Haemaglobinopathies Flashcards
What is a haemoglobinopathy
The normal production of structurally abnormal globin chains
What may cause haemoglobinopathies
(4)
Point mutations
Deletion of triplets
Mutation of a stop codon
A fusion mutation or due to an insertion
Give some examples of haemoglobin varients
Hb C
Hb S/C
Hb E
Hb D
What is Hb C
B6 Glu>lys
Results in mild haemolytic anaemia
What is Hb S/C
This accounts for 20-50% of patients with sickle cell disease
Causes less severe vaso-occlucive complications
What is Hb E
(3)
Most common mutation
B26 Glu.lys
Homozygotes suffer from mild anaemia
What is Hb D
(2)
B121 Glu>Gln
Mostly asymptomatic
What percentage of west Africans have sickle cell disease
1 in 4
Comment on the severity of sickle cell disease
(6)
HbS
Heterozygotes are asymptomatic
Homozygotes have SCD
Haemolytic anaemia of varying severity punctuated by painful sickling crises
Reduced life expectancy with most people suffering in Nigeria dying before 21 years of age
Approximately 20% of children die under 5 in Africa
How does sickle cell disease confer protection against malaria
(3)
Hb S shape is difficult for the malaria parasite to use
Macrophages will also continuously break down these sickle cells
Parasites are unable to get to the end of their life cycle before being phagocytosed along with the sickle cell
What causes structural/qualitative defects of Hb
Occurs when the amino acid sequence of one globin chain is altered
Mutation may arise due to addition, substitution, deletion or fusion to the base sequence in the globin chain -> this leads to a change in the amino acid sequence
Leads to sickle cell anaemia
How does sickle cell anaemia come about
(5)
Due to a change in the base sequence of the B chain
An A is changed to a T
GAG»_space;> GTG
This results in the replacement of glutamic acid by valine at position 6 on the B chain
This causes a change in the overall charge and polarity of the molecule
How is HbS diagnosed in the lab
(7)
Positive solubility test
Blood film
Full Blood Count
DNA analysis to confirm genetic mutation
Electrophoresis
HPLC
General bloods
How is a blood film used to diagnosed HbS
(7)
Sickle cells
Target cells
Polychromasia
Hypochromic microcytes
Reticulocytes
NRBC
Howel Jolly bodies if hyposplenic
How is a full blood count used to diagnose HbS
Haemoglobin is low
MCV is low
MCH is low
How is High Performance Liquid Chromatography used to diagnose Hb
(4)
Hb A is absent
Hb S 80-100%
Hb F 5-15%
Increased HbA2
How is the Bio-Rad Varient II used in the primary screening of B-thalassaemia
(6)
Analyser utilises ion-exchange HPLC to investigate B-thalassaemia
A B-thalassaemia short program test kit is used with the analyser
Samples are diluted and mixed at the sampling station and then injected into the analytical cartridge
Hb is fractioned by type, based on its ionic interactions with the reagent
Results are shown as a chromatogram
The analyser is usually interfaced to the Laboratory Information System (LIS)
How is the Sebia Capillarys 2 Flex Piercing - Confirmatory Testing used
(3)
Analyser uses capillary electrophoresis to confirm thalassaemia and other haemoglobinopathies
Works by separating charged molecules by their electrophoretic mobility in an alkaline buffer when voltage is applied
The Hb fractions are detected at 415nm at the cathode of the capillary and an electrophoretogram is produced, together with quantified fractions of Hb
How is sickle cell anaemia treated
(4)
Drug treatments
Allogenic transplant
Ex vivo gene therapy
In vivo gene therapy
How are drugs used to treat sickle cell anaemia
These aim to allow more cells to transit the microcirculation before sickling
How are allogeneic transplants used to treat sickle cell anaemia
Bone marrow stem cells from a donor without SCD are transplanted
How will Ex vivo gene therapy be used in the future to treat SCD
The patients bone marrow cells are modified by adding a B-globin gene using a retroviral vector or with gene editing
This will reactivate foetal haemoglobin (HbF) or correct the disease mutation
How will Ex vivo gene therapy be used in the future to treat SCD
The patients bone marrow cells will be modified by adding a B-globin gene
This will be done using either a retroviral vector or with gene editing
This will reactivate foetal haemoglobin or correct the disease mutation
How will In vivo gene therapy be used in the future to treat SCD
Direct gene editing in patients could circumvent the need for transplantation of modified patient cells if sufficient efficiency and safety can be achieved
What are quantitative abnormalities of Hb
Heterogenous group of genetic disorders which result from a reduced rate of synthesis of the alpha or Beta globin chains
What causes quantitative abnormalities
May arise because of:
Inadequate amount of mRNA
Unstable mRNA
mRNA that contains untranslatable nonsense message
What happens when there is quantitative abnormalities of Hb (Thalassaemias)?
There is a decrease in one of the globin chains
So the other chain is in excess and thus the molecule is unbalanced and the chain in excess tries to form tetramers and precipitates within the cell
What are quantitative abnormalities of Hb called
Thalassaemias
Write a note on the types of thalassaemia
(2)
Some mutations cause a complete absence of globin chain synthesis -> a0 or B0 thalassaemias
In other mutations the globin chain is produced at a reduced rate and these are known as a+ or B+ thalassaemias
What are the three groups of thalassaemias
Thalassaemia major
Thalassaemia intermedia
Thalassaemia minor
What is thalassaemia major
Severe anaemia
Transfusion dependant
What is thalassaemia intermedia
Phenotype ranging from severe anaemia to mild hypochromic microcytic anaemia
What is thalassaemia minor
Clinically asymptomatic carrier state
What is the most sever thalassaemia genetically
B0 B0
What haemoglobin is affected by a Thalassaemia?
HbA (aB)
A2 (aB)
F (ay)
What haemoglobin is affected by B Thalassaemia?
HbA (aB)
What haemoglobin is affected by y Thalassaemia?
HbF (ay)
What haemoglobin is affected by d Thalassaemia?
HbA2 (ad)
What happens in beta thalassaemia
(4)
Excess alpha chain produced which are highly unstable and precipitate in red cells
Degradation of excess a chains produce deleterious effects on RC membrane and electrolyte homeostasis - shortened RC survival
alpha chain inclusions in red cell results in haemolysis in spleen
Anaemia becomes apparent after 3-6 months of age when they switch from gamma to beta chain production occurs
How is B Thalassaemia diagnosed in the lab
(6)
Full Blood Count
Blood film
HPLC
Reticulocytes
DNA analysis to confirm genetic mutation
Family studies
How is B thalassaemia diagnosed on a blood film
(7)
Hypochromic microcytes
Target cells
Anisopoikloctosis
Polychromasia
NRBC
Pappenheimer bodies
basophilic stippling
How is a FBC used to diagnose B Thalassaemia
Haemoglobin down
MCV down
MCH down
How is HPLC used to diagnose B thalassaemia
Reduced or absent HbA peak
Increased Hb F
Increased Hb A2
How is B thalassaemia treated
(4)
Conventional therapies
Pharmaceutical induction of y-globin
Allogenic HSC transplantation
Gene and cell therapy
What conventional therapies are used to treat B thalassamia
Transfusions and iron chelation therapy are used
Frequent transfusions are needed which result in high iron which needs to be chelated to prevent overload
How is pharmaceutical induction of y-globin used to treat Thalassaemia
Drugs given to induce the production of y-globin
How is gene and cell therapy used to treat Thalassaemia
B or y-globin vectors to bring about the production of the genes
How is allogeic HSC transplantation used to treat thalassaemia
Haematopoeitic stem cell transplant
What percentage of people does thalassaemia affect
7%
What percentage of thalassaemias are alpha thalassaemia
3%
What are the different types of a thalassaemia
(4)
Carrier: asymptomatic
a-thal minor: only 2 alpha genes -> asymptomatic or mild microcytic anaemia
Hb H disease -> only 1 working a gene -> haemolytic and microcytic anaemia -> splenomegaly
no working alpha gene -> incompatible with life -> hydrops fetalis -> most common occurring type -> excess fluid builds up in the body before birth
How is alpha thalassaemia diagnosed in the lab
(8)
FBC
Blood film
Reticulocytes
H Prep
HPLC
Family studies
DNA analysis
General blood
How is FBC used to diagnose a- thalassaemia
Hb down
MCV down
MCH down
How is blood film used to diagnose a- thalassaemia
Hypochromic microcytes
Target cells
Anispoikilocytosis
Polychromasia
NRBC
Pappenheimer bodies
Basophilic stippling
How is general bloods used to diagnose a- thalassaemia
LDH and bilirubin up
Haptoglobin down
How is H prep used to diagnose a- thalassaemia
Incubation with brilliant Cresyl Blue typical golf ball appearance (30-90%)
How is HPLC used to diagnose alpha thalassaemia
Hb H peak
Hb Barts peak
What type of inclusions are seen in thalassaemias
Inclusion body
Heinz body
Howell-Jolly body
What is an inclusion body and how does it come about
(3)
Seen in HbH disease (a type of a thal)
Excess B chains precipitating as haemoglobin H inclusion bodies in the cell
In B thalassaemia major, excess a chains can also precipitate as inclusion bodies
What is a Heinz body and how does it come about
(4)
Type of inclusion body containing denatured haemoglobin
Classically associated with G6DP deficiency but can be found in thalassaemias as well
Typically larger than inclusion bodies
If functional spleen is present these will lead to bite cells
What are Howell-Jolly bodies and how does it come about
(3)
Type of inclusion body containing DNA
Usually removed by splenic macrophages like Heinz bodies
Can be seen when red cells fail to mature or when a functional spleen is absent
What is hereditary persistence of foetal Hb
(3)
Rare, benign condition characterised by continued synthesis of HbF in adults
No clinical symptoms of thalassaemia
Little significance except when combined with other forms of thalassaemia or Haemoglobinopathies
What happens if hereditary persistence of foetal Hb is combined with sickle cell anaemi
It produces a milder form of disease due to the presence of Hb F
What is the one benefit of HbF
Its more resistant to denaturation than Hb A
How do we identify hereditary persistence of foetal Hb in the lab
Can be demonstrated on blood smears using Kleihauer Betke stain
Only cells containing HbF will stain
Classify hereditary persistence of foetal Hb
Classified according to distribution of Hb F among red cells
Pancellular HPFH
Heterocellular HPFH
What is pancellular HPFH
Hb F uniformly distributed throughout red cells
What is heterocellular HPFH
Hb F found in only small number of cells
