Haem 4 - Haemoglobin molecule and thalassaemia

Question 1

What the approximate number of red blood cells in our bodies?

Answer 1

3.505 x10^12/L

Question 2

How many molecules of Hb are there in a RBC?

Answer 2

640 million molecules

Note: Hb is found exclusively in the RBC. They are very toxic if in the blood stream - can activate free radicles.

Question 3

Numerical facts about Hb?

Answer 3

Normal concentration in adults: 120-165g/L
Approximately 90mg/kg produced and destroyed in the body everyday
Each gram of Hb contains 3.4mg of Fe

Question 4

Describe the synthesis of Hb?

Answer 4

Synthesis occurs during development of RBC and begins in pro-erythroblast:

• 65% erythroblast stage
• 35% reticulocyte stage

Question 5

Where is haem and globin synthesised?

Answer 5

haem - mitochondria

globin - ribosomes

Question 6

Describe the structure and synthesis of haem

Answer 6

1) Also contained in other proteins e.g. myoglobin, cytochromes, peroxidases, catalases, tryptophan
2) Same in all types of Hb
3) Combination of protoporphyrin ring with central iron atom (ferroprotoporphyrin). Iron usually in ferrous form (Fe2+)
4) Able to combine reversibly with oxygen
5) Synthesised mainly in mitochondria which contain the enzyme ALAS. Regulation - ALAS is part of negative feedback mechanism which controls haem synthesis.

Question 7

Describe the synthesis of globin

Answer 7

Various types which combine with haem to form different haemoglobin molecules

Eight functional globin chains, arranged in two clusters:
b- cluster (b, g, d and e globin genes - e globin genes are only important in embryogenesis) on 11p (short arm of chromosome 11)

a- cluster (a and z globin genes - zeta globin chains are only important in embryogenesis) on 16p (short arm of chromosome 16) 16. a1 and a2

See graphs which show the varying levels of globin

Question 8

What is the normal proportions of Hb in adults?

Answer 8

See table
Hb A (adult) - 96-98%
HbA2 - 1.5-3.2%
Hb F (foetal) - 0.5-0.8%

Question 9

What is the globin structure?

Answer 9

Primary:

1) α 141 amino acids
2) Non- α 146 amino acids

Secondary:
1) 75% α and b chains-helical arrangement

Tertiary:

1) Approximate sphere
2) Hydrophilic surface (charged polar side chains), hydrophobic core
3) Haem pocket

See diagram - oxygenated Hb adopts a more open state with the haem pocket holding the O2.

Question 10

Draw the oxygen-haemoglobin dissociation curve

Answer 10

See graph

Sigmoid state - cooperativity

Question 11

What it the P50?

Answer 11

Partial pressure of O2 at which Hb is half saturated with O2. About 26.6mmHg

Question 12

What happens when the oxygen-haemoglobin dissociation curve shifts to the left?

Answer 12

Means the oxygen carrying capacity is greater - caused by increased pH, Hb F and decreases 2,3-DPG

Left shift (give up oxygen less readily):

• Low 2,3-DPG
• HbF

Question 13

What happens when the oxygen-haemoglobin dissociation curve shifts to the right?

Answer 13

Means the oxygen carrying capacity is decreased - caused by decreased pH, Hb S and increased 2,3-DPG

Right shift (easy oxygen delivery):
High 2,3-DPG - it binds more readily to deoxy Hb decreasing its affinity for oxygen.
High H+
High CO2
HbS

Metabolising tissues produce the lower pH (CO2 produced) and 2,3-DPG causing the curve to shift to the right meaning oxygen is released more readily; at these sites of respiring tissue where they need oxygen. Opposite effect as well

Question 14

What does the normal position of the oxygen-dissociation curve depend on?

Answer 14

1) Concentration of 2,3-DPG
2) H+ ion concentration (pH)
3) CO2 in red blood cells
4) Structure of Hb

Question 15

What are thalassaemias?

Answer 15

Genetic disorders characterized by a defect of globin chain synthesis

Most common inherited single gene disorder worldwide - see map

Question 16

How can you classify thalassaemia?

Answer 16

Globin chain affected and [clinical severity (minor (trait), intermedia or major)]

Clinical severity not really used anymore - normally classified as transfusion dependent or non-transfusion dependent

Question 17

What is the definition of beta thalassaemia?

Answer 17

Deletion or mutation in b globin gene(s)

Resulting reduced or absent production of b globin chains

Question 18

How is beta thalassaemia inherited?

Answer 18

It is inherited via the recessive mandelian inheritance. See inheritance tree.

The clinical classification of Minor, Intermedia and Major shows how the mutations vary in severity. This can be explained by the degree of suppression of globin chain synthesis, some mutations result in no globin production (b0) where as others have decreased levels of production (b+). Inheritance of 2 b0 genes will give rise to a Major where as inheritance of 2 b+ genes will give rise to an intermedia (a clinically milder form)

Question 19

What is the laboratory diagnosis of beta thalassemia?

Answer 19

1) FBC- Microcytic Hypochromic indices (reduction in MCV and MCHC), increased RBCs relative to Hb
2) Film- Target cells, poikilocytosis but no anisocytosis
3) Hb EPS (electrophoresis) / HPLC
alpha - thal- Normal HbA2 and HbF, +/- HbH
beta - thal- Raised HbA2 and raised HbF
4) Globin Chain synthesis/ DNA studies - Genetic analysis for β-thalassaemia mutations and XmnI polymorphism (in β-thalassaemias) and α-thalassaemia genotype (in all cases)

For a-thalassaemia DNA analysis will be carried out.
See diagram for beta thalassemia trait graph

Question 20

Describe beta thalassaemia major?

Answer 20

Carry 2 abnormal copies of the beta globin gene
Severe anaemia, incompatible with life without regular blood transfusions
Clinical presentation usually after 4-6 months of life

See pictures - see lecture

Question 21

What are the clinical presentations of beta thalassaemia major? What clinical features are there?

Answer 21

Severe anaemia usually presenting after 4-6 months
Hepatosplenomegaly
Blood film shows gross hypochromia, poikilocytosis and many NRBCs
Bone marrow - erythroid hyperplasia
Extra-medullary haematopoiesis

Beta Thalassaemia Major presents at 6-12 months of age as the synthesis of g globin chains is reduced and the b globin chains would be expected to be increasing toward normal adult levels but in the case of beta thalassaemia major the beta genes are suppressed.
As a result of the gross anaemia the marrow becomes hyperplastic in an attempt to redress the anaemia. Should this persist the bone marrow expands giving rise to characteristic facial changes, frontal bossing and thickening of the maxillary bones
The patient will show splenomegaly as extramedullary haemopoesis (erythropoesis outside the bone marrow) develops.

Chronic fatigue
Failure to thrive
Jaundice
Delay in growth and puberty
Skeletal deformity
Splenomegaly
Iron overload

Question 22

What are other complications of beta thalassaemia major?

Answer 22

Cholelithiasis and biliary sepsis - caused by chronic haemoloysis
Cardiac failure - iron overload
Endocrinopathies - iron overload
Liver failure - iron overload

Question 23

What is the most common cause of death from beta thalassaemia?

Answer 23

Cardiac disease

Question 24

How do you treat beta thalassaemia major treatment?

Answer 24

Regular blood transfusions - main treatments
Iron chelation therapy - main treatments
Splenectomy
Supportive medical care (for cardiac problems, ironover load etc)
Hormone therapy
Hydroxyurea to boost HbF
Bone marrow transplant

Usually the child will be identified before they were even born if they risked being beta thalassaemia.

Question 25

What is required from blood transfusions?

Answer 25

Phenotyped red cells - aim to get similar RBC
Aim to keep the Hb concentration at the pre-transfusion Hb of 95-100g/L - this is enough to suppress extramedullary haematopoeisis
Regular transfusion 2-4 weekly
If high requirement, consider splenectomy

Question 26

What infections can occur from beta thalassaemia?

Answer 26

Yersinia
Other Gram negative sepsis - they thrive off the iron

Prophylaxis in splenectomised patients – immunisation and antibiotics. Protected from mainly encapsulated organisms

Question 27

Describe iron chelation therapy (reduce iron concentrations)?

Answer 27

Start after 10-2 blood transfusions or when the serum ferritin >1000 mcg/l
Audiology and ophthalmology screening prior to starting

Question 28

Compare currently available iron chelating agents?

Answer 28

See notes/lecture

Deferasirox (Exjade), Desferrioxamine (Desferal), Deferiprone (Ferriprox).

Question 29

How do you monitor iron overload?

Answer 29

Serum ferritin - >2500 associated with significantly increased complications (not accurate), Acute phase protein, Check 3 monthy if transfused otherwise annually
Liver biopsy - Rarely performed
T2* cardiac and hepatic MRI - <20ms – increased risk of impaired LF function, Check annually or 3-6monthly if cardiac dysfunction
Ferriscan - R2 MRI
1) Non-invasive quantitation of LIC (liver iron concentration)
2) Not affected by inflammation or cirrhosis (unlike standard MRI)
3)<3mg/g normal
4) >15mg/g associated with cardic disease
5) Check annually or 6monthly if result >20

Question 30

Describe alpha thalassaemia

Answer 30

Deletion or mutation in a globin gene(s)
Reduced or absent production of a globin chains
Affects both foetus and adult
Excess b and g chains form tetramers of HbH and Hb Barts respectively
Severity depends on number of a globin genes affected

Question 31

What is a thalassaemia carrier?

Answer 31

Also known as Thalassaemia minor / trait
Carry a single abnormal copy of the beta globin gene
Usually asymptomatic
Mild anaemia