Red Cell Membrane Disorders Flashcards
Write about the structure of the red cell membrane
(5)
The membrane is semi-permeable
There is a submembranous filamentous protein meshwork that is attached to the inner surface of the RC membrane, called the membrane cytoskeleton
The four main proteins in this cytoskeleton are spectrin, actin, protein 4.1 and ankyrin
The cytoskeleton is important for maintaining the normal biconcave shape of the cell
The cholesterol content adds rigidity and reflects the plasma concentration
What are the four main proteins in the cytoskeleton
Spectrin
Actin
Protein 4.1
Ankyrin
What is the point of the cytoskeleton
Important for maintaining the normal biconcave shape of the red cell
What is the point of cholesterol
Adds rigidity to cell
Cholesterol content of the membrane also reflects the plasma concentration
Describe the chemical structure of the red cell
(4)
Composed of a trilaminar structure consisting of approximately 50% protein, 40% lipid and 10% carbohydrate
The outer hydrophilic region consists of glycolipid, glycoprotein and protein
The central hydrophilic layer contains protein and functions as an internal cytoskeletal scaffold
Proteins are integral and internal to the membrane
What are some characteristics of the red cell membrane?
(7)
Flexible and elastic and is capable of responding to fluid force and stress
The inner membrane composition is responsible for deformability
A normal 8uM rbc can deform to pass through a 3uM blood vessel lumen
The rbc can deform so that its length increases by 250% whereas an increase in surface area of only 3-4% is likely to lead to cell lysis
Biconcave disk shape allows the cell to have a maximum surface area ration to its volume/size
This maximises the transfer of gases in and out of the cell
It also gives the RBC its deformability, which allows the RBC to transverse the microvasculature
List the characteristics of the red cell membrane, in your own words
(4)
Flexible and elastic to respond to fluid force and stress
Ability to deform - stretch out its length to fit through vasculature
Biconcave shape for maximum surface area for gas diffusion
Biconcave shape to also aid deformability
Write about the permeability of the red cell
Freely permeable to H2O anions, chloride Cl- and bicarbonate HCO2-
Exchange occurs through exchange channels formed by integral proteins
Relatively permeable to cations such as Na+ and K+
Control of Na+ and K+ concentration gradient controls the red cell volume and water homeostasis
This Na+/K+ gradient is controlled by an energy requiring system known as the sodium potassium pump
This system is found in the plasma membrane of virtually every human cell and is common to all cellular life. It helps maintain cell potential and regulate cellular volume
What anions is the red blood cell freely permeable to?
H2O
Chloride (Cl-)
Bicarbonate (HCO2-)
Where does rbc exchange for anions occur?
Through exchange channels formed by integral proteins
What cations is the red blood cell relatively impermeable to
Na+
K+
Why is control of the Na+ and K+ concentration in red blood cells important?
Na+ and K+ concentration gradient controls the red cell volume and water homeostasis
What controls red cell Na+/K+ concentration
Controlled by an energy requiring system known as the sodium potassium pump
This mechanism is found on all human cells
It maintains cell potential and regulates cell volume
How do red cells maintain their cell potential
In order to maintain cell potential they must keep a low concentration of sodium ions and high levels of potassium ions within the cell (intracellular)
Outside the cells there are high concentrations of sodium and low concentrations of potassium, so diffusion occurs through ion channels in the plasma membrane
What happens if the Na+/k+ regulation breaks down
Sodium will leak into the cell and bring H2O with it causing the cell to rupture
How is Ca++ regulated by the red blood cell
Ca++ is actively pumped from the interior of the cell, by a Ca++ ATP-ase pump
Write about the sodium potassium pump
(5)
The pump, with bound ATP, bind 3 intracellular Na+ ions
ATP is hydrolysed, leading to phosphorylation of the pump at a highly conserved aspartate residue and the subsequent release of ADP
A conformational change in the pump exposes the Na+ ions to the outside
The phosphorylated form of the pump has a low affinity for sodium ions, so they are released
The pump binds 2 extracellular K+ ions, leading to the dephosphorylation of the pump
ATP binds, and the pump reorients to release potassium ions inside the cell so the pump is ready to go again
What happens when the sodium potassium pump hydrolyses ATP
(4)
This causes the phosphorylation of the pump at a highly conserved aspartate residue
This causes a subsequent release of ADP
There is also a conformational change in the pump which exposes the Na+ ions to the outside
The phosphorylated form of the pump has a low affinity for sodium ions so they are released
How does the pump become dephosphorylated?
The pump binds 2 extracellular K+ ions
What happens when the NA/K pump is dephosphorylated?
