliferation of bone marrow stem cells leads to diseases including leukaemia (see p. 451), polycythaemia vera (see p. 402), myelofibrosis (see p. 404), paroxysmal nocturnal haemoglobinuria (see p. 401). Failure of stem cell growth leads to aplastic anaemia (see p. 385).
Peripheral blood
The mean corpuscular volume (MCV) of red cells is a useful index and is used to classify anaemia (see p. 376).
The red cell distribution width (RDW) is calculated by dividing the standard deviation of the red cell width by the mean cell width × 100. An elevated RDW suggests variation in red cell size, i.e. anisocytosis, and this is seen in iron deficiency. In β-thalassaemia trait, the RDW is usually normal.
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.
Reticulocytes are young red cells and usually comprise <2% of the red cells. 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.
Erythrocyte sedimentation rate (ESR) 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, with red cells clumping together and therefore falling more rapidly. The ESR increases with age, and is higher in females than in males.
Plasma viscosity is a measurement used instead of the ESR in some laboratories. It is also dependent on the concentration of large molecules such as fibrinogen and immunoglobulins. It is not affected by the level of Hb.
C-reactive protein (CRP) 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. The CRP level rises with fever (possibly triggered by IL-1, IL-6 and TNF-α and other cytokines), in inflammatory conditions and after trauma. It follows the clinical state of the patient much more rapidly than 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. The measurement of CRP is easy and quick to perform using an immunoassay that can be automated. High-sensitivity assays have shown that increased levels may predict future cardiovascular disease (see p. 728).
Table 8.1 Normal values for peripheral blood
Male | Female | |
---|---|---|
Hb (g/L) | 135–175 | 115–160 |
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/L) | 320–360 | |
RDW (%) | 11–15 | |
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) |
ESR, erythrocyte sedimentation rate; Hb, haemoglobin; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration; MCV, mean corpuscular volume of red cells; PCV, packed cell volume; RCC, red cell count; RDW, red blood cell distribution width; WBC, white blood count.
The red cell
Erythropoiesis
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. 413).
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 30 400 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, e.g. 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 occurs in certain tumours such as renal cell carcinoma and other causes (see Table 8.15).
Haemoglobin synthesis
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.
Anaemia
Anaemia is present when there is a decrease in Hb in the blood below the reference level for the age and sex of the individual (Table 8.1). Alterations in the Hb may occur as a result of changes in the plasma volume, as shown in Figure 8.6. 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 (see p. 404). A raised plasma volume produces a spurious anaemia, even when combined with a small increase in red cell volume as occurs in pregnancy.
Clinical features
Investigations
Peripheral blood
A low Hb should always be evaluated with:
The white blood cell (WBC) count
The reticulocyte count (as this indicates marrow activity)
The blood film, as abnormal red cell morphology (see Fig. 8.9) 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
Techniques for obtaining bone marrow are shown in Practical Box 8.1.
Practical Box 8.1
Techniques for obtaining bone marrow
The technique should be explained to the patient and consent obtained.
Type of erythropoiesis (e.g. normoblastic or megaloblastic)
Cellularity of the various cell lines
Infiltration of the marrow, i.e. presence of non-haematopoietic cells such as cancer cells
Microcytic anaemia
Iron
Absorption
Factors influencing iron and haem iron absorption (Fig. 8.8) are shown in Table 8.2.
Table 8.2 Factors influencing iron absorption
Iron deficiency
Clinical features
Spoon-shaped nails (koilonychia)
Atrophy of the papillae of the tongue
A syndrome of dysphagia and glossitis (Plummer–Vinson or Paterson–Brown–Kelly syndrome; see p. 243).
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).
Anaemia of chronic disease
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.
Macrocytic anaemias
Megaloblastic anaemia
Vitamin B12 deficiency or abnormal vitamin B12 metabolism
Folic acid deficiency or abnormal folate metabolism
Other defects of DNA synthesis, such as congenital enzyme deficiencies in DNA synthesis (e.g. orotic aciduria), or resulting from therapy with drugs interfering with DNA synthesis (e.g. hydroxycarbamide (hydroxyurea), azathioprine, zidovudine – AZT)
Haematological findings
Anaemia may be present. The MCV is characteristically >96 fL unless there is a co-existing cause of microcytosis when there may be a dimorphic picture with a normal/low average MCV.
The peripheral blood film shows oval macrocytes with hypersegmented polymorphs with six or more lobes in the nucleus (Fig. 8.11).