Anaemia, Haemolysis and Haematinic Deficiencies

Question 1

Define anaemia.

Answer 1

• Anaemia is a reduction in red cell count, haemoglobin or haematocrit.
• For men, non anaemia is defined as >130 hb g/L.
• For women, >120 hb g/L.
• N.B. values at sea level.

Question 2

Where are red cells produced?

What is their lifespan?

What are their precursor cells?

What controls their differentiation?

Answer 2

• Red cells are produced in the bone marrow.
• The lifespan is ~120 days.
• They differentiate from the myeloid stem cell lineage.
• Their differentiation is regulated by erythropoietin.

Question 3

List the types of anaemia.

List the causes of each type.

Answer 3

• Anaemia is divided into microcytic, normocytic and macrocytic.

Microcytic (MCV <80fL):

1 - Iron deficiency (more on this later in the lecture).

2 - Thalassaemia.

• Most common in African people, involves mutation to alpha or beta chain of haemoglobin resulting in impaired binding (more on this on another lecture).

3 - Anaemia of chronic disease (more on this later in the lecture).

4 - Sideroblastic anaemia.

• Most common in alcoholics, inability of RBCs to use iron to produce haemoglobin, resulting in accumulation of iron in mitochondria, giving a ringed appearance.

5 - Sickle cell anaemia (more on this on another lecture).

Normocytic (MCV 80-95 fL):

1 - Anaemia of chronic disease.

2 - Acute blood loss.

3 - Mixed haematinic deficiencies.

4 - Bone marrow failure (e.g. aplastic anaemia or chemotherapy drugs).

Macrocytic (MCV >95fL):

1 - Megaloblastic anaemia.

• Caused by B12 and folate deficiency, which are required for development of early red blood cells. Megaloblastic RBCs are therefore immature and structurally abnormal.

2 - Myelodysplasia.

• A precursor to AML, myelodysplasia is a cancer preventing development of RBCs, producing ring sideroblastic RBCs.

3 - Haemolytic anaemia.

4 - Liver disease.

5 - Excessive alcohol consumption.

6 - Drugs, esp. antiepileptics and chemotherapy drugs such as hydroxycarbamide.

7 - Hypothyroidism.

8 - Pregnancy.

Question 4

Describe the changes to the haemoglobin concentration, iron stores and RBC characteristics following bleeding.

Answer 4

• Acutely, bleeding leads to loss of both red cells and plasma, meaning the haemoglobin concentration stays the same.
• The plasma volume then expands to maintain blood pressure, leading to dilution of red cells.
• Chronically, iron stores are depleted due to making more red cells.
• When the iron stores are depleted, the red cells being produced become microcytic and hypochromic.

Question 5

Describe the two routes of iron metabolism.

What is the role of hepcidin in anaemia?

Answer 5

• The route of iron metabolism depends on whether the source of iron is haem-bound or non-haem-bound.
• Non-haem-bound iron carries a charge of +3, and must be converted by duodenal cytochrome B (DCYTB) and ascorbic acid to Fe2+.
• This is then taken up into duodenal enterocyte by divalent metal transporter 1 (DMT1).
• Haem-bound iron (Fe2+) can be directly taken up into the enterocyte by haem carrier protein (HCP).
• In the enterocyte, Fe2+ can either be stored with ferritin or secreted into the blood via the basolateral ferroportin 1. In the blood, it will circulate bound to transferrin.
• The action of ferroportin is governed by hepcidin, which blocks secretion of Fe2+ into the blood.
• In anaemia of chronic disease, hepcidin levels are high, preventing entry of iron into the blood.

Question 6

List the signs and symptoms of anaemia.

Answer 6

Symptoms of anaemia:

1 - Breathlessness.

2 - Palpitations.

3 - Fatigue.

4 - Ability to hear heartbeat.

5 - Restless legs.

Signs of anaemia:

1 - Pallor, esp. conjunctival.

2 - Tachycardia.

3 - Flow murmur.

4 - Hyperdynamic circulation.

5 - Koilonychia (in iron deficiency).

6 - Pica (in iron deficiency - eating non-food items high in iron - common in children).

Question 7

List the causes of iron deficiency anaemia.

Answer 7

• For men and postmenopausal women, the cause is assumed to be a GI malignancy until proven otherwise.

Other causes include:

• Poor diet intake.
• Poor absorption due to coeliac / Crohn’s disease.
• Pregnancy.
• Iron loss through bleeding, which may be due to:

1 - Trauma.

2 - Angiodysplasia (swollen, fragile blood vessels in the colon).

3 - Cancer.

4 - Ulcers.

5 - Inflammation, esp. gastritis.

6 - Menorrhagia.

Question 8

How is iron deficiency diagnosed?

Answer 8

Diagnosis of iron deficiency:

1 - History and examination.

• Diet.
• Bleeding:
• Begin with oesopho-gastro-duodenoscopy (OGD) and colonoscopy unless the patient is a menstruating woman with no features of malignancy or family history.
• Unless OGD shows gastric malignancy or coeliac disease, proceed to lower GI investigation.

