Haemoglobinopathies

Question 1

Describe Hb structure and synthesis

Answer 1

Composed of four polypeptide globin chains, each having a single haem group containing ferrous iron.
Haem is synthesised by liver, muscles, erythroblasts and nucleated RBC’s.
Globin is synthesised by nucleated RBC’s.

Question 2

Describe the different types of globins produced by humans at different stages of life.

Answer 2

Embryonic Hb: exists for first 12 weeks, consists of alpha, zeta, epsilon and gamma globulin chains, which make up Gower 1, Portland and Gower 2.

Foetal Hb: composed of 2 alpha and 2 gamma chains.
HbF has higher oxygen affinity, allowing foetus to obtain oxygen from mothers circulation.

Adult Hb: switch occurs after 6 months.
Beta globulin replaces gamma globulin.
95% is HbA, consisting of 2 alpha and 2 beta chains.
2-3% is HbA2, consisting of 2 alpha and 2 delta chains.
0.2-1% is HbF.

Question 3

Describe qualitative haemoglobinopathies.

Answer 3

Production of abnormal Hb structure, due to point mutations that alter amino acid composition of proteins.

SCD is an example disease.

Question 4

Describe quantitative haemoglobinopathies.

Answer 4

Decreased production of normal alpha or beta chains.

For example, alpha and beta thalassaemia’s.

Question 5

Describe the antenatal screening programme.

Answer 5

Recommends all pregnant women in England to be offered screening for haemoglobinopathies and routine FBC’s.
Furthermore, identifies women living in high prevalence areas and those from ethnic minorities that are at a higher risk.
Women are offered screening by 10 weeks of pregnancy, and definitely before 12 weeks.
This allows individuals to be treated early on and supports pregnancies, thus avoiding deaths.
Until 2018, >730,000 women were screened, of which 15,000 were positive.

Question 6

Describe the general characteristics of thalassaemia.

Answer 6

Decreased synthesis of α or β chains.
Causes an imbalance of alpha and beta chain synthesis, resulting in an excess in one chain over the other.
Unmatched globin chains aggregate, causing apoptosis of erythroid precursors.
Abnormal erythrocytes are removed by macrophages of reticuloendothelial system.

Question 7

Describe alpha thalassaemia silent carrier.

Answer 7

1 α-globin gene deletion (-α/αα).
Asymptomatic, silent carriers.

Haematology lab:
There may be mild anaemia during illness or pregnancy.
Difficult to identify in lab.
Can do DNA analysis using Hb electrophoresis.

Question 8

Describe alpha thalassaemia trait.

Answer 8

2 α-globin gene deletions (–/αα or -α/-α).
Typically asymptomatic.

Haematology lab:
FBC: Normal or low Hb, decreased MCV and MCH, increased RBCC (compensatory mechanism).
Blood film: Microcytic, hypochromic RBC’s.

Question 9

Describe haemoglobin H disease.

Answer 9

3 α-globin gene deletions (–/-α).
Excess β-globin chains form tetramers.
Splenomegaly, hepatomegaly, jaundice, bone marrow hyperplasia.

Haematology lab:
FBC: Moderate decrease in Hb; decreased MCV and MC; increased RBCC.
Blood film: Microcytic, hypochromic cells; nucleated RBC’s; increased reticulocytes; target cells; poikilocytes and anisocytes; inclusion bodies can be seen with supravital staining.

Question 10

Explain why target cells arise in Hb H disease.

Answer 10

Hb H disease is characterised by decreased Hb synthesis, so RBC has less Hb, resulting in dips in the cell membrane, giving rise to target cell formation.

Question 11

Describe Bart’s hydrops fetalis.

Answer 11

4 α-globin chain deletions (-/-/-/-).
Incompatible with life as neither HbF or HbA can be synthesised.
Forms γ tetramers, which have a very high affinity for oxygen so release oxygen to tissues poorly.
Death occurs in utero.
Hepatomegaly, jaundice, ascites (huge swollen stomach).

Question 12

What chains are produced in excess in alpha thalassaemia?

Answer 12

Excess gamma chains and beta chain are produced.
Excess gamma chains form tetramers called HB Bart.
Excess beta chains form tetramers called HB H.

Question 13

What treatment can be given to patients with SCD?

Answer 13

Occasional blood transfusion if mild.
In severe cases, can give regular transfusions; splenectomy, for patients with increased transfusion demands, or splenomegaly; iron chelation, to remove excess iron as more iron is introduced into body.
Iron levels are monitored by serum ferritin levels and liver MRI.

Question 14

Describe the abnormality associated with beta thalassaemia.

