The Haemoglobin Molecule and Thalassemia Flashcards
Why are RBCs unable to divide?
- Anuclear
- No mitochondria
What is the molecular weight of Hb?
64-64.5 kDa
What is the normal [Hb] in adults?
120-165g/L
At what stages and to what degree does Hb synthesis occur during development of the RBC?
- Begins in pro-erythroblast
- 65% - erythroblast stage
- 35% - reticulocyte stage
1) Where is Haem synthesised within the RBC
2) What substance and what enzyme is haem synthesis dependent upon?
1)
- In the mitochondria
2)
- Depedent upon Fe2+ incorporation into the cell
- Enzyme = ALAS
Where is Globin synthesised within the RBC?
- Cytoplasmic ribosomes
Describe the structure of Hb
- Tetramer of 4 globin chains
- Ferroprotoporphyrin (porphyrin ring with central iron atom) within each globin molecule within the tetramer
Describe the structure of HbA
- 2 alpha and 2 beta chains
- Each chain has a haem molecule at its centre
Give some examples of other proteins that haem can be found in (doesn’t matter if you can’t name all the ones in the answers)
- Myoglobin
- Cytochromes
- Peroxidases
- Catalases
- Tryptophan
Describe the structure and arrangement of globin molecules
- They combine with haem wro form different haemoglobin molecules
- 8 functional chains
- 2 clusters:
- ß-cluster - (beta, gamma, delta and epsilon globin genes) on the short arm of chromosome 11
- α-cluster - (alpha and zeta globin genes) on the short arm of chromosome 16
What and where are the genes coding for the globin chains?
- α-cluster - chromosome 16 - α1,2 and zeta globin genes
- ß-cluster - chromsome 11 - beta, gamma, delta, epsilon
Where is the site of production of RBCs
1) During early embryogenesis?
2) In foetal life?
3) After birth?
1) Mainly in the yolk sac
2) Liver and spleen
3) Bone marrow
Describe the chronology of different globin chain production (except delta chain) and thus when alpha and beta thalassaemia manifest
- Zeta and epsilon chains are produced until 6-8 weeks, after this there is a switch to alpha chain production
- Therefore alpha thalassaemia major manifests early in embryonic life, after the zeta/epsilon-alpha switch
- Gamma globin chain production persists until 3-6 months in life, after which beta chains take over
- Therefore beta thalassaemia manifests after birth, after the gamma-beta switch
1) List the 3 types of Hb molecules present in normal adults in order of how common they are, what 4 globin chains are these made up of and finally what is a common feature of all these aside from the haem molecules?
2) How to detect the Hb - and which is the only type you can detect by this method and why?
1)
- HbA - α2ß2
- HbA2 - α2d2
- HbF - α2g2
- Each are glycated
2)
- HPLC chromatography
- Only HbA - the only type in sufficient quantity to be visible
Describe the primary (number of amino acids), secondary, and tertiary (including properties within the tertiary structure) of globin chains
- Primary: α-globin chains: 141 A.As , non-α globin chains: 146 A.As
- Secondary: 75% α and beta chain clusters form a helical arrangement
- Tertiary: Approximately spherical, hydrophilic surface (charged, polar side chains), hydrophobic core, haem pocket
What is the principle of Hb-O2 cooperativity?
The binding of one O2 molecule facilitates the second molecule binding
Define P50 and then state what this value is approx. normally
- PO2 at which Hb is half saturated with O2
- Approx. 26.2 mmHg
1) What is the PO2 in arterial blood and what is the saturation of Hb with O2 at this PO2?
2) What is the PO2 in arterial blood and what is the saturation of Hb with O2 at this PO2?
1) 14kPa - 100% saturation
2) 5.5kPa - 75% saturation
1) What does a right-shifted oxygen dissociation curve mean for the function of Hb?
2) List 4 factors that cause a right-shifted oxygen dissociation curve
1) Lower affinity of Hb for O2 - therefore easier O2 unloading at active tissue
2)
- High CO2
- Low pH (high acidity)
- High [2,3-DPG]
- High temp
1) What does a left-shifted oxygen dissociation curve mean for the function of Hb?
