The Haemoglobin Molecule and Thalassaemia Flashcards by Anas A

Red blood cells don’t contain what?

Mitochondria and nucleus

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What is the normal concentration of haemoglobin in adults?

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

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

65% erythroblast stage

35% reticulocyte stage

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

Haem is synthesised in mitochondria

Globin is synthesised in ribosomes

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

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

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Summarise the classification of the globin components of haemoglobin

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.

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State and describe each of the different types of haemoglobin

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

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State the haemoglobin types present at different developmental stages

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

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

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

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

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

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

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)

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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.

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

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

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

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

Structural variants of haemoglobin (e.g. HbS)

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

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Give some epidemiological facts about thalssaemia

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)

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How is thalassaimia classified?

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

Describe the inheritance of beta thalassaemia

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

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

Full blood count:
- Microcytic hypochromic blood picture in the absence of iron deficiency
- RBC count is relatively high relative to haemoglobin
Blood film:
- Target cells, poikilocytosis but no anisocytosis
Hb electrophoresis/HPLC:
- In beta thal = Raised HbA2 & raised HbF
- In alpha thal = Normal HbA2 & HbF, +/- HbH
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.

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

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

List the clinical features of beta thalassaemia

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)

What about alpha thalasaemia?

Likely fatal in utero

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

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

Describe how the face changes with beta thalassaemia

Thicker maxillary bones

Teeth separate due to extra mandibular haematopoisesis

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

Regular blood transfusions

Iron chelation therapy

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

- 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

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

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

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

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

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

Compliance with iron chelation therapy determines survival

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

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

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

- 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

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

- Oral (3 times daily) - Effective in reducing myocardial iron SE: GI disturbance, hepatic impairment, neutropenia, agranulocytosis, arthropathy

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

- Oral (Once daily) SE: rash, GI symptoms, hepatitis, renal impairment

May need combination iron chelation therapy in some patients?

Yes

What are the ways in which iron overload is monitored?

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

Describe the appearance of a sickle beta thalassaemia blood film

Sickled cells, target cells, microcytosis and hypochromia. | HbS will be the dominant haemoglobin and will precipitate

What is HbE beta thalassaemia?

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

What are the different types of alpha thalassaemia?

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)

Describe the HbH blood film and HPLC chromatogram

Blood film will show haemolysis, microcytosis, anisocytosis, and poikilocytosis On chromatogram, you see peaks due to dimers and tetramers of beta globin chains forming

List some Problems Associated with Treatment in Developing Countries

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