Haematopoiesis and red blood cells

BASIC SCIENCE

Haematopoiesis and red blood cells

proportion of these stem cells are quiescent in the G0 phase of the cell cycle. Entry into the active part of the cycle is controlled by a series of protein enzymes which control DNA synthesis and subsequent progression into mitosis. This is driven by a protein called E2F and inhibited by phosphorylated retinoblastoma protein. Haematopoiesis is nurtured by adjacent stromal cells and soluble growth factors which together help ensure there is appropriate replacement of mature circulating blood cells. An erythrocyte’s average lifespan is 120 days, platelets 9e11 days and granulocytes survive barely a few hours. During the average human lifespan, approximately 5000 kg of blood cells will be manufactured.1,2

Caroline Ebdon Paul Batty Graham Smith

Abstract Our understanding of the formation of blood cells and its control has evolved over the last 40 years from work originally undertaken in murine and subsequently human marrow cells. This led to the concept of haematopoietic stem cells and more recently these same cells have been shown to be capable of generating other cell types such as liver, pancreas and neural tissue. This topic is huge and the authors have focussed on some of the background and clinical aspects which are of importance to surgeons in training.

Erythropoiesis and erythropoietin (EPO) Erythropoiesis is the production of mature erythrocytes from stem cells. It is influenced by multiple growth factors at various stages of maturation. The most important of these is erythropoietin (EPO), a heavily glycosylated 34-kDa protein produced by the kidney (90%) and liver (10%). Erythropoietin concentration is controlled by the oxygen tension in the kidney, with hypoxia leading to an increase in EPO concentration, stimulating erythropoiesis. It has a short plasma half life (6e9 hours), allowing rapid response to alterations in tissue oxygenation EPO exerts its effect by binding to its receptor EPO-R located on ‘burst forming unit-erythroid’ (BFU-E) and ‘colony forming unit-erythroid’ (CFU-E) progenitors (see Figure 2). As EPO levels increase there is reduced apoptosis within these precursors which increases proliferation and terminal differentiation into mature erythrocytes. Once EPO-R is stimulated it undergoes conformational change activating pre-bound cytoplasmic tyrosine kinase, JAK2.1,2 In 2005, a single nucleotide substitution within JAK2, V617F, was discovered. This causes continual stimulus of affected cells resulting in myeloproliferative disease.3 It occurs in 35e57% cases of idiopathic myelofibrosis, 25e57% of essential thrombocythaemia and >95% of polycythaemia vera (PV). By definition PV is defined as a haematocrit >0.52 in males and >0.48 in females.3e5 This can also occur secondarily to a decrease of plasma volume or increased erythropoietin production. PV is associated with arterial and venous thrombosis and thus a patient may present with an ischaemic limb. Other manifestations include hypertension, gout, bleeding, erythrolmyalgia (burning sensations in fingers and toes) and transformation to acute leukaemia. Treatment involves aspirin to reduce the risk of arterial thrombosis and venesection to reduce the haematocrit to <0.45. Cytoreductive therapy (for example hydroxycarbamide or interferon) is indicated if venesection is inappropriate or not tolerated. Patients requiring elective surgery should have their haematocrit controlled for 3 months prior to surgery.2e5

Keywords Anaemia; erythropoietin; haematopoeisis; haemochromatosis; haemoglobin; iron; polycythaemia; sickle cell; thalassaemia; transfusion

Haematopoiesis Haematopoiesis is the production of mature blood cells from stem cells, the principal site of which in adults is the bone marrow. At the start of fetal development however, the primary site is the yolk sac with the involvement of the liver and then the spleen thereafter by the third to seventh month of gestation. At birth haematopoietic tissue is almost entirely located in the bone marrow cavity with the liver and spleen resuming activity only if required.1 Haematopoiesis is a highly orchestrated cellular process, characterized by a stepwise pattern of commitment and differentiation that restricts the potential and the proliferation capacity of cells as they progress through a lineage-specific program of gene expression (Figure 1). Stem cells are capable of self-renewal and develop into multiple cell lines depending on physiological need. They can be identified within the marrow and peripheral blood via expression of specific markers, particularly CD34. In health, a significant

Caroline Ebdon BSc(Hons) MBBS MRCP is a Specialist Registrar in Haematology at Frimley Park Hospital, Frimley, Surrey, UK. Conflicts of interest: none declared. Paul Batty BSc(Hons) MBBS MRCP is a Specialist Registrar in Haematology at the Royal Surrey County Hospital, Guildford, UK. Conflicts of interest: none declared.