Answer 14

More than 200 different mutations in beta globin gene, present on chromosome 11.
Cause decreased production of beta globin genes.
Results in increased production of gamma globulin chains, which pair with alpha globulin chains to form foetal Hb.
Mutations are mostly point mutations.
Autosomal recessive disorder.

Question 15

Describe beta thalassaemia minor.

Answer 15

Silent carrier disorder
β/β+, reduced synthesis of one beta globin gene.
Low MCV

Clinical presentation:
Hypochromic, microcytic cells
High RBCC >5.5 x10^12/L.
Mild anaemia that doesn’t respond to iron supplements.
Hb 10-12g/Dl.
>3.5% of HbA2

Question 16

Describe beta thalassaemia trait.

Answer 16

β/β*, no synthesis of one beta globin gene.
FBC: Slight decrease in Hb; low MCV; increased RBCC.
Blood film: Microcytic, hypochromic cells; target cells.
Mild anaemia

Question 17

Describe beta thalassaemia intermedia.

Answer 17

β+/β+, decreased synthesis of both genes.
Low MCV
Moderate anaemia

Question 18

Describe beta thalassaemia major.

Answer 18

β/β, no beta chain synthesis.
Causes excess of alpha chains.
FBC: low MCV and MCH.
Blood film: Target cells; nucleated RBC’s; hypochromic, microcytic cells; reticulocytes; basophilic stippling; teardrop cells; cell fragments; poikilocytes and anisocytes.
Severe anaemia, hepatomegaly, splenomegaly, bone marrow hyperplasia, ‘hair on end’ appearance in skull x-ray.

Question 19

Describe alpha chain synthesis in beta thalassaemia major.

Answer 19

Causes excess alpha chain synthesis, which precipitate out into Heinz bodies.
They produce reactive cytotoxic oxidant species.
This results in ineffective erythropoiesis and chronic haemolysis.
These cells are removed by macrophages.
Excess alpha chain affect membrane lipid and protein composition, resulting in rigid RBC’s that have a shortened lifespan.

Question 20

Describe treatments for beta thalassaemia.

Answer 20

Regular transfusions:
To treat the anaemia and maintain Hb levels over 10g/Dl.
Prevent complications like skeletal deformation.

Folic acid:
Haemolysis may cause folate deficiency.

Iron chelation therapy:
Small molecules (chelators) bind tightly to metal ions.
Removes excess iron that results from chronic blood transfusions.

Hepatitis immunisation

Splenectomy - Removal of spleen.

Endocrine therapy: Growth hormone replacement therapy for children who have short stature and growth hormone deficiency.

Allogeneic stem cell transplantation.

Question 21

Describe the abnormality in SCD.

Answer 21

○ Autosomal recessive mutation of beta globin gene.
○ Inheritance of sickle beta globin gene (HbS).
○ Mutation causes substitution of glutamine for valine at position 6 in Hb chain.
○ Base change results in replacement of adenine by thymine, causing amino acid change from glutamic acid to valine.
○ Causes sickle beta globin abnormality.

Question 22

Describe the pathophysiology of SCD.

Answer 22

○In oxygenated conditions, HbS is fully oxygenated and remains in soluble form.
○In deoxygenated conditions, HbS is insoluble and forms crystals.
○HbS polymerises into long fibres made up of seven intertwined double strands with cross-links.
○This denatured fibrous Hb damages membrane of RBC’s, resulting in the formation of sickle-shaped RBC’s.
○Sickle-shaped RBC’s block vessels, causing infarction of organs.
○This is initially reversible as oxygen causes RBC shape to return back to normal.
○RBC’s become irreversibly sickled after many cycles of oxygenation and deoxygenation.
○Conditions that cause sickling include hypoxia, high temp (ex. Fever) and acidosis.
○ Further obstruction is caused by high adhesion molecules on RBC surface, that bind to vascular endothelium and the sticky RBC’s adhere to endothelium cells, causing thrombosis.
○RBC’s develop increased stickiness, causing RBC’s to adhere to endothelial cells and increasing risk of thrombosis.
○Sickle cells are removed by reticuloendothelial system, which can lead to chronic haemolytic anaemia.

Question 23

Describe two clinical manifestations of SCD.

Answer 23

Hb AS:
○Carrier sickle cell trait.
○Patients are generally asymptomatic but diagnosis is important to avoid anoxia.
○Anoxia could arise due to infections, dehydration and acidosis.
○Can cause a sickling crisis.

Hb SS:
○Sickle cell disease.
○Inheritance of two sickle cell genes.
○Chronic haemolytic anaemia.
○Symptoms develop in second six months of life as HbF levels decrease and HbS increases.

Question 24

Describe some clinical features of SCD.