ATP binds, and the pump reorients to release potassium ions inside the cell so the pump is ready to go again
What is spectrin
The major component of the red cell cytoskeleton
It consists of two intercoiled non-identical filamentous subunits, which form heterodimers
The head of each chain/dimer pair bind with the opposite subunit head of another dimer and form a tetramer
The tails of spectrin tetramers bind to a protein cluster of short actin microfilaments
This binding is enhanced by protein 4.1
What does protein 4.1 do
Enhances the binding between the tails of spectrin tetramers to a protein cluster of short actin microfilaments
What does protein 4.2 do
The protein forms a two dimensional web that is secured to the overlying lipid bilayer by means of ankyrin
What does ankyrin do
It anchors spectrin to the cytoplasmic domain of the anion transporter
What is the function of spectrin?
(3)
Composed of alpha and beta chains
Principle structural element of the cell membrane
Plays a major role in cytoskeleton membrane organisation
What is the function of ankyrin
Involved in the attachment of the lipid bilayer
What is the function of adducin
Promotes the association of spectrin with F actin
What is the function of band 3
Its a major transmembrane protein and integral protein
Responsible for chloride bicarbonate exchange
Contains binding sites for ankyrin, band 4.1, 4.2 and glycolytic enzymes
What is the function of band 4.1
Consists of 5% of the rbc mass
Promotes affinity of spectrin to actin
Promotes the linkage of the skeleton to the membrane
What is the function of band 4.2
Associates with band 3
Deficiency of this protein is associated with haemolytic anaemia
What is a red cell membrane disorder
Absence/abnormality of the membrane proteins or lipids or defective protein interactions resulting in deformability and premature destruction of the red cell (haemolytic anaemia)
What causes anaemia
When the rate of destruction exceeds the capacity of the marrow to produce RBCs
What is normal RBC survival time
110-120 days
What are the two main red cell membrane defects
Hereditary spherocytosis (HS)
Hereditary elliptocytosis (HE)
What is hereditary spherocytosis
(6)
Membrane defect whereby the cell takes on the classical spherical shape
Autosomal dominant inheritance
20% spontaneous mutation
Presentation may be at any age but the more severely affected tend to present early in life
Clinical severity ranges from no symptoms to fatigue to severe anaemia and jaundice
Can be an accidental finding in those with no symptoms
What are the main symptoms of HS
Anaemia
Jaundice
Splenectomy
Comment on the prevalence of HS
Most common hereditary haemolytic anaemia among Northern Europeans
Affects 1/2000-5000 Northern European
Found in most ethnic groups
Write about the pathology of hereditary spherocytosis
(4)
Spherocytes are less deformable
Spherocytes get trapped, engulfed and destroyed by splenic macrophages leading to reduced lifespan of rbc (20 days)
The quantitative deficiency of spectrin will relate to the degree of haemolysis and clinical severity
In some cases the degree of haemolysis will be compensated by the increased production of rbcs
Write about the clinical finding of HS
(6)
Anaemia
Family history
Mild jaundice
Pallor
Splenomegaly
Haemoglobin fluctuates
10% of anaemia cases will have severe low haemoglobin and require blood transfusions
What causes hereditary spherocytosis and how does this happen
A deficiency in spectrin, ankyrin or band 3
These deficiencies cause uncoupling in the vertical interactions of the lipid bilayer skeleton and the loss of membrane microvesicles
What does defects in vertical stabilisation of the phospholipid bilayer do?
(4)
This causes separation of the spectrin-phospholipid bilayer
This causes a loss of lipid
Loss of lipid results in a reduction of surface area and thus cases the older cells to become microspherocytes
Repeated passage through the spleen aggravates the spherocytic change
Write about ankyrin-1 deficiency in HS
(3)
Mild to moderate severity
Seen in 50-67% of patients
Dominant and recessive inheritance
Write about band 3 deficiency in HS
Mild to moderate severity
Seen in 15-20% of patients
Mostly dominant inheritance
Write about B spectrin deficiency in HS
Mild to moderate severity
Seen in 15-20 % of patients
Dominant inheritance
Write about alpha spectrin deficiency in HS
Severe disease
seen in less than 5% of patients
Recessive inheritance
Write about protein 4.2 deficiency in HS
Mild to moderate severity
Less than 5% HS
Recessive inheritance
What deficiencies can cause HS
Ankyrin-1
Band 3
B Spectrin
A spectrin
Protein 4.2
Write about the relationship between parvovirus B19 and HS
(4)
Patients may be asymptomatic until they contract parvovirus B19 infection with resultant aplastic crisis
Virus directly attacks erythroid precursors in the bone marrow and results in erythroid aplasia for about 10 days
Rapidly progressive anaemia during this period of absent erythropoiesis and typically present with acute onset of marked pallor, lethargy and fever
This infection may unmask hitherto undiagnosed HS in a family
How does a neonate present with HS
Hydrops fetalis (rare)
Neonatal anaemia (rare)
Neonatal jaundice
How does a young child present with HS
Severe haemolytic anaemia
How does a child present with HS?
