Haemolytic anaemias + Haemoglobinopathies

Question 1

what are haemoglobinopathies

Answer 1

inherited disorders where expression of one or more of the globin chains of haemoglobin is abnormal

Question 2

two main categories of haemoglobinopathies

Answer 2

abnormal haemoglobin variants
- result of mutations in genes for α or β chains
- alter stability and/or function of haemoglobin
- eg sickle cell disease

thalassaemias
- reduced or absent expression of normal globin chains
- imbalance in composition of haemoglobin tetramer
- reduced level of haemoglobin

Question 3

inheritance pattern of haemoglobinopathies

Answer 3

• typically autosomal recessive
• heterozygotes show mild or no symptoms
• homozygotes show symptoms of disease

Question 4

structure of haemoglobin

Answer 4

• tetramer of 4 globin polypeptide chains
• 2 alpha (α) chains and 2 non-alpha chains (β
  , δ or γ) held together by noncovalent interactions
• each globin chain complexed with an oxygen binding haem group

Question 5

changes in haemoglobin types

Answer 5

• different haemoglobins expressed during developent as an adaptive response to variations in oxygen requirements
• several embryonic forms expressed early in development
• fetal haemoglobin (HbF) main form just before birth
• HbA commences before birth and steadily increases to become dominant after birth

Question 6

3 main types of haemoglobin in adults

Answer 6

• HbA 2α+2β ~95%
• HbA2 2α+2δ ~3%
• HbF 2α+2γ <1%

Question 7

where are the globin gene clusters located

Answer 7

• α globin genes on chromosome 16
• γ, δ, β globin genes on chromosome 11
• humans have 4 α genes and 2 β genes

Question 8

expression of globin genes

Answer 8

• expression of these genes under tight control to ensure 1:1 ratio of α:non-α chains
• defects in regulation of expression of globin genes can result in abnormalities in absolute and relative amounts of globin chains resulting in thalassaemia
• defects in coding regions result in abnormal variants with structural defects that alter stability and/or function of haemoglobin

Question 9

thalassaemias

Answer 9

• heterogeneous group of genetic disorders
• more prevalent in South Asian, Mediterranean, Middle east and Far east
• result from decreased or absent α or β globin chain production resulting in an imbalance in composition of a2b2 tetramer

Question 10

α thalassaemia

Answer 10

deletion or loss of function of one or more of the four α globin genes
1- silent carrier state
- asymptomatic
- carrier of disease

2- α thalassaemia trait
- minimal or no anaemia
- microcytosis and hypochromia
- resembles β thalassaemia minor

3- Haemoglobin H disease (HbH)
- moderately severe anaemia
- tetramers of β globin (HbH) form
- microcytic, hypochromic anaemia
- target cells and Heinz bodies
- resembles β thalassaemia intermedia

4- hydrops fetalis
- severe anaemia, in utero death
- excess γ globin forms tetramers in foetus (Hb Bart) that can’t deliver oxygen to tissues

Question 11

β thalassaemia

Answer 11

mutation in one or both β globin genes leading to reduction or absence of β globin
β thalassaemia minor/trait
- asymptomatic with mild anaemia
- microcytic and hypochromic RBC
- resembles α thalassaemia trait
- heterozygous

β thalassaemia intermedia
- severe anaemia
- mild variants of homozygous (reduction)
- some compound heterozygous states

β thalassaemia major
- severe transfusion dependent anaemia
- manifests 6-9 months after birth when synthesis switches from HbF to HbA
- homozygous

Question 12

thalassaemia phenotypes

Answer 12

• thalassaemia major transfusion dependent
• thalassaemia intermedia require transfusions intermittently
• thalassaemia minor require no transfusions

Question 13

peripheral blood smear results of thalassaemia

Answer 13

• hypochromic and microcytic RBC
• anisopoikilocytosis
• frequent target cells
• nucleated RBC
• Heinz bodies

Question 14

how is thalassaemia a form of haemolytic anaemia

Answer 14

• relative excess of the unaffected globin chain contributes to defective nature of RBC eg. insoluble aggregates of α chains
• haemoglobin aggregates get oxidised
• premature death of erythroid precursors within bone marrow leading to ineffective eryhtropoiesis
• excessive destruction of mature red cells in spleen leading to shortened RBC survival

Question 15

consequences of thalassaemia

Answer 15

• extramedullary haemopoiesis to compensate but results in splenomegaly, hepatomegaly and expansion of haemopoiesis into bone cortex imparing growth and skeletal abnormalities
• reduced oxygen delivery leads to stimulation of EPO which further contributes to drive to make more defective RBC
• iron overload due to repeated blood transfusions to treat anaemia and the excessive absorption of dietary iron due to ineffective haemopoiesis
• reduced life expectancy

Question 16

treatment for thalassaemias

Answer 16

• red cell transfusion from childhood
• iron chelation to delay iron overload
• folic acid to support erythropoiesis
• immunisation
• holistic care - cardiology, endocrine, psychological, ophthalmology input to manage complications
• stem cell transplantation to replace defective red cell production
• pre-conception counselling for at risk couples and antenatal screening

Question 17

what is sickle cell disease

Answer 17

• autosomal recessive disease due to mutation of β globin gene causing HbS variant
• GAG changed to GTG so uncharged valine instead of charged glutamic acid at position 6 of β globin
• HbS variant common in black africans, arab, mediterranean and south asian
• heterozygous HbS (sickle cell trait) causes mild anaemia and resistance to malaria due to changes in RBC making it hard for Falciparum parasite to grow
• homozygous HbSS develop sickle cell disease
• HbS can be co-inherited with another abnormal Hb e.g. HbC or β thalassaemia

Question 18

mechanism of sickle cell anaemia

Answer 18

• HbS forms** tetramers under normal oxygen tension** so readily gives up oxygen compared to HbA
• HbS forms polymers under low oxygen tension causing cell to form sickle shape
• repeated episodes of sickling causes red cell membrane to lose its elasticity causing irreversibly sickled red blood cells

Question 19

consequences of sickle cell formation

Answer 19

vaso-occlusive episodes due to occlusion of small capillaries from trapped sickle cells
- recurrent acute pain
- painful bone crises
- stroke
- acute chest syndrome
- chronic kidney disease
- avascular encrosis - joint damage

anaemia
- sickle cells undergoing haemolysis
- shortened erythrocyte lifespan from ~120 days to ~20-30 days

jaundice and gallstones
- increased bilirubin from chronic haemolysis

splenic atrophy
- splenic infarction
- susceptibility to infection by encapsulated bacteria (streptococcus pneumoniae, streptococcus meningitidis)

aplastic crises
- often triggered by parvovirus

Question 20

treatment for sickle cell anaemia

Answer 20

haemopoietic stem cell transplantation is only cure but rarely performed
treatment concentrated on reducing symptoms with regular medical care to prevent complications:
- folic acid
- penicilin
- vaccinations
- hydroxycarbamide - increases HbF levels
- red cell exchange

Question 21

what is haemolytic anaemia

Answer 21

• results from abnormal breakdown of RBC in blood vessels (intravascular haemolysis) or spleen (extravascular haemolysis)
• normal RBC lifespan ~120 days
• bone marrow can compensate for decrease in lifespan by increasing production up to a point (5-6 fold)
• if haemolysis exceeds capacity of marrow then rate of destruction exceeds rate of production and anaemia develops

Question 22

consequences of haemolytic anaemia

Answer 22

• severity of anaemia typically worse than chronic disease if Hb very low or sudden fall in Hb
• accumulation of bilirubin leading to jaundice and pigment gallstones
• overworking of red pulp leading to splenomegaly
• massive sudden haemolysis can cause cardiac arrest due to lack of oxygen delivery to tissues & hyperkalaemia due to release of intracellular contents

Question 23

key laboratory findings in haemolytic anaemias

Answer 23

• raised reticulocytes as marrow tries to compensate
• raised bilirubin due to breakdown of Haem
• raised LDH as red cells rich in this enzyme

Question 24

types of haemolytic anaemis

Answer 24

inherited defective gene
- glycolysis defect (pyruvate kinase deficiency)
- pentose phosphate pathway (G6PDH deficiency)
- membrane protein (eg hereditary spherocytosis)
- haemoglobin defect (eg sickle cell)

acquired damage to cells
- mechanical damage (eg microangiopathic anaemia)
- antibody damage (autoimmune haemolytic damage)
- oxidant damage (exposure to chemicals or oxidants)
- heat damage (severe burns)
- enzymatic damage (snake venom)

Question 25

microangiopathic haemolytic anaemias (MAHA)

Answer 25

**mechanical damage of red cells** - **shear stress** as cells pass through defective heart valve eg. in aortic valve stenosis - **cells snagging on fibrin** strands in small vessels where there's increased activation of clotting cascade eg. in DIC **heat damage from severe burns** **osmotic damage**

Question 26

what is disseminated intravascular coagulation (DIC)

