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1

Describe the life cycle of an erythrocyte and what's involve in haematopoesis

Life Cycle of an Erythrocyte

  •     The basic life cycle of a red blood cell involves erythropoiesis and haemoglobin synthesis in the bone marrow, in the peripheral circulation and functioning for oxygen and carbon dioxide transportation, and then removal by the reticulo-endothelial system (mainly by the spleen)

Haematopoesis

  •     It all begins with a multipotential haematopoietic stem cell (hemocytoblast).
  •     Common myeloid progenitor can give rise to megakaryocyte (=> thrombocytes (platelets)), erythrocytes, mast cells and myeloblasts.
  • A myeloblast could give rise to basophils, neutrophils, eosinophils and monocytes (=> macrophages).
  •     A hemocytoblast could also differentiate into a common lymphoid progenitor.
  •     A common lymphoid progenitor could give rise to a small lymphocyte (B cells/ T cells) or natural killer cell (large granular lymphocyte)

2

Describe erythropoesis

    Stem Cell (Hemocyte) => Proerythroblast (committed cell) => enters developmental pathway.

  • Phase 1: ribosome synthesis (occurs in early erythroblast)
  • Phase 2: haemoglobin accumulation (late erythroblast => normoblast)
  • Phase 3: reticulocyte (immature red blood cells without a nucleus)
  • Finally: erythrocyte

NB: note in the blood film that the bigger nuclei are most immature. They get smaller as they mature and gain cytoplasm (accumulate Hb etc)

    Bone marrow produces 10^12 new red cells daily.

    Regulated by erythropoietin (90% produced in kidney, hypoxia stimulates production)

    Erythropoietin increases the number of erythroid progenitor cells, which proliferate, differentiate and produce Hb.

    Plasma Epo levels can be measured (but this is not frequently done in clinical practice)

3

Describe the negative feedback loop in erythropoiesis

    Erythropoiesis involves a negative feedback loop:

  • Kidney senses hypoxia (anaemia) and increases endogenous erythropoietin production
  • Erythropoietin acts on the E-progenitor cells in the bone marrow to produce new red blood cells
  • Kidney senses increased tissue oxygenation
  • Kidney decreases erythropoietin production.

4

Describe the structure of Hb

    Tetramer of 2 pairs of globin chains each with its own haem group

    Exists in 2 configurations

  • Oxyhaemoglobin: relaxed binding structure
  • Deoxyhaemoglobin: tight binding structure
  •     Increasing H+ (pCO2), increasing 2,3-DPG and falling pO2 causes haemoglobin to be in the Tense state (deoxyhaemoglobin – doesn’t bind to oxygen, lower affinity)
  •     Increasing pO2 and CO causes haemoglobin to be in the Relaxed state (oxyhaemoglobin – binds to oxygen tightly, high affinity)

    Different haemoglobins (mostly HbA present but a small amount of HbA2 as well).

    HbF has gamma globin chains instead of beta chains.

5

Describe the functions of haemoglobin and iron

Haemoglobin – Function

  •     Carriage of O2 from lungs to tissues and CO2 between tissues and lungs.

Functions of iron:

    Essential for the formation of:

  • Hb: carries oxygen to the tissues
  • Myoglobin: facilitates oxygen use and storage in muscles
  • Cytochromes: transport electrons within cells

    An integral part of enzyme reactions in various tissues.

6

Give examples of how anaemia can arise due to problems making Hb

    Mutations in the genes that encode the globin proteins can lead to anaemia (haemolytic):

  • Thalassaemia
  • Sickle cell disease

    Lack of iron leads to anaemia

  • Iron deficiency

Anaema of Chronic disease (functional lack of iron – body is not able to use iron, seen in some chronic infections, RA, chronic inflammation etc)

    Deficiency in building blocks for DNA synthesis leads to anaemia (megaloblastic)

  • Vit B12
  • Folate
  • They can also cause pancytopenia

7

Describe the normal red cell structure and  two ways anaemia can arise due to abnormal red cells 

Anaemia can arise due to problems associated with the red cell membrane (abnormalities of the structural proteins making up the membrane) or metabolism within red cells => leading to haemolytic anaemia (shortened lifespan, premature destruction of erythrocytes)

    Normal red cell structure: biconcave flexible disc 8 micrometres diameter

  • Facilitates passage through the microcirculation which has a minimum diameter of 3.5 micrometres

8

What do abnormal red cells look like?