2 - FBC and film.

• Look for microcytic hypochromic anaemia.
• Look for target and pencil cells.

3 - Blood tests.

• Low ferritin (N.B. is also raised with inflammation and so can be falsely normal).
• Low serum iron.
• High transferrin (~TIBC).
• Low transferrin saturation.

Question 9

How is iron deficiency distinguished from anaemia of chronic disease using a blood test?

Answer 9

• In iron deficiency:
• Ferritin is low.
• Serum iron is low.
• Transferrin (~TIBC) is high.
• Transferrin saturation is low.
• In anaemia of chronic disease, serum iron and transferrin saturation are also low but:
• Ferritin is normal. This is because iron is still being taken up into enterocytes and stored with ferritin (the problem is secretion of iron into the blood).
• Transferrin is low. This is due to reduced transferrin synthesis related to chronic disease (see card 10).

Question 10

Describe the pathophysiology of anaemia of chronic disease due to infection.

Answer 10

• Anaemia of chronic disease due to infection is an evolutionary mechanism designed to keep iron away from pathogens during infection / inflammation.
• It involves:

1 - Increasing stores of iron.

2 - Decreasing iron absorption by upregulating hepcidin block at the enterocyte basolateral membrane.

3 - Reducing erythropoietin production / release.

• This is a type of functional iron deficiency.

Question 11

What is the drug treatment for iron deficiency anaemia (after addressing any causes of bleeding)?

What is the main challenge associated with this drug and how can it be managed?

Answer 11

• The most common drug treatment for iron deficiency anaemia is 100-200mg oral iron.
• It is generally poorly tolerated due to failure to absorb.
• Ways to improve adherence and absorption include:

1 - Reducing the dose (e.g. consider taking every other day).

• High doses of oral iron can lead to an upregulation of hepcidin, leading to reduced absorption and further accumulation in the gut. Reducing the dose may therefore increase iron absorption and reduce side effects.

2 - Taking iron on an empty stomach.

3 - Taking oral iron with orange juice.

• Orange juice reduces Fe3+ to Fe2+, aiding absorption.
• Intravenous iron is available to those who show poor tolerance or require a faster increase in iron (e.g. with severe anaemia or for an impending surgery / birth).

Question 12

What is the role of vitamin B12 and folate in the production of DNA?

What are the daily requirements of B12 and folate and what is the total body store?

What is the implication of this requirement : store ratio?

Which anaemia is caused by B12 / folate deficiency?

Answer 12

• Vitamin B12 converts methyl-tetrahydrofolate (derived from dietary folates) into tetrahydrofolate.
• Tetrahydrofolate is required for the conversion of uracil into thymine, which is then used to synthesise DNA.
• The daily requirements are 5 micrograms of B12 and 100 micrograms of folate.
• The total body store is 15-25mg (4 months stores) of folate and 2-3mg of B12 (3-4 years stores). This is because folate and B12 are tightly bound intracellularly until cell death.
• This means that folate / B12 deficiencies don’t occur quickly.
• Vitamin B12 / folate deficiency causes megaloblastic anaemia.

Question 13

List causes of folate deficiency.

Answer 13

Causes of folate deficiency include:

1 - Low intake.

• Dietary.
• Pernicious anaemia, preventing absorption of vitamin B12 due to autoimmune response against parietal cells producing intrinsic factor (more on B12 absorption in card 18) (pernicious anaemia is a type of megaloblastic anaemia).

2 - Impaired absorption.

• E.g. coeliac / Crohn’s and drugs such as cholestyramine.

3 - Increased requirement.

• E.g. pregnancy, prematurity, inflammation and homocystinuria.

4 - Increased excretion / loss.

• E.g. haemolysis, dialysis and drugs such as dihydrofolate reductase inhibitors such as methotrexate.

Question 14

List the symptoms of vitamin B12 / folate deficiency.

Answer 14

Symptoms of vitamin B12 / folate deficiency include:

1 - Symptoms of anaemia.

2 - Jaundice (unconjugated - due to haemolysis resulting from malformed RBCs).

2 - Glossitis (swollen, shiny tongue).

3 - Angular cheilitis.

4 - Mild fever.

5 - Skin hyperpigmentation.

6 - Infection and bleeding (because B12 deficiency causes pancytopaenia rather than just megaloblastic anaemia).

7 - Infertility.

8 - Neural tube defects.

Question 15

How is B12 / folate deficiency anaemia (megaloblastic anaemia) seen histologically on a blood film?

Answer 15

Characteristics of B12 / folate deficiency anaemia (megaloblastic anaemia) on a blood film include:

1 - Macrocytic RBCs.

2 - Oval shaped RBCs.

3 - Hypersegmented neutrophil nuclei (>5 lobes).

Question 16

How are folate levels measured?

How does B12 deficiency affect these measurements of folate?

Answer 16

• Folate deficiency is measured using an ELISA technique on blood samples.
• Serum ELISAs are representative of recent intake.
• In B12 deficiency, folate is high in the serum because the folate cannot be processed to enter the cell.
• RBC ELISAs are representative of relatively more long term folate intake.
• In B12 deficiency, folate is low in the RBC (for same reason as above), however the result can also be falsely normal with recent transfusion or high reticulocyte count.