2) List 4 factors that cause a left-shifted oxygen dissociation curve
1) Higher affinity for O2 - therefore easier loading of O2 at exchange surfaces
2)
- Low CO2
- High pH (low acidity)
- Low [2,3-DPG]
- Low temperature
Which type of haemoglobin is right and which is left shifted from normal HbA oxygen-dissociation curve?
- HbS (sickle-cell) is right-shifted
- HbF (foetal) is left-shifted
2 ways to classify thalassaemia?
- Type of globin chain affected
- Severity (minor-‘trait’, intermedia, major)
How many alpha-like genes are there for globin, and what are they?
- 3 functional: α1, α2, zeta
- 3 pseudo genes (w) and one more that does not produce a detectable protein
How many ß-like genes are there for globin?
- 5 - all functional
- ß
- Ggamma
- Agamma
- Delta
- Epsilon
What is the principle pathophysiological basis for ß-thalassaemia?
Deletion or mutation of ß-globin genes resulting in reduced or absent production of ß-globin chains
What is the inheritance pattern of ß-thalassaemia?
- Recessive Mendelian
Carriers of ß-thalassaemia are asymptomatic save for the ….. ….. …..
Microcytic Hypochromic Indices
What is the pathophysiological basis for the classification of ß-thalassaemia by severity, including the symbolic representation
So then how would you symbolically represent trait (mild), intermedia and major ß-thalassaemia?
1)
- Severity of the mutations - indicate the degree of suppression of globin chain synthesis
- Some mutations result in no globin production (ß0)
- Others have just decreased globin production (ß+)
2)
- Trait = ßß0 / ßß+ /
- Intermedia = ß+ß0
- Major = ß0ß0
Diagnostic features of ß-thalassaemia, including the main feature of ß-thalassaemia, type of Hb present, other features and findings in the peripheral film?
How can you diagnose it in the lab - 2 techniques
1)
- Main feature: microcytic, hypochromic blood in the absence of iron deficiency
- RBC high when compared to Hb
- HbA2 is raised in relation to the severity of the mutation and raised HbF
- Peripheral film shows microcytosis, hypochromia, target cells, poikilocytosis but NO anisocytosis (RDW is normal)
2)
- Hb electrophoresis
- Globin chain DNA studies
1) Why is there no simple diagnostic process to diagnose alpha-thalassaemia trait?
2) So give 2 ways you can diagnose it?
1) Because Hb electrophoresis yields no definite diagnostic features
2)
- A presumptive diagnosis of α-thalassaemia can be made if microcytosis and hypochromia is seen in the absence of iron deficiency
- Globin chain DNA studies
What is the gold standard diagnostic test for ß and α thalassaemia and what results will you yield?
- Globin chain DNA studies
- Tests for ß-thalassaemia mutations and Xnml polymorphism for ß-thalassaemia
- Tests for α-thalassaemia genotype
What will you see on a blood smear for ß-thalassaemia trait and why?
- Microcytosis
- Hypochromia - due to reduced Hb
- Occasional basophilic stippling (basophilic granules found spread across some erythrocte cytoplasms)
- Poikilocytosis - some target cells
What will the HPLC plot for ß-thalassaemia trait show - in regards to the types of Hb present?
- HbA is still predominant, but still increased levels of HbA2 and HbF are present
- In ß-thalassaemia trait, the ability to produce some HbA is preserved
1) What is the basic pathophysiological basis for ß-thalassaemia and why is this condition incompatible with life?
2) When does the clinical presentation of ß-thalassaemia major arise?
1)
- 2 abnormal copies of the ß-globin gene (ß0ß0)
- Leads to severe anaemia - incompatible with life
2)
- 4-6 months after birth
1) What does the blood film look like for ß-thalassaemia major and explain why (minus the RBC inclusions)?
2) What 2 RBC inclusions will you see in the blood film of someone with ß-thalassaemia major and why (also what stain will you need to see one of them)?
1)
- Extreme hypochromia - due to v. reduced Hb
- Extreme microcytosis
- Extreme poikilocytosis - Howell-Joly bodies and nucleated RBCs present (a result of splenectophy and a hyperplastic bone marrow)
2)
- α-globin precipitates - alpha globin chains are unstable and precipitate readily
- Pappenheimer bodies - Perl’s stain needed - appear as inky blue haemosiderin granules due to iron overload due to blood transfusion treatment
1) What is the main treatment for ß-thalassaemia major?
2) What can be an adverse side-effect of this treatment?
3) How to manage this side-effect?
4) How can the presence of this side-effect be detected in a blood film?
1)
- Regular blood transfusions (2-3 units per month)
2)
- Iron overload
3)
- Iron chelation therapy
4)
- Pappenheimer bodies (iron deposits) may be seen as coarse blue granules in the RBCs
What gives ß and α-thalassaemia their haemolytic component / makes the cells rigid?
- α-chains are unstable and readily precipitate
- These cause the cells to be more rigid and in effect give alpha thalassaemia major its haemolytic component
What will haemoglobin electrophoresis show for ß-thalassaemia major (i.e. thus what types of haemoglobin are present)?
- Little to no HbA
- Lots of HbA2
Give the clinical presentations of ß-thalassaemia major, including the blood film basics and why these arise (9)
- Severe anaemia (so chronic fatigue) - due to reduced [Hb]
- Failure to thrive (grow)
- Hepatosplenomegaly - due to extramedullary haemoatopoeisis
- Splenomegaly - due to extramedullary haematopoiesis
- Blood film - gross hypochromia (low [Hb]), poikilocytosis, many nucleated RBCs
- Bone marrow shows erythroid hyperplasia - as it attempts to redress the anaemia
- Frontal bossing, maxillary thickening etc due to bone marrow hyperplasia
- Jaundice - as body works overtime to produce loads of RBCs to redress the anaemia, so lots of RBC breakdown too - so high bilirubin, leading to jaundice. Also due to liver failure.
- Iron overload - due to ineffective erythropoiesis and iron overload due to transfusion therapy)
Give some other complications of ß-thalassaemia apart from the main signs and symptoms
- Cholelithiasis and biliary sepsis
- Cardiac failure
- Endocrinopathies
- Liver failure
By what means can ß-thalassaemia cause death?
Due to
- Cardiac disease
- Infections
- Liver failure
- etc
What are the treatments for ß-thalassaemia major (another flashcard incudes the major one, but there are others too, include the major treatment in your answer), including also the curative treatment?
- Regular blood transfusions every 2-3 months and then iron chelation therapy
- Splenectomy
- Hormone therapy
- Hydroxyurea to boost HbF
- Bone marrow transplant - CURATIVE
1) What infections are people with ß-thalassaemia at risk of and explain why (where applicable)?
2) How to manage this risk?
1)
- Yersinia infection prone - yersinia is a siderophilic (iron loving) bacterium so thrives in patients with iron overload as a result of the blood transfusion treatments
- Patients at risk from other gram-negative sepsis
2)
- Prophylaxis is splenectomised patients (mores susceptible group) - immunisation and antibiotics
1) List 3 current iron chelators and their routes / when they are administered
2) One of them is particularly good at something - what is it and which one is it?
- DFO - sc or i.v - 5 days a week (8-12 hrs infusion)
- Deferiprone - orally - 3 times a day
- Deferasirox - orally - once a day
- Deferiprone is the most effective treatment in reducing iron in cardiac iron overload
4 ways to monitor for iron overload and describe how and how often they are used further?