Haemoglobin Haemoglobin (Hb) is the oxygen-carrying molecule of erythrocytes, consisting of a tetramer of four polypeptide globin chains, each of which contains an iron (Fe2þ) containing haem group. Adult Hb is predominantly HbA, consisting of two alpha-globin chains and two beta-globin chains (a2b2). Globin is synthesized

J Graham Smith BSc MD FRCP FRCPath is a Professor of Haematology at the University of Surrey and Consultant in Haematology at Frimley Park Hospital, Frimley, Surrey, UK. Conflicts of interest: none declared.

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Overview of haematopoiesis Multipotent stem cell

Common myeloid progenitor

MKs

RBCs

Basophil

Common lymphoid progenitor

Mast cells

Neutrophil

Myeloblasts

Eosinophil

NK cells

Monocyte

Lymphocytes

T cells

B cells

Platelets Macrophages

Plasma cells

RBC, red blood cell; MK, megakaryocyte; NK, natural killer Figure 1

Erythropoietic pathway showing points of erythropoietin action (red arrows) (adapted from Postgraduate Haematology 5th Edition) Progenitors HSC

Precursors CFU-GEMM

BFU-E Early

BFU-E Late

CFU-E

Pro

Bas

Early Pol

Late Ort

Retic

RBC

Maturation HSC, haemopoietic stem cell; CFU-GEMM, colony forming units granulocytic/erythroid/monocytic/megakaryocytic (multilineage colony); BFU-E, burst forming unit erythroid; CFU-E, colony forming unit erythroid; Pro, pronormoblasts; Bas, basophilic erythroblasts; Early Pol, polychromatic erythroblasts; Ort, orthochromatic erythroblasts; Retic, reticulocytes; RBC, mature red blood cells

Figure 2

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in polyribosomes and haem in the mitochondria of erythrocytes. Around 65% of Hb synthesis occurs at the erythroblast stage of development with the remainder being synthesized in reticulocytes. No haemoglobin is synthesized in mature erythrocytes.6 During adult life very small amounts of fetal haemoglobin (Hb F) and HbA2 (a2d2) are detectable. Alpha-globin production is controlled by two genes on both copies of chromosome 16 (four genes), and beta-globin single genes located on each chromosome 11 (two genes).6 Abnormalities can therefore occur because of altered amounts of globin chain produced (for example thalassaemia) or the structure of Hb produced, for example sickle cell anaemia (vide infra).

Ferric iron is transported out of the enterocyte via basolateral ferroportin-1 transporter before binding to transferrin. The ferricironetransferrin complex then travels to the liver via the portal circulation. In the presence of adequate iron stores, these complexes bind to hepatic transferrin-receptor-2 which is associated with the HFE protein causing increased secretion of the protein hepcidin. This small peptide (20e25 amino acids) exerts a negative effect on iron absorption by binding to ferroportin-1 and accelerating its destruction, thus blocking the passage of iron through the enterocyte into the plasma. Hepcidin is downregulated when iron stores are low and upregulated when they are high or in states of inflammation such as is seen with anaemia of chronic disease where hepcidin production is increased 100-fold by IL-6.2

Anaemia Anaemia is a reduction in haemoglobin concentration of the blood, defined in adult males as <13.5 g/dl and <11.5 g/dl in females. Symptoms include fatigue, dyspnoea and dizziness, although many patients are asymptomatic at presentation.6 Anaemias are subclassified either on the basis of morphological criteria and red cell size (mean cell volume (MCV)) or aetiology. Common causes of anaemia are shown in Table 1.

Iron deficiency Iron deficiency can occur through blood loss (for example gastrointestinal blood loss), dietary deficiency (vegan diet), malabsorption (coeliac disease, gastrectomy) or increased physiological requirements (pregnancy). Treatment involves identifying and treating any underlying cause, and replacement of body iron stores. Most patients achieve this by using oral iron preparations, ferrous sulphate 200 mg three times daily, being the most commonly used preparation and dose. An increase in Hb by 2 g/dl every 3 weeks would be expected, taking up to 6 months for iron stores to replenish. In patients unable to tolerate or absorb oral preparations, parenteral (intravenous) preparations can be used. These have similar rates of increase in Hb levels compared to oral forms.1,6

Iron physiology Iron is required for oxygen transport, but excessive amounts can be toxic to the body. As a result there are strict regulatory mechanisms to ensure homeostasis is achieved, with dysregulation resulting in disease. The total iron content of an adult is approximately 4e5 g, most of which is bound with Hb in circulating erythrocytes (approx. 2.5 g). Haem iron is continually recycled following the destruction of senescent erythrocytes by tissue macrophages releasing 25e30 mg of iron per day, the daily requirement for erythropoiesis.1,2 Dietary iron is predominantly absorbed in the duodenum by conversion of insoluble Fe3þ ions to the ferrous (Fe2þ) form by action of gastric acid and the enzyme duodenal cytochrome B (Dcytb) within enterocyte walls. Ferrous iron enters the enterocyte via the divalent metal transporter 1 (DMT1) and is reconverted into the ferric form by ferrioxidases (for example caeruloplasmin).1,2