Question 17

How is folate deficiency treated?

What is a risk of this treatment?

Answer 17

• Folate deficiency is treated by (usually oral) folate replacement, typically 5mg/day.
• A risk of excessive folate supplementation is overwhelming the body’s remaining vitamin B12.

Question 18

List the routes of absorption of B12.

Answer 18

Routes of absorption of B12 include:

Passive routes:

1 - Duodenum and ileum (accounts for <1% of total absorption).

Active routes:

1 - Hepatocorins (saliva).

2 - Intrinsic factor (stomach).

3 - Cubilin receptor (ileum).

4 - Transcobalamin (circulation).

5 - Enterohepatic circulation.

Question 19

List the neurological signs and symptoms of B12 deficiency.

Answer 19

Neurological signs and symptoms of B12 deficiency include:

Symptoms:

1 - Parasthesiae.

2 - Muscle weakness.

3 - Difficulty walking.

4 - Confusion.

Signs:

1 - Peripheral neuropathy.

2 - Long tract demyelination.

3 - Dementia.

4 - Psychosis.

Question 20

List 5 causes of B12 deficiency.

Answer 20

Causes of B12 deficiency:

1 - Poor dietary intake (fish, chicken, eggs, cheese and milk).

2 - Gastric malabsorption (e.g. pernicious anaemia).

3 - Ileal malabsorption.

4 - Drugs, e.g. metformin.

5 - Nitrous oxide, which interferes with B12 metabolism.

*Apparent deficiency in oral contraceptive pill / HRT / pregnancy. This is because the test for B12 measure total content (active B12 + inactive / bound B12) rather than just the active B12 (the physiologically relevant component), and the hormonal changes associated with pregnancy / these treatments reduce the bound B12. Hence, this is usually not genuine B12 deficiency.

Question 21

How is vitamin B12 deficiency treated?

Answer 21

• Vitamin B12 deficiency is treated with oral B12 supplements if there is no evidence of pernicious anaemia / malabsorption.
• Even if there is evidence of PA / malabsorption, very high doses can still be given and will be absorbed via the passive route.
• Otherwise hydroxocobalamin (synthetic B12) can be given intramuscularly in 1mg doses 3 times a week for two weeks, and then once every 3 months unless there is a clear reversible cause.

Question 22

List 7 investigations involved in the diagnosis of haemolytic anaemia.

Answer 22

Investigations involved in the diagnosis of haemolytic anaemia:

1 - Full blood count.

2 - Blood film, revealing:

• Spherocytes - especially in immune haemolytic anaemia as the proteins required to maintain the biconcave shape are destroyed.
• Schistocytes.
• Red cell fragments.
• Polychromatophilic cells.

3 - Lactate dehydrogenase (LDH) - a measure of cell turnover.

4 - Haptoglobin - a measure of free haem.

5 - Unconjugated bilirubin - a breakdown product of Hb.

6 - Reticulocyte count - young red cells.

7 - Direct antiglobulin test - tells you whether it is an immune cause by testing for autoantibodies (e.g. caused by recent transfusion).

• This is only useful after confirmed haemolysis because 50% of people test positive for this antibody.

Question 23

List the types of immune haemolytic anaemia.

What is the difference between them?

Briefly give an overview of non-immune haemolytic anaemia.

Answer 23

Immune haemolytic anaemia can be warm, cold or mixed.

1 - IgG autoantibodies are associated with ‘warm’ haemolytic anaemia, which is active at physiological temperatures. This form of anaemia is associated with spherocytes, as the antibodies target the structural proteins of the RBC as they pass through the spleen, leading to loss of the biconcave disk shape.

• Warm haemolytic anaemia is associated with immune thrombocytopenic purpura (ITP).

2 - IgM autoantibodies are associated with ‘cold’ haemolytic anaemia, which is only active at cold temperatures. In this case, RBCs adhere to the pentameric structure of the IgM autoantibody, resulting in agglutination.

• Cold haemolytic anaemia is associated with Epstein-Barr virus, mycoplasma pneumonia and lymphoma.

3 - Mixed immune haemolytic anaemia shows signs of both of the above types.

• Non-immune haemolytic anaemia can be congenital or acquired.
• Congenital causes include membrane structure defects, enzymopathy and haemoglobinopathy.
• Acquired causes include physical breakdowns of RBCs.
• These can be either macrovascular, e.g. stenotic valves causing high pressures that burst RBCs, known as valve haemolysis or after repetitive impacts on the body, known as march haemolysis.
• Or they can be microvascular, e.g. upregulation of the clotting cascade, leading to fibrin strands in the microcirculation which cut the RBCs into pieces.

Question 24

List 3 treatments for immune haemolytic anaemia.

Answer 24

Treatments of immune haemolytic anaemia include:

1 - Folic acid supplementation (to support RBC synthesis).

2 - Splenectomy (to reduce immune response).

3 - Transfusion.