Spherocytes: small dense cells without a normal area of central pallor such as seen in hereditary spherocytosis

  • - Hereditary Spherocytosis: in the common dominant form, Spectrin may be depleted by 40-50% (mutation results in less protein production leading to cytoskeleton being less intact. Erythrocytes round up and become much less resistant to lysis during passage through the capillaries, so are cleared by spleen. The shortened survival of red cells and the inability of the bone marrow to compensate for their reduced lifespan leads to haemolytic anaemia).

Elliptocytes: cigar-shaped cells seen in hereditary elliptocytosis. It’s a common defect resulting in Spectrin molecules unable to form heterotetramers, resulting in fragile elliptoid cells, which lead to haemolytic anaemia.

Acanthocytes: usually found in reactive problems (cells have lots of protrusions). They arise from either alterations in membrane lipids (e.g. in liver dysfunction) or alterations in membrane structural proteins.

Target cells: typical in liver disease, heavy alchol intake, thalassaemias. The cells have an abnormal density in the area of pallor (bull’s eye target appearance)

9

What are the two main metabolic pathways in red cells?

1. Embden-Meyerhof Pathway

  • Glucose metabolised to lactate
  • ATP generated

2. Hexose Monophosphate Pathway

  • Glucose-6-phosphate metabolised
  • Generates NADPH

    Abnormalities within these pathways lead to anaemia (haemolytic) such as in

Glucose-6-phosphate dehydrogenase deficiency

Pyruvate-kinase deficiency 

10

Describe erythrocyte removal

    Reticuloendothelial System (predominanrtly spleen)

    Removal of aged, damaged or antibody-coated red cells.

    Excess removal can occur in autoimmune haemolytic anaemia, mistakes in blood transfusion etc

    Enlargement of the spleen can cause anaemia (blood is spending more time in the splenic circulation than in the normal circulation and splenomegaly also leads to increased RBCs being broken down => anaemia) e.g. liver disease, malaria, malignancy.

11

What kind of things could potentially go wrong to lead to anaemia?

    Erythropoiesis: bone marrow failure, haematinic (iron) deficiency, erythropoietin deficiency

    Haemoglobin synthesis: thalassaemias, structural Hb variants, porphyrias (so haem is not made properly)

    Structure: hereditary spherocytosis, hereditary elliptocytosis

    Metabolism: G6PD deficiency, pyruvate-kinase deficiency

    Circulation: blood loss, intravascular haemolysis (rupture/lysis of erythrocytes within the circulation).

    Removal (Reticuloendothelial system): extravascular haemolysis, hypersplenism

12

What is the definition of anaemia?

Definition of Anaemia

  •     Reduced haemoglobin concentration in the blood

    References range vary between labs but generally

  • <130g/L adult men (normal 135-180)
  • <115g/L adult women (normal 115-160)
  • <110g/L children (3/12 – puberty)
  • <150g/L new-borns

Abnormal results: more often reactive rather than reflective of an underlying haematological disorder

    Interpret in light of clinical context and previous FBC (if known)

    Always ask…does it fit with the clinical scenario?

13

What are the physiological adaptations to anaemia?

   Cardiovascular

  • Increased cardiac output
  • Increased stroke volume
  • Tachycardia (partly because of volume loss, partly because of anaemia)

    Shift in Hb-oxygen dissociation curve

    Increased erythropoiesis (Epo stimulated)

Adaptations depend on speed of onset, severity and age

14

What are the clinical features of anaemia? Symptoms, signs, specific signs

   Symptoms

  • Shortness of breath, reduced exercise tolerance
  • Weakness, lethargy, pain in muscles upon exertion
  • Palpitations
  • Headaches
  • Angina, heart failure, claudication, confusion