- Serum ferritin - if ferritin >2500, associated with increased complications. Check quarterly if patients are transfused or annually if not
- Liver biopsy - rarely performed
- T2 cardiac and hepatic MRI - if relaxation time of cardiac fibres is <20ms, this is suggestive of Fe deposition in the heart. Check annually or 3-6 monthly if patient has cardiac dysfunction
- Ferriscan R2 MRI - measures liver [iron] non-invasively. Normal liver [iron] is <3mg/g. If .15mg/g - associated with severe complications (hepatic,cardiac disease). Check annually or every 6 months
Why is serum ferritin a slightly unreliable method for monitoring iron overload?
Because ferritin is an acute phase protein so it goes up in other situations e.g. inflammation
Why is liver biopsy a slightly unreliable method for monitoring iron overload?
Because it can be affected by inflammation or cirrhosis
Why is the ferriscan R2 MRI a superior method for monitoring iron overload in comparison to the serum ferritin or liver biopsy methods?
- Unaffected by inflammation like both the serum ferritin tests and the liver biopsy tests
- Unaffected by cirrhosis like in the liver biopsy test
1) What is sickle beta thalassaemia and what does it lead to?
2) What will the blood film show in sickle beta thalassaemia?
3) What type of haemoglobin will be present and why?
1)
- Coinheritance of sickle beta globin molecule with beta thalassaemia
- Leads to a sickling disorder, rather than a thalassaemia
2)
- Sickle cells
- Target cells
- Microcytosis
- Hypochromia
3)
- Little to no HbA production
- HbS production dominant - and precipitates as it does in homozygote sickle cell patients
1) Where is HbE ß-thalassaemia particularly prevalent and why?
2) How does its expression vary?
3) Why can it be said that HbE has thalassaemic properties?
1)
- Common combination in SE Asia (HbE highly prevalent here)
2)
- Clinically variable in expression - can be as severe as ß-thalassaemia major
3)
- It leads to a reduction in globin chain production
What is the basic principle of alpha thalassaemia?
Deletion / mutation in alpha globin genes leading to reduced or absent production of alpha globin chains
In alpha thalassamia, what globin chains pick up the slack for the lack of alpha globin chains and what does this lead to structurally for the haemoglobin molecule?
Excess ß and gamma chains - leads to development of HbH and Hb Barts tetramers respectively
What does the severity of alpha thalassaemia depend on?
- The number of globin genes affected - there are 4 alpha globin genes in total
1) What is another term for being a thalassaemia carrier?
2) What does it mean pathophyiologically to be a thalassaemia carrier with respect to ß-thalassaemia?
3) What does it mean pathophysiologically to be a thalassaemia carrier with respect to alpha thalassaemia?
4) How are thalassaemia carriers diagnosed as being as such?
1) Thalassaemia minor trait
2) Carry a single abnormal trait of the ß-globin gene
3) Carry either one or two abnormal copies of the alpha globin chain
4) Usually asymptomatic but diagnosed on the basis of mild anaemia
1) The peripheral blood film of HbH disease has a ….. element to it
2) List 4 signs you would see in the peripheral blood film for HbH disease
1) The peripheral blood film of HbH disease has a haemolytic element to it
2)
- Microcytosis
- Anisocytosis
- Poikilocytosis
- ‘Puddling’ of haemoglobin within the RBC
List 5 problems associated with treatment of thalassaemia in developing countries
- Lack of awareness of the problem
- Lack of experience of health care providers
- Lack of availability of safe, screened blood
- Cost and poor compliance with iron chelation therapy
- Lack of availability of and very high cost of bone marrow transplant
List 5 ways to prevent and screen for thalassaemia?
- Counselling and education for thalassaemics, family and general public
- Extended family screening to identify carriers
- Pre-marital screening
- Discourage marrying between relatives
- Antenatal diagosis (CVS/amniocentesis)
What does electrophoresis by HPLC show when it comes to HbH disease and when is it more easily seen?
- Shows a fast band which is probably more easily seen shortly after the strip has started to run
The amount of Hb Barts seen in the ….. does not directly correlate with the amount of ….. present in the …..
The amount of Hb Barts seen in the neonate does not directly correlate with the amount of HbH present in the adult