Answer 26

**bleeding and clotting occur at the same time in the patient** eg. malignancy, obstetric complications, trauma, sepsis

Question 27

what is thrombotic thrombocytopenic purpura

Answer 27

syndrome where small thrombi form within the microvasculature

Question 28

what is haemolytic uraemic syndrome (HUS)

Answer 28

**clots in vessels of kidney** common in children after developing E coli diarrhoea

Question 29

what are schistocytes

Answer 29

- fragments of RBC resulting from mechanical damage - good indicator that some form of pathology is present

Question 30

autoimmune haemolytic anaemias

Answer 30

- caused by **autoantibodies** binding to red cell membrane proteins - result from **infections**, **lymphoproliferative** disorders and reactions to **drugs** (eg. cephalosporins) - classified as **warm (IgG)** or **cold (IgM)** based on the temperature the autoantibodies react best under laboratory conditions - **macrophages** in the **spleen** recognises antibody bound cells as abnormal and destroys them - red cell **lifespan reduced** resulting in anaemia

Question 31

warm (IgG) autoimmune haemolytic anaemia

Answer 31

- **IgG antibodies** recognise epitopes on red cell membrane - leads to **macrophages** in spleen recognising antibody-coated red cells and destroying cell by **phagocytosis** or **nibbling** a bit off - some membrane is lost so red cells form a **spherocyte** - **splenomegaly** often occurs as spleen is doing extra work

Question 32

cold (IgM) autoimmune haemolytic anaemia

Answer 32

- **IgM autoantibodies** recognise red cell epitopes and **complement** fixed to patient's red cells - bind best at cooler temps so bind in **distal parts** of body like fingertips - IgM autoantibodies span **several red cells** creating large **agglutinates** that block small capillaries - creates **ischaemic** conditions in peripheral body parts causing numb fingertips, pallor, blue discolouration, gangrene - **IgM falls off** in warmer parts of body and agglutination disappears - **complement** binding to RBCs directly create **holes** in membrane and cause **macrophages** in spleen to recognise and destroy cells

Question 33

what is the direct Coombs test

Answer 33

- used to **detect antibodies or complement** bound to surface of RBCs - patient's red cells mixed with **anti-human globulin antibody** that will attach to antibodies on red cell making them **clump** together - suggests patient's haemolysis is **immune related**

Question 34

how does pyruvate kinase deficiency cause haemolytic anaemia

Answer 34

- mutations in **PKLR gene** causing deficiency in pyruvate kinase which catalyses final step in **glycolysis** and **produces ATP** - **red blood cells lack mitochondria** so deficiency inhibits their only metabolic pathway that **supplies ATP** for cellular processes - **sodium potassium ATPase** pump activity inhibited from insufficient ATP so red cells **lose potassium** to plasma - **water moves down** concentration gradient causing cells to shrink resulting in **cellular death** and haemolytic anaemia - mild deficiency doesn't require treatment, severe deficiency require **regular blood transfusions**

Question 35

how does G6PDH deficiency cause haemolytic anaemia

Answer 35

- G6PDH is rate limiting enzyme of **pentose phosphate pathway** which supplies reducing energy by maintaining **NADPH** levels - NADPH protects against **oxidative stress** by maintaining level of **reduced glutathione** - pentose phosphate pathway **only source** of reduced glutathione in RBCs - risk of haemolytic anaemia in states of **oxidative stress** such as infection or certain chemicals and medications - damaged red cells are **phagocytosed in spleen** and metabolism of excess Hb to bilirubin can lead to **jaundice**

Question 36

hereditary spherocytosis

Answer 36

- inherited **autosomal dominant** disease - abnormalities in erythrocyte membrane proteins causing cells to be **spherical** which make cells **less flexible and more easily damaged** - **ankyrin, spectrin, protein 4.2 or Band 3** defects disrupt vertical **interactions** between cytoskeleton and plasma membrane - poor deformability of spherocytes causes them to be **trapped and damaged** as they pass through **spleen** resulting in reduced lifespan and haemolytic anaemia - symptoms include anaemia, jaundice, splenomegaly and **Howell-Jolly bodies** in blood - severe symptoms improved with partial or full **splenectomy**

Question 37

hereditary eliptocytosis

Answer 37

- many cells elliptical rather than biconcave disc shape - spectrin defect most common - also defects in band 4.1, band 3 and glycophorin C proteins

Question 38

hereditary pyropoikilocytosis

Answer 38

- spectrin defect - severe form of hereditary elliptocytosis - abnormal sensitivity of red cells to heat - similar morphology to thermal burns