    Signs: majority have no signs but could have

  • Pallor
  • Tachycardia
  • Systolic flow murmur
  • Heart failure

    Specific Signs

  • Koilonychia (spooning of nails, could indicate iron-deficiency anaemia)
  • Glossitis (inflammation of tongue, could indicate Vit B12 and/or folate deficiency)
  • Leg ulceration: in thalassaemia, expansion of areas that don’t normally produce bone marrow => skeletal deformities. (Doesn’t happen these days due to treatment regimes)
  • Angular stomatitis: could indicate vitamin B12 and/or folate deficiency

15

Describe the evaluation of anaemia

    Reduced production could be due to haematinic deficiency or bone marrow failure.

    Increased removal could be due to bleeding or destruction e.g. immune haemolysis, mechanical haemolysis

    Reticulocyte count: measure of young early red cells reduced into the circulation (low in reduced production, high in increased removal)

    If the WCC and platelet count are abnormal as well, consider bone marrow infiltration/failure. 

16

What are reticulocytes?

    Named due to staining of ribosomal RNA.

    Normal reticulocyte count is 0.5-1.5% total red cells (20-80)

    The bone marrow can produce erythrocytes at 6-8 x normal rate.

    The absence of an appropriate reticulocytosis in the setting of anaemia suggests RBCs are not being produced appropriate. 

17

Describe the classification of anaemias

    Low reticulocytes: low, normal or high mean cell volume?

    High reticulocytes

18

Describe anaemias with elevated reticulocytes (reticulocytosis/i.e. appropriate reticulocyte response)

If there is an appropriate reticulocyte response, look for evidence of haemolysis (identify cause) or if there is no evidence of haemolysis, look for evidence of bleeding

    Anaemia with reticulocytosis

  • Bleeding
  • Splenic sequestration
  • Haemolysis
  • Immune haemolytic anaemia
  • Mechanical e.g. heart valves,
  • Haemoglobinopathies
  • Enzyme defects
  • Membrane defects

19

What are possible hereditary and acquired anaemias with reticulocytosis

   Hereditary

  • Red cell membrane: HS, He
  • Red cell enzymopathies: G6PD, PK
  • Abnormal Hb – unstable haemoglobins, sickle cell, thalassaemias

   Acquired

  • Alloimmune: - haemolytic disease of the newborn, incompatible transfusion
  • Autoimmune: Warm Antibody Haemolytic Anaemia (idiopathic, URTI, lupus, RA, chronic lymphocytic leukaemia), Col (mycoplasma, Epstein-barr virus), Cold Agglutinin disease, lymphoma, paroxysmal cold haemoglobinuria (rare, typically after exposure to cold temperatures).
  • Non immune: MAHA (microangiopathic haemolytic anaemia (formation of a fibrin mesh due to increased activation of coagulation => the proteins cut erythrocytes)), thrombotic thrombocytopenic purpura, hypersplenish, prosthetic heart valves, sepsis, malaria, paroxysmal nocturnal haemoglobinuria (rare, acquired due to new mutations in the PIGA gene)

20

How do you exclude haemolysis?

Haemolysis screen

    Blood film: spherocytes (small red cells), red cell fragments (schistocytes), polychromasia

    DCT (Direct Coombs Test – uses an antibody-antibody (Coombs reagent) that binds and detects antibodies or complement proteins that are bound to the surface of red blood cells). It is a direct antiglobulin test.

    Bilirubin (if increased RBC destruction)

    Raised LDH (very sensitive to haemolytic anaemia)

    Haptogloin – carrier protein for Hb in the blood, mopping up Hb so if decreased, indicates haemolytic anaemia

    Reticulocyte count

    +/- urinary haemosiderin

21

What if reticulocyte response is not appropriate?

If reticulocyte count is not appropriate (i.e. normal or low), need to look at RBC indices – mean cell volume.

    Microcytic <80fl

    Macrocytic >100fl

    Normocytic

22

What kind of things could cause microcytic anaemias?

    If white cell count and platelet count are low, consider bone marrow failure. But microcytic anaemia is also seen with vit B12 deficiency.

    Microcytic anaemia: TAILS

  • Thalasaemia
  • Anaemia of chronic disease
  • Iron deficiency – commonest cause
  • Lead and others
  • Sideroblastic anaemia (ring shaped erythorcytes because the body is unable to incorporate iron into HB, either genetic disorder or part of myelodysplastic syndrome)

23

How would you confirm and investigate iron deficiency?

Confirm suspicion of iron deficiency

  • Ferritin: acute phase protein, low in iron deficiency, may be raised in reactive states e.g. malignancy. It cannot be used to exclude iron deficiency in people with inflammation – ferritin falsely elevated). Need to do other tests such as
  • Low serum iron/Low % transferrin saturation.

Find out cause of iron deficiency

  • Inadequate intake (vegetarian)
  • Increased loss (GI investigations; CT scan, gastro endoscopy looking for colon cancers). Iron deficiency anaemia is often first presentation of a GI malignancy.
  • Excessive use (pregnancy).

24

Describe the treatment of iron deficiency and dietary iron

    Treatment of iron deficiency

Dietary advice

  • 3x daily Oral iron supplements (take with orange juice/ascorbic acid, do not take with chelaters such as tea), may get abdominal cramps, constipation, black stools
  • Intramuscular iron injections (only if severe – e.g. cardiac complications, oral Fe supplements required afterwards)
  • Intravenous iron
  • Transfusion – not unless there is severe anaemia with imminent cardiac compromise

    Dietary Iron

  • 10-20mg in diet
  • 10-20% is absorbed
  • The bioavailability of iron depends on its chemical form.
  • Haem iron in blood, muscle meat includes fish, chicken (haem iron is better absorbed)

25

Describe the absorption of iron supplements and response to iron supplements

    Absorption of iron supplements:

  • Duodenum and jejunum
  • Ferrous state
  • Maximal absorption in first few weeks of treatment (works best initially)
  • Absorption inhibited by antacids, H2 blockers, PPIs, tetracyclines, calcium, phytates
  • Absorption enhanced by ascorbic acid
  • Take between meals

    Response to oral iron

  • Increase in Hb by 20g/l in 3 weeks (if not as high as expected, suggests ongoing blood loss somewhere)
  • Clinical improvement in symptoms
  • Changes in reticulocyte count
  • Increase in Mean Cell Volume
  • Increase in serum ferritin

26

Describe causes of macrocytic anaemia with hypersegmented neutrophils

Macrocytosis (megaloblastic anaemia) with hypersegmented (multilobular) neutrophils – (usually caused by first two)

B12 deficiency

  • Many years to become Vit B12 deficient (compared to iron deficiency); patients adapt => present late
  • Vegetarians / vegans
  • Gastric (pernicious anaemia) and intestinal causes – Schilling test (used to determine pernicious anaemia)
  • NB: pernicious anaemia’s proper meaning is megaloblastic anaemia caused by atrophic gastritis, parietal cell loss and lack of intrinsic factor only; pernicious anaemia involves the presence of an autoantibody to intrinsic factor required to absorb Vit B12.
  • Oral (if no malabsorption) or IM hydroxycobalamine repacement

Folate deficiency

  • Months to become deficiency
  • Diet
  • Malabsorption
  • Excess use e.g. sickle cell/hereditary spherocytosis
  • Drugs e.g. anticonvulsants, co-trimoxazole

Myelodysplasia-other dysplastic features on film

27

What are other possible causes of macrocytic anaemia?

    Check B12/folate

    Check thyroid function tests/ liver function tests (can occur in hypothyroidism, liver failure, heavy alcohol – can be detected by a Gamma-glutamyl transpeptidase blood test)

    Check immunoglobulins (monoclonal gammopathy of undetermined significance – M protein, myeloma)

    Consider referral for Bone Marrow exam…MDS/other (myelodysplastic syndrome)

28

What are possible causes of normocytic normochromic anaemia

    Anaemia of chronic disease

  • Associated with chronic inflammation/infection
  • Cytokine driven reduction in serum iron (causes increased hepcidin carrier molecule => reduced gut iron absorption + recycled iron pushed into stores i.e. none for red cells).
  • Reduction in erythropoietin production
  • Reduction in lifespan of red cells
  • Can be normocytic normochromic anaemia or microcytic anaemia
  • Ferritin normal or raised
  • Serum iron reduced
  • Normal transferrin saturation (if reduced indicates associated iron deficiency)