(4)
Anaemia
Jaundice
Parvovirus infection
Incidental finding on blood count
How does an adult present with HS
(4)
Parvovirus infection
Incidental finding on blood count
Extra-medullary haemopoiesis
Anaemia unmasked by pregnancy
What are the four characteristics of mild HS
Normal Hb
Little or no splenomegaly
Haemolytic episode triggered by infection
20-30% of cases, autosomal dominant
What are the five characteristics of Moderate HS
Mild to moderate anaemia
Moderate splenomegaly
Episodes of jaundice
Increased occurrence of gallstones
60 to 75% of cases are autosomal dominant
What are the four characteristics of severe HS
Chronic jaundice
Enlarged spleen
Serious haemolytic anaemia needing transfusion
5% of cases, autosomal recessive
What are the main laboratory findings of HS
(6)
Hb between 30 and 60g/L
Increased reticulocyte count (not always)
Normal or reduced MCV
Increased MCHC
Increased plasma bilirubin
Characteristic finding in HS is the small spherocyte on blood film which lacks a central area of pallor and appears densely haemoglobinised
When might you see increased reticulocytes in HS?
During the recovery phase
Following recovery from an aplastic crisis
Permanent immunity usually results
What do we need to rule out in order to diagnose hereditary spherocytosis
Autoantibodies
Eosin 5-malemimide (EMA)
Autoimmune haemolytic anaemia (AIHA)
Haemolysis caused by irregular maternal igG antibodies
How do we rule out autoantibodies
Direct antiglobulin test -> needs to be negative to rule out
How do we rule out EMA
Flow cytometry
What does EMA stand for
Eosin 5-maleimide
How do we test for EMA?
(3)
EMA binds to band 3 protein, lysine 430
This accounts for 80% of the fluorescence produced in flow cytometry
The Rhesus complex is often bound however in HS positive patients, both of these proteins are often decreased fur to gene mutations
Explain in your own words how we carry out EMA testing in Hereditary Spherocytosis
Flow cytometry for EMA binding
Healthy patient has a mean fluorescence intensity (MFI) of 523 while HS patient has a MFI of 442
There is less EMA binding in HS patient due to mutations in the band 3 protein, lysine 430 and Rhesus complex (less expression)
This test may also be abnormal in other red-cell disorders such as CDA-II
What is CDA-II
Congenital dyserythropoietic anaemia type II
When is additional testing used in HS?
(3)
When the clinical phenotype is more severe than expected from the red cell appearances
The red-cell abnormalities are more sever than seen in the one known affected parent
Splenectomy is considered and the morphology is atypical
What additional testing may be carried out for abnormal cases of HS
Quantitative protein analysis by SDS-PAGE may be undertaken in these atypical cases
Osmotic fragility test -> confirmatory test
What is quantitative protein analysis by SDS -PAGE
Analysis of red-cell membrane content
Establishes which membrane protein is deficient by demonstrating protein bands for spectrin, ankyrin, band 3 and protein 4.2
What is the osmotic fragility test
Test to measure the increased sensitivity of spherocytes to lysis in a gradient of sodium chloride concentrations compared with normal red cells
Why is the osmotic fragility test not carried out anymore
Although the sensitivity is improved by incubating the blood at 37 degree for 24 hours, this test is not specific, showing increased OF in any condition in which there are spherocytes present
Time consuming and may give false negative results in infants and in mild cases. About 20% of cases of mild HS are missed by this test, so no longer recommended
How is HS treated
Splenectomy is the treatment for symptomatic moderate or sever HS, this eliminates the primary location of haemolysis
Why is splenectomy discouraged
(2)
Discouraged in mild because the risks associated with the resultant immunocompromise outweigh the risk of haemolytic complications
In patients with moderate to severe HS the risk-benefit ratio is inverted because splenectomy substantially diminishes haemolysis and the incidence of pigment gallstones
What is Hereditary Elliptocytosis
Rare, autosomal dominant disorder
HE presents with discoidal elliptocytes
Elliptocytosis affects about 1 in every 2,500 people
Mutations in alpha or B spectrin most common which leads to defective spectrin dimer formation
Deficiency or dysfunction of protein 4.1 or band 3
Severity of haemolysis depends on the degree of spectrin deficiency
The % of eliptocytes present reflects HE severity
What causes HE
Mutations in alpha or B spectrin most common which leads to defective spectrin dimer formation