Vitamin B12 deficiency Vitamin B12 is absorbed in the terminal ileum complexed with intrinsic factor (produced by gastric parietal cells). Deficiencies occur due to pernicious anaemia, ileal disease (Crohn’s disease, ileal resection), dietary deficiency (vegans), blind loop syndromes (diverticulae) or malabsorption (coeliac disease, tropical sprue). Treatment involves reversing any underlying cause and replacement of body vitamin B12 stores. This is achieved with intramuscular hydroxocobalamin 1 mg every 2e3 days for 2 weeks (total six doses) and then further 1 mg doses of hydroxocobalamin every 2e3 months for life.1,6

Causes of anaemia (Adapted from a table in Essential Haematology 5th edition6) Microcytic anaemia MCV <80 fl Iron deficiency Thalassaemia Sideroblastic anaemia Lead poisoning Anaemia of chronic disease

Normocytic anaemia MCV 80e95 fl Acute blood loss Haemolytic anaemia Anaemia of chronic disease Mixed deficiency (e.g. B12 and iron) Bone marrow failure/infiltration Renal disease

Macrocytic anaemia MCV >95 fl B12 deficiency Folate deficiency Alcohol Hypothyroidism Cytotoxic drugs (e.g. hydroxycarbamide) Pregnancy Multiple myeloma Myelodysplasia Liver disease

MCV, mean cell volume

Table 1

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Folic acid deficiency

Sickle cell anaemia

Folate absorption occurs in the proximal jejunum. Deficiencies may result from dietary deficiency, increased requirements/loss (pregnancy, haemolysis, exfoliative dermatitis, renal dialysis), malabsorption (coeliac disease, Crohn’s disease, tropical sprue), alcohol, antifolate drugs (methotrexate, trimethoprim) and other drugs (phenytoin, sodium valproate, oral contraceptive pill). Treatment is with oral folic acid replacement, 5 mg once daily. Prior to starting treatment, B12 levels must be checked as replacement therapy in a B12 deficient patient may precipitate subacute combined degeneration of the spinal cord. In situations of increased folate demand, prophylactic folic acid is recommended.1,6

Sickle cell anaemia (HbSS) is a chronic haemolytic anaemia characterized by episodes of symptomatic vaso-occlusive crises. It arises from a point mutation where valine is substituted for glutamine at position 6 on the b-globin chain. The abnormal Hb forms insoluble polymers in hypoxic states leading to deformation of erythrocytes into the characteristic ‘sickle’ shape, reduced red cell survival and increased blood viscosity. Inheritance is autosomal recessive with heterozygote carriers (HbAS) being asymptomatic. If co-inherited with another haemoglobin variant (for example HbSC) this may lead to a sickle syndrome. Sickle cell anaemia is a phenotypically heterogeneous condition with variable microvascular infarction. Patients are functionally hyposplenic by their 20s with increased risk of infection from encapsulated organisms (Haemophilus influenzae, Streptococcus pneumonia, Salmonella typhimurium, and Neisseria meningitides). Prophylaxis with penicillin is therefore advised.1,5,8

Iron overload Excess iron is removed from the circulation and stored by hepatocytes. When supply exceeds demand and there is a sustained increase in the amount of iron being accumulated either through genetic predisposition, diet or parenterally (multiple blood transfusions) potentially fatal tissue damage can ensue. Hereditary haemochromatosis (HH) is an autosomal recessive condition caused by the C282Y mutation of the HFE gene on chromosome 6. Approximately one in eight people in the UK are heterozygotes for this mutation with around one in 200 being affected.4 It has been demonstrated that release of hepcidin from the liver requires expression of HFE protein. Defects within its gene lead to reduction of the negative regulator of iron absorption resulting in uncontrolled iron absorption via upregulation of the iron transport protein ferroportin-1. Hepcidin also controls iron release from macrophages, and in HH macrophages demonstrate very little iron storage.2 In HH, symptoms usually develop in the fifth to sixth decade when body iron stores of more than 15e20 g have accumulated. Clinical expression is 10 times more common in males. Environmental factors such as alcohol consumption and menstruation in females affect the rate of accumulation and age at presentation. Iron accumulation affects multiple organ systems notably the heart (cardiomyopathy/dysrhythmias), liver (chronic hepatitis, fibrosis, cirrhosis, hepatocellular carcinoma (20e30%)) and endocrine organs (diabetes mellitus, hypothyroidism, hypoparathyroidism, adrenal insufficiency, hypogonadism).4 The earliest indicator of iron overload is an increase in the percentage saturation of plasma transferrin (iron binding capacity). Ferritin levels will also be elevated but can be affected by concurrent infection, inflammation and neoplasia. The aim of treatment is to initially lower the serum ferritin concentration by weekly venesections to <20 mg/litre and transferrin saturation to <16%. Once achieved, transferrin saturations should be maintained at <50% and serum ferritin <50 mg/litre. Family members should be offered counselling and genetic screening once an index case has been identified. Individuals should be carefully monitored for complications, especially cirrhosis and hepatocellular carcinoma (HCC). Surveillance imaging and monitoring of a-fetaprotein (elevated in HCC) should thus be adopted.7