    Mixed deficiency of iron and Vit B12/folate (normal MCV, high red cell distribution width)

    BM failure  - normally causes macrocytic but some cause normocytic

29

Describe the metabolism of iron including the two forms it's found in the body

Iron transports and stores oxygen

Iron is an integral part of many enzymes including:

  • Energy metabolism
  • Neurotransmitter production
  • Collagen formation
  • Immune system Function

BUT the human body does not have a mechanism for excreting iron so the quantity of body iron has to be carefully controlled (excess iron can be harmful)

In the body, iron is found in two forms:

1. Active Iron: Predominantly haemoglobin (carries oxygen to tissues) but also

  • Myoglobin (oxygen reserve in muscles)
  • Tissue Iron: (enzyme systems cytochromes)
  • Transported iron: ‘serum iron’

2. Iron stores: there are 2 types

  • Ferritin within the blood, soluble
  • Haemosiderin is macrophage iron and insoluble; iron deposits e.g. in bone marrow

30

Describe the daily requirements of iron

As there is no pathway for excretion of iron, the concentration of iron can only be regulated by absorption to match losses.

Absorption needs to match losses to avoid iron overload or deficiency.

1-2mg of iron enters and leaves the body each day.

Daily iron requirements vary between ages:

  • Iron is present in breast milk
  • Breast milk does not contain a high concentration of iron but it is very well absorbed - ~50% breast milk iron is absorbed, compared to only a 7% absorption from formula
  • By about 6 months of age, however, infants should be introduced to foods containing iron.
  • Iron requirement is very high during pregnancy – very common for pregnant women to become iron deficient as the foetus requires a lot of iron.

31

What are the two types of iron found in our diet?

 

In our diet, two types of iron are found: Haem (e.g. meat, seafood) vs non-Haem iron (e.g. spinach, beans, cereal, lentils)

Need 10-15mg of iron per day in diet

Iron supplementation of diet e.g. cereals

Haem iron is the better iron because it is already in ferrous form (Fe2+) – absorbed more readily across the gut border.

Non-Haem Iron:

  • Mainly exists in the Fe3+ (= ferric)
  • Reduced to ferrous iron before being transported across the intestinal epithelium

Different proteins involved in absorbing haem vs non-haem iron.

32

How is iron absorbed and transported?

Iron is absorbed across the apical surface of the duodenum and upper jejunum.

When it is transported in the blood, it is attached to Transferrin

Transported in the blood to liver, macrophages and bone marrow.

Ferrorportin transports Fe2+ across the border

Once Fe2+ inside the enterocyte, it can either be:

  • Stored as ferritin or
  • Transported into the blood stream

Iron is exported out of the cell by ferroportin.

Fetal enterocytes (cells of the intestinal lining) have receptors for Lactoferrin - primary source of iron in infants. 

When taking iron supplements or eating iron, it is very important how you take it or what you take it with:

Vitamin C present in e.g. orange juice, enhances absorption of iron as ascorbic acid reduces Fe3+ to Fe2+ so it more readily absorbed and transported across the membrane

Tea, chapattis and antacids amongst others inhibit absorption of iron as they precipitate/chelate iron. 

33

How is iron taken into cells?

Iron is taken into cells e.g. Red Blood Cells by binding of iron-transferrin complex to transferring receptor (TfR)

Erythroid cells contain the highest number of TfRs.

A soluble form, soluble TfR (sTfR) is a good indicatior of functional iron levels.

20% of iron might be lost e.g. in bleeding, skin and hair and nails.

80% of iron is recycled in the body

Macrophages are a good place to store iron and deliver iron to RBCs when needed

34

Describe the regulation of iron in the body

Depends on dietary factors, body iron stores and erythropoiesis.