Deficiency or dysfunction of protein 4.1 or band 3
What are the three main types of eliptocytosis
Common HE
Spherocytic HE
Southeast Asian ovalocytosis
What is common HE
Minimal to severe haemolysis
Eliptocytes
What is spherocytic HE
Haemolysis present
Spherocytes and fat elliptocytes
What is southeast asian ovalocytosis
Mild or absent haemolysis
Roundish elliptocytes that are also stomatocytic
When is there haemolysis in HE
Severly dysfunctional proteins cause membrane fragmentation (haemolysis)
What is notable about the permeability of HE
The cells are abnormally permeable to Na+ which demands an increase in ATP to run cation pump and maintain osmotic equilibrium
What are the laboratory findings of HE
(5)
90% have no signs of haemolysis
RC survival can be decreased
Haemolysis is very mild or compensated for by the BM
Increased reticulocytes and bilirubin
Surgery to remove the spleen may decrease the rate of RBC damage
How is HE diagnosed
Diagnosis depends on % of eliptocytes
Positive diagnosis if 25-90% eliptocytes present with positive family history
Oval cells with long diameter > 2 times the short diameter
Comment on the severity of HE
Its frequently harmless
In mild cases, fewer than 15% of RBCs are elliptical shaped
Some people may have crises in which the RBCs rupture, especially if they have a viral infection - can develop anaemia, jaundice and gallstones
What are the laboratory findings of HE
If asymptomatic, only symptom might by elliptocytes
Hb levels higher than 120 g/L
Negative DAT
Reticulocytes elevated up to 4%
If haemolytic Hb between 90 and 100 g/L
Reticulocytes elevated to as high as 20%
Bone marrow shows erythroid hyperplasia with normal maturation
Microelliptocytes, poikilocytes, schistocytes, spherocytes evident
Write about PNH
(6)
Paroxysmal Nocturnal Haemoglobinuria
Rare, acquired haematopoietic stem cell defect
Somatic mutation of the PIG-A gene
Prevents assembly of the GPI-anchor protein and results in partial or complete deficiency of glycosylphosphatidylinositol (GPI)
Deficiency seen in both WBC and RBC but WBC not affected by the GPI-deficiency
RBC vulnerable to complement-mediated lysis
How is PNH characterised
Continuous destruction of PNH RBCs
Often occurs in bone marrow failures
How is PNH characterised
Continuous destruction of PNH RBCs
Often occurs in bone marrow failures
Write about the diagnosis of PNH
Delays in diagnosis range from 1 to 10 years
Diagnosis often delayed or missed due to variable clinical manifestations e.g. lack of classical signs or changing appearance and symptoms of the disorder over time
Increased mortality due to thromboembolism, severe marrow failure, small risk of clonal evolution to MDS/leukaemia
What can PNH cause
Chronic haemolytic anaemia with episodic crises -> due to complement-mediated intravascular haemolysis
Propensity to thromboembolisms -> often at unusual sites e.g. cerebral, hepatic, splenic veins
Bone marrow failure -> cytopenia and possible overlap with AA
What are the symptoms of PNH
Haemoglobulinuria
Anaemia
Fatigue
Abdominal pain
Thrombocytopenia
Dysphagia
Dyspnoea
What are the clinical signs of PHN
Acute renal failure
CKD
Stroke
Cardiac dysfunction
Pulmonary hypotension
Hepatic failure
DVT
How is PNH diagnosed
(3)
Flow cytometry using GPI linked antibodies CD55, CD59, CD14, CD16, CD24, CD66b and more recently FLAER
At least 2 GPI linked antibodies must be absent for the diagnosis
Cells are analysed to detect PNH clones
Explain what cause PNH
Lack of expression of two GPI-anchored proteins involved in the regulation of complement renders PNH erythrocytes susceptible to complement-mediated lysis
How is PNH treated
Eculizumab
A humanised monoclonal antibody directed against the terminal complement protein C5
Has resulted in dramatic improvement of survival and reduction in complications
What does FLAER stand for
Fluorescein-labelled proaerolysin
How is FLAER used in diagnosing PNH
Aerolysin is a bacterial toxin which binds to RBCs via GPI anchor and initiates haemolysis
We can modify this aerolysin into a nonhaemolytic form of fluorescently labelled molecule to detect PNH cells
This is the most specific test for PNH, as FLAER binds the GPI anchor specifically
Lack of FLAER binding is sufficient for diagnosis of PNH
What are some requirements for PHN testing
Need to evaluate more than one cell line for deficiency
Request quantitative results using gold standard testing: high-sensitivity analysis > 0.1%
Request reports that provide clone sizes on all cell lines tested
Ensure that more than one reagent agaisnt GPI anchor has been used
Use FLAER
What happens in PNH
Lack of complement inhