The Haemoglobin Molecule and Thalassaemia

Question 1

Red blood cells don’t contain what?

Answer 1

Mitochondria and nucleus

Question 2

What is the normal concentration of haemoglobin in adults?

Answer 2

120-165g/L (each gram contains 3.4mg Fe)

Question 3

In which developmental stages of a RBC does synthesis of haemoglobin occur?

Answer 3

65% erythroblast stage

35% reticulocyte stage

Question 4

Where in the cell are the haem and globin components synthesised?

Answer 4

Haem is synthesised in mitochondria

Globin is synthesised in ribosomes

Question 5

Briefly summarise the synthesis of the haem component of haemoglobin and others*

Answer 5

• Iron bound to transferrin is brought into the cell by endocytosis and transported in vesicle to the mitochondrion.
• Combination of protoporphyrin ring with central iron atom => ferroprotoporphyrin
• The iron is now usually in the ferrous form (Fe2+) so it is able to combine reversibly with oxygen
• The synthesis is regulated by the enzyme ALAS contained in the mitochondrion (by negative feedback mechanism)

*Also contained in other proteins e.g. myoglobin, cytochromes, peroxidases, catalases, tryptophan

Question 6

Summarise the classification of the globin components of haemoglobin

Answer 6

There are eight functional types of globin genes arranged into two clusters:

• 𝜷-cluster = 𝜺 gene, 𝜹 gene, 𝜸-A gene, 𝜸-G gene, 𝜷 gene; located on the short arm of chromosome 11
• 𝜶-cluster = 𝜶1 gene, 𝜶2 gene, 𝜻 gene; located on the short arm of chromosome 16

These give rise to the 𝜻, 𝜺, 𝜹, 𝜸, 𝜷, 𝜶 globin chains which combine in different ways to form different haemoglobin molecules.

Question 7

State and describe each of the different types of haemoglobin

Answer 7

Hb Gower I = 2𝜻, 2𝜺
Hb Gower II = 2𝜺, 2𝜶
Hb Portland = 2𝜻, 2𝜸
Hb F = 2𝜶, 2𝜸 
Hb A = 2𝜶, 2𝜷
Hb A2 = 2𝜶, 2𝜹

Question 8

State the haemoglobin types present at different developmental stages

Answer 8

Embryonic stage: Hb Gower I, Hb Gower II, Hb Portland
Foetal stage (mainly): Hb F
Adult: Hb A (96-98%), Hb A2 (1.5-3.2%), Hb F (0.5-0.8%)

In early emrbyonic life (during the first trimester) you mainly get synthesis of 𝜻 and 𝜺 globin chains, but these quickly go down, due to switching to 𝜶 and 𝜸 globin chain synthesis (i.e. HbF). HbF persists from approx. the 2nd trimester to the first 3-6 months after birth.
In adulthood, 𝜷 globin chain synthesis takes over from 𝜸

Question 9

How would you test to see what Hb’s a person has and in what proportion?

Answer 9

Using HPLC
Note that each of HbA, HbA2 and Hb F has aa glycate fraction but only glycated HbA is normally present in sufficient quantity to be visible on a HPLC chromatogram

Question 10

Describe the primary, secondary and tertiary structure of the globin chains

Answer 10

Primary: 141 amino acids in the 𝜶 chain, 146 amino acids in non-𝜶 globin chains

Secondary: 75% 𝜶 and 𝜷 chains have a helical structure

Tertiary:

• Approximate sphere
• Hydrophilic surface (charged polar side chains)
• Hydrophobic core
• Haem pocket

Question 11

Briefly explian why the oxygen-haemoglobin dissociation curve is sigmoid shaped, and define P50

Answer 11

Binding of one oxygen molecule to haemoglobin facilitates the second molecule binding due to a change in conformation (co-operative binding)

P50 = partial pressure of oxygen at which Hb is half saturated with oxygen (= 26.6mmHg)

Question 12

State the factors which determine the position of the curve, and hence state the set of conditions that shift the curve to right, and left.

Answer 12

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

Right shift (decreased oxygen affinity):

• High 2,3-DPG
• High H+
• High CO2
• HbS (sickle)

Left shift (increased oxygen affinity):

• Low 2,3-DPG
• HbF

Question 13

What effect does 2,3-DPG have on oxygen delivery?

Answer 13

It facilitates oxygen delivery by making the haemoglobin molecule less flexible and pushing out the oxygen.

Question 14

Haemoglobinopathies can be divided into two categories. What are they?

Answer 14

1. Structural variants of haemoglobin (e.g. HbS)

2. Defects in globin chain synthesis (e.g. thalassaemia)

Question 15

Give some epidemiological facts about thalssaemia

Answer 15

• Caused by an inherited single gene disorder which is the most common worldwide
• Worldwide distribution of thalassaemia cases tend to be around areas where you have malaria and areas by the sea (e.g. Mediterranean countries, Arabian peninsula, Iran, Indian subcontinent, Africa, Southern China, South-East Asia)

Question 16

How is thalassaimia classified?

Answer 16

1. By the type of globin chain affected
2. By the clinical severity:
   - minor/“trait” (asymptomatic but carrier)
   - intermedia (non-transfusion dependent)
   - major (transfusion dependent)

Question 17

Describe the inheritance of beta thalassaemia

Answer 17

• Autosomal recessive
• Caused by a deletion or mutation in the beta globin gene(s) on chromosome 11 => reduced or absent production of b globin chains
• Beta thalassaemia trait means you have one completely functional gene and one completely non-functional gene (no globin production), so you’re a carrier
• Beta thalassaemia intermedia means you have one gene with partial function (produces less beta chains) and the other is completely non-functional
• Beta thalassaemia major means you have both genes completely non-functional

Question 18

How may beta (and alpha) thalassaemia be diagnosed in the laboratory?

Answer 18

1. Full blood count:
   - Microcytic hypochromic blood picture in the absence of iron deficiency
   - RBC count is relatively high relative to haemoglobin
2. Blood film:
   - Target cells, poikilocytosis but no anisocytosis
3. Hb electrophoresis/HPLC:
   - In beta thal = Raised HbA2 & raised HbF
   - In alpha thal = Normal HbA2 & HbF, +/- HbH
4. Globin Chain synthesis/DNA studies:
   - Genetic analysis for β-thalassaemia mutations and XmnI polymorphism, and α-thalassaemia genotype
   - Note that DNA analysis is the only reliable way of diagnosing 95% of alpha thalassaemias.

Question 19

State the important characteristics of thalassaemia major - including time of presentation and appearance of the blood film

Answer 19

• 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 (because gamma to beta globin chain switch)

BLOOD FILM:
- shows extreme hypochromia, microcytosis and poikilocytosis
- Often Howell Jolly bodies and nucleated RBC’s will be present as a result of splenectophy and a hyper plastic bone marrow
- In patients that have been on long term transfusion regimens iron overload is common and pappenheimer bodies (iron deposits) may be seen as coarse blue granules in the RBCs; see this better with Perls stain
- 𝜶 chain precipitates may also be seen (as alpha globin chains are unstable)
Note that alpha globin precipitates and pappenheimer bodies are called inclusion bodies

Question 20

List the clinical features of beta thalassaemia

Answer 20

• Chronic fatigue (anaemia)
• Failure to thrive
• Jaundice
• Delay in growth and puberty
• Skeletal deformity
• Hepatosplenomegaly
• Iron overload
• Bone marrow: erythroid hyperplasia (i.e. bone marrow working extra hard and expands giving rise to characteristic facial changes
• Extra-medullary haematopoiesis (i.e. making RBC outside of bone marrow)

Question 21

What about alpha thalasaemia?

Answer 21

Likely fatal in utero

Question 22

List the complications associated with beta thalassaemia? State which results in the most deaths

Answer 22

• Cholelithiasis (gall stone formation) and biliary sepsis
• Cardiac failure (due to iron overload)
• Endocrinopathies
• Liver failure (due to iron overload)

Most die from cardiac disease,
then infections and liver disease

Question 23

Describe how the face changes with beta thalassaemia

Answer 23

Thicker maxillary bones

Teeth separate due to extra mandibular haematopoisesis

Question 24

What are the two major treatment options for an individual with beta thalassaemia?

Answer 24

Regular blood transfusions

Iron chelation therapy

Question 25

What are the requirements that are to be met regarding transfusions?

Answer 25

• Phenotyped red cells (donor matches recipient)
• Aim for pre-transfusion Hb 95-100g/L
• Regular transfusion 2-4 weekly
• If high requirement of transfusion, consider splenectomy

Question 26

What are patients undergoing transfusions susceptible to? What is the prophylaxis in splenectomised patients?

Answer 26

Infections caused by bacteria which thrive in an iron overloaded environment e.g. Yersinia and other Gram negative sepsis

If you remove spleen, patients will be immunocompromised: so you need to immunise them with vaccines and gives prophylactic antibiotics

Question 27

When do you start iron chelation therapy? Why do you need to do audiology and ophthalmology screening before starting treatment?

Answer 27

Done after certain number of transfusions or when serum ferritin >1000 mcg/l

Because drugs that remove iron from blood are toxic to eyes and ears, and you want to measure baseline

Question 28

Iron chelation therapy improves survival rates for patients with beta thalassaemia. In terms of iron chelation therapy, what affects this survival rate?

Answer 28

Compliance with iron chelation therapy determines survival

Question 29

List the three currently available iron chelators (in order of least to most recently licensed)

Answer 29

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

Question 30

State some important facts about desferrioxamine (to include dosage, ROA, SE)

Answer 30

• sc infusion (or iv in cardiac iron overload)
• Very challenging for patient: sit in hospital for hours 5 days a week => bad compliance

SE: vertebral dysplasia, pseudo-rickets, genu valgum, retinopathy, high tone sensorineural loss, increased risk of Klebsiella and Yersinia infection

Question 31

State some important facts about Deferiprone (to include ROA, SE)

Answer 31

• Oral (3 times daily)
• Effective in reducing myocardial iron

SE: GI disturbance, hepatic impairment, neutropenia, agranulocytosis, arthropathy

Question 32

State some important facts about Deferasirox (to include ROA, SE)

Answer 32

• Oral (Once daily)

SE: rash, GI symptoms, hepatitis, renal impairment

Question 33

May need combination iron chelation therapy in some patients?

Answer 33

Yes

Question 34

What are the ways in which iron overload is monitored?

Answer 34

Monitor serum ferritin

• If it goes too high, increased risk of complications
• May go up for other reasons ie infection, so not always accurate

Liver biopsy (rarely performed)

Cardiac and hepatic MRI

Ferriscan - R2 MRI
- New non-invasive way to measure liver iron concentration

Question 35

Describe the appearance of a sickle beta thalassaemia blood film

Answer 35

Sickled cells, target cells, microcytosis and hypochromia.

HbS will be the dominant haemoglobin and will precipitate

Question 36

What is HbE beta thalassaemia?

Answer 36

Hemoglobin E (HbE) is an extremely common structural hemoglobin variant that occurs at high frequencies throughout many Asian countries (has the phenotype of a mild form of β thalassemia)

Its coinheritance with β thalassemia, a condition called hemoglobin E β thalassemia, is by far the most common severe form of β thalassemia in Asia

Question 37

What are the different types of alpha thalassaemia?

Answer 37

Loss of function of three alpha globin genes => excess beta chains form tetramers = HbH disease

Loss of function of four alpha globin genes => excess gamma chains form tetramers = Hb Barts (fatal in utero because alpha globin is needed to make HbF)

Question 38

Describe the HbH blood film and HPLC chromatogram

Answer 38

Blood film will show haemolysis, microcytosis, anisocytosis, and poikilocytosis

On chromatogram, you see peaks due to dimers and tetramers of beta globin chains forming

Question 39

List some Problems Associated with Treatment in Developing Countries

Answer 39

Lack of awareness of the problems
Lack of experience of health care providers
Availability of blood
Cost and compliance with iron chelation therapy
Availability of and very high cost of bone marrow transplant