Dietary iron levels are sensed by the villi of enterocytes

Control mechanisms:

  • Regulation of transporters
  • Expression of receptors e.g. HFE and Tf receptor
  • Other humoral and tissue derived factors like hepcidin and cytokines
  • Crosstalk between the epithelial cells and other cells like macrophages

35

What is the role of Hepcidin?

Secreted by the liver and excreted by the kidneys

Synthesis increased in iron overload

  • Transgenic mice constructed to over-express hepcidin died shortly after birth with iron deficiency (they were not able to absorb iron from the gut)
  • Hepcidin production is decreased by high erythropoietic activity.
  • It degrades ferroportin, preventing iron from be released into the blood stream.

REMEMBER most (80%) of active iron comes recycling within the body e.g. breakdown of RBC and NOT gut absorption

36

How is iron recycled?

v    Macrophages ‘eat’ old senescent RBCs, releasing iron during Hb breakdown. Iron is then recycled.

-                Mainly splenic macrophages and Kupfer cells of the liver

-                95% of stored iron in liver tissue is found in hepatocytes as ferritin.

-                Hemosiderin constitutes the remaining 5% and is found predominately in Kupffer cells.

v    Ferritin and transferrin receptor expression are regulated mainly at the post transcriptional level by iron regulatory proteins (IRP1 and IRP2)

37

What could cause iron deficiency?

Iron deficiency is a symptom not a diagnosis so you always need to consider the CAUSE.

  • Insufficient intake (e.g. vegetarians and vegans are at risk of insufficient intake of iron in particular)/poor absorption (e.g. due to a GI disorder)
  • Increased use physiological e.g. pregnancy.
  • Increased use pathological e.g. bleeding
  • Rough estimates of the cause:
    • 25% Menstruation
    • 13% AIDS/NSAID use
    • Colonic carcinoma
    • Gastric carcinoma
    • Benign gastric ulceration
    • 5% blood donation
    • 5% of celiac disease
    • 15% other

38

How would you confirm this iron deficiency?

Tests would reveal:

  • Low Hb
  • Small Red blood cells (low mean cell volume)
  • RBCs change shape and size e..g become pencil cells, hypochrmia – very pale), microcytosis, target cells.
  • Low serum ferritin, serum iron and %transferrin saturation, raised TIBC (Total Iron Binding Capacity)
  • Serum ferritin is produced y the liver and is the single most important measure of iron status, particularly iron store
  • Reduced levels indicate iron deficiency

BUT Limitation: acute or chronic inflammation, malignancy, liver disease and alcoholism can cause increased serum ferritin levels

SO normal or increased levels of serum ferritin do not exclude iron deficiency (as liver could be damaged)

39

Consider iron replacement

If you suspect iron deficiency, investigate the cause:

  • Dietary history
  • GI investigations

Iron replacement:

Dietary advice (e.g. eat iron with orange juice not tea etc)

Supplements: give advice on how to take such as take on an empty stomach but consider what factors affect absorption and often side effects might be strong – cause constipation, diarrhoea, nausea.

Intravenous: there is the rsk of anaphylaxis and death

Intramuscular: risks of anaphylaxis, unpredictable absorption and local complications (e.g. pain, staining of the skin, sarcoma formation).

40

What happens when there is iron excess? what is meant by haemochromatosis?

Iron excess:

When the amount of iron exceeds binding capacity of transferring

Free iron is dangerous as it acts as a free radical and can damage cells

Reduced Fe (Fe2+) can produce highly reactive hydroxyl and lipid radicals, which can damage lipid membranes, nucleic acids and protein

Excess iron is deposited in tissues – Haemosiderin is deposited in liver, pancreas, heart, joints and skin. Very hard to eliminate. 

 

41

How would you treat hereditary haemochromatosis?

42

Describe transfusion associated haemosiderosis

Transfusion associated haemosiderosis occurs in haematology patients, especially ones with chronic anaemia and are dependent on regular transfusions

Desferrioxamine is a liquid drug given over a 16 hour period through a pump (not very convenient) or pleasant).

Oral iron chelating agents (bind, prevent iron absorption) are available but not as good as desferrioxamine therefore can only be used in combination rather than as a sole treatment

Close monitoring is required.