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Sickle cell anaemia and surgery Patients with sickle cell anaemia may present to a surgeon with variable manifestations including osteomyelitis, avascular necrosis, chronic leg ulcers, priapism, haematuria (secondary to medullary infarction) or an acute abdomen (differential diagnoses listed below):8  cholelithiasis (pigmented gallstones)/cholangitis  mesenteric ischaemia  hepatic/splenic infarction  intra-abdominal abscess  renal/hepatic vein thrombosis  constipation. Renal medullary carcinoma almost exclusively presents in patients with sickle cell anaemia or sickle cell carriers. This is an aggressive neoplasm that presents with haematuria, flank pain, fever and weight loss.8 Sickle cell patients are at increased risk of mortality and complications during the perioperative period. Prior to elective surgery all patients from at-risk ethnic groups should be screened for sickle cell anaemia using high performance liquid chromatography (HPLC). All sickle cell patients should have a peri-operative treatment plan made in conjunction with the treating haematologist. Some patients may require either red cell exchange transfusions or ‘top up’ blood transfusions prior to surgery. If a patient is febrile or having a sickle cell crisis, elective surgery should be postponed until this has resolved. Prior to surgery, patients who are nil by mouth should receive intravenous hydration. During surgery patients should be kept normothermic in an oxygen-rich environment. In the postoperative period oxygen saturations should be closely monitored with patients undergoing major surgery often best managed in a high dependency unit.8

Thalassaemia The thalassaemias are a group of conditions associated with a defect in production in one or more of the globin chains. The alpha thalassaemias are a spectrum of illness where there is deletion of one or more of the alpha-globin genes. Excess betaglobin chains tetramerise forming HbH which does not damage the red cell membrane.5

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Risks of blood transfusion. Number in brackets represents the estimated risk per unit transfused9e11 Acute life threatening Delayed complications Acute haemolytic reaction (1/250,000 to 1/1 million) Delayed haemolytic transfusion reaction (1/1000)

Infusion of bacterial contaminated blood Transfusion-related acute lung Injury (TRALI) Acute fluid overload Severe allergic reaction/anaphylaxis

Post-transfusion purpura Transfusion-associated graft versus host disease Iron overload

Transmissible infections Hepatitis A Hepatitis B (1/910,000) Hepatitis C (1/71.92 million) HIV (<1/5.38 million) HTLV 1 (1/23.89 million) Parvovirus B19 Malaria (only five cases in past 25 years)

HIV, human immunodeficiency virus; HTLV, human T-lymphotropic virus

Table 2

 Silent alpha thalassaemia (deletion of 1 alpha-globin genes): undetectable.  Alpha thalassaemia trait (deletion of 2 alpha-globin genes): asymptomatic, Hb concentrations are normal or reduced with a low MCV/MCH. No treatment is required.  Haemoglobin H disease (deletion of 3 alpha-globin genes): moderate anaemia with low MCV/MCH. Signs include hepatosplenomegaly, chronic leg ulcers and jaundice (chronic haemolysis). Management includes prompt treatment of infection and folic acid replacement. Transfusion is seldom required. Splenectomy may be indicated for increasing transfusion demands or massive splenomegaly.  Haemoglobin Bart’s hydrops fetalis (deletion of four alphaglobin genes): tetramers of Hb Bart’s (g4) which binds oxygen tightly are formed leading to organ hypoxia. This is incompatible with life and the fetus either dies in utero or soon after birth. The beta thalassaemias have a deletion in one or more betaglobin gene. Resultant excess of alpha-globin chains cannot tetramerise and alpha-chain monomers damage the red cell membrane resulting in a greater degree of microcytosis and anaemia.5  Beta thalassaemia trait (1 dysfunctional beta-globin gene): asymptomatic with a mild anaemia (Hb not <10) with low MCV and an elevation in HbA2 concentration. Treatment is seldom required.  Beta thalassaemia major (dysfunction of both beta-globin genes): presents in childhood with a moderately severe anaemia (Hb 3e9 g/dl) with low MCV and MCHC. It does not clinically manifest at birth due to persistent production of HbF in the first 6 months. The lack of beta chain formation results in the majority of Hb formed being HbF (a2g2). Affected individuals suffer frequent infections, hepatosplenomegaly, extramedullary haematopoieisis and skeletal deformities. The mainstay of treatment is regular transfusion with adjuvant iron chelation to prevent/reduce iron overload. Splenectomy may be indicated if there are increasing transfusion requirements or massive splenomegaly.

per year. On collection all blood is tested for syphilis, human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV) and hepatitis B and C and other infections based on the donor’s travel history. Red blood cells are separated from the platelets and white cells and then re-suspended in a solution containing saline, adenine, glucose and mannitol (SAGM) to make a red cell concentrate. The average volume of which is 282 ml. Red cell transfusions are given over a period of 2e3 hours, but may be infused faster (5e10 minutes) in the case of massive haemorrhage. Red cell transfusion must be completed within 4 hours of removal from controlled temperature storage. Whole blood is no longer produced as there is no evidence to suggest that this is associated with better outcomes compared with red cell concentrates.9 The indications and risks of blood transfusion are shown below and in Table 2 respectively.  Acute blood loss B <30% blood loss: resuscitation may be required with intravenous crystalloid/synthetic colloids. Red cell transfusion is advised only if there is pre-existing anaemia or the patient is unable to compensate for this degree of blood loss (for example severe cardiac/respiratory disease) or continued haemorrhage. B 30e40% blood loss: rapid replacement with crystalloid or synthetic colloids. Blood transfusion is often required. B 40% blood loss: rapid volume replacement including red cell transfusion.10  Haemoglobin concentration B Hb >10 g/dl: transfusion is not indicated B Hb 7e10 g/dl: transfusion may be required if anaemia is poorly tolerated (for example elderly, severe cardiac/ respiratory disease) B Hb <7 g/dl: transfusion is indicated in relation to ongoing red cell loss.10  Perioperative transfusion: the aim should be to manage the patient such that transfusion can be avoided, by identifying and treating the cause of anaemia preoperatively and to minimize blood loss (for example discontinuing antiplatelet agents). Transfusion up to a ‘normal’ haemoglobin level either prior to or after surgery is not indicated.10  Chronic anaemia: transfusion should not be used if there is an effective alternative treatment. If no alternative exists

Blood transfusion Blood is collected by voluntary donations, with approximately 450e600 ml of blood collected from each donor up to three times

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5 Provan D, Singer CRJ, Baglin T, Lilleyman J. Oxford handbook of clinical haematology. 2nd edn. Oxford University Press, 2004. 6 Hoffbrand AV, Moss PAH, Pettit JE. Essential haematology. 5th edn. Wiley-Blackwell Publishing, 2006. 7 Guidelines on the diagnosis and therapy of genetic haemochromatosis. British Committee for standards in Haematology, British Society of Haematology, 2000. 8 Standards for the clinical care of adults with sickle cell disease in the UK. Sickle Cell Society, 2008. 9 McClelland DBL. Handbook of transfusion medicine. 4th edn. The Stationery Office (TSO), 2007. 10 Murphy MF, Wallington TB, Kelsey P, et al. Guidelines for the clinical use of red cell transfusions. Br J Haematol 2001; 113: 24e31. 11 Safe supplies: testing the nation. Annual report from the NHS Blood and Transplant/Health Protection Agency Centre for Infections Epidemiology Unit, 2008. London, October 2009.

transfusion is given at intervals to maintain the Hb level just above the level that will lead to symptoms.10

A REFERENCES 1 Hoffbrand AV, Catovsky D, Tuddenham EGD. Postgraduate haematology, 5th edn. Blackwell Publishing. 2 Beaumont C, Beris P, Beuzard Y, Brugnara C. Disorders of iron homeostasis, erythrocytes, erythropoiesis. European School of Haematology, 2006. 3 McMullin MF, Reilly JT, Campbell P, et al. Amendment to the guideline for diagnosis and investigation of polycythaemia/erythrocytosis. Br J Haematol 2007; 138: 821e2. 4 McMullin MF, Bareford D, Campbell P, et al. Guidelines for the diagnosis, investigation and management of polycythaemia/ erythrocytosis. Br J Haematol 2005; 130: 174e95.

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