Answer 24

○Mild anaemia, as HbS releases oxygen more readily than HbA.
○Ulcers
○Bone marrow hyperplasia
○Renal complications
○Splenomegaly
○Pulmonary defects, such as pneumonia
○Cerebral complications, such as stroke and mental retardation
○Cardiovascular complications

Question 25

How can painful vaso-occlusive crisis occur in patients with SCD.

Answer 25

○Ischemia can occur in spleen, bones, lungs and brain due to oxygen starvation of tissue, caused by blood vessel blockage.
○Ischemia in brain can cause stroke.

○Young children may experience ‘hand foot syndrome’, where the digits of their fingers or toes have varying length.
○A result of infarction in fingers, due to oxygen blockage, preventing growth of finger affected.

○Can be treated with analgesics, such as morphine, and hydration.

Question 26

Describe visceral sequestration crisis, seen in SCD.

Answer 26

Sickling causes RBC’s to pool in the liver, spleen or lungs, which can be fatal.

Question 27

Describe aplastic crisis, as seen in SCD.

Answer 27

○Can be caused by Parvovirus or folic acid deficiency.
○Characterised by a sudden decrease in Hb levels.
○Blood transfusion can be given to patient.

Question 28

Describe haemolytic crisis, as seen in SCD.

Answer 28

○Characterised by increased haemolysis rate, decreased Hb levels and increased reticulocyte count.
○Involves lower leg ulcers, liver damage and kidney damage.
○Liver damage occurs as it’s trying to remove all the defective RBC’s.

Question 29

What would be seen in a FBC of a patient with SCD?

Answer 29

○Microcytic anaemia
○MCV <80fl
○MCH <27pg
○Hb in SCD 6-9g/dl
○High reticulocyte count >5.5 x10^12/L

Question 30

What would be seen in a blood film of a patient with SCD?

Answer 30

○Sickled cells - irregular, flattened, half moon-shaped.
○Target cells
○Microcytic and hypochromic cells

Question 31

Describe the association of malaria with SCD.

Answer 31

○Caused by Plasmodium parasites, the most common parasite being P.falciparum.
○Parasite remodels the host actin to survive within RBC, however mutations in human Hb prevent this remodelling as a result of evolutionary pressure.
○In countries where malaria is endemic, having sickle cell trait can be advantageous, as they produce enough Hb to be largely asymptomatic, and the parasite cannot survive in erythrocytes of people with sickle cell trait.

Question 32

What can HPLC be used to determine?

Answer 32

○Gold standard to determine Hb variants and thalassaemia’s.
○Quantifies HbA, HbA2 and HbF.

Question 33

Describe the process of HPLC.

Answer 33

○A form of column chromatography:
1) Sample mixture is pumped in a solvent.
2) The sample is pumped under high pressure through a column containing chromatographic packing material.
3) Molecules with positive charge (Hb) are separated in a column by their adsorption onto a negatively charged surface.
4) A liquid of increasing cation concentration is used to elute the molecules.
5) Different haemoglobins will elute differently, as they have different structure and charge, which can be detected optically.

Question 34

What are the advantages of HPLC?

Answer 34

○Quick - can obtain results in five minutes.
○Inexpensive.
○Accurate.
○Only a small sample is used.

Question 35

What results does HPLC generate?

Answer 35

○Detects the different times Hb variants elute and using this data generates a chart, with minutes on X-axis and amount in % on Y-axis.
○The retention time of Hb, from injection to the peak’s maximum point, is calculated and plotted.

Question 36

Describe Hb electrophoresis.

Answer 36

○HbS and HbC are two abnormal Hb variants commonly found by electrophoresis.
○This technique separates normal and abnormal Hb variants in blood using an electric current.
○Hb variants migrate at different speeds through the column as they have different electrical charges.
○Measures the amount of each Hb variant in the current.
○Carried out at acid pH, because at alkaline pH certain Hb variants co-migrate.
○Can indicate presence of disease if abnormal variant is detected in blood, or if there are abnormal amounts of normal Hb in blood.

Question 37

What Hb variants can HPLC and Hb electrophoresis detect?

Answer 37

○HbS - found in SCD.
○ HbC - has low oxygen carrying capacity.
○HbF - foetal Hb.
○HbH - found in alpha thalassaemia.

Question 38

Describe the sickle solubility urgent test.

Answer 38

○Patients blood is mixed with saponin and sodium dithionite.
○Saponin haemolyses RBC’s.
○Sodium dithionite is a reducing agent that binds to and removes oxygen from sample, thus reducing oxygen tension.
○In deoxygenation, HbS polymerises and becomes insoluble.
○Precipitate is formed in the high molarity phosphate buffer solution.
○The precipitate contains tactoids, which are liquid crystals that cause the solution to appear turbid by refracting and deflecting light.
○A positive sample forms a turbid solution, while a negative sample forms a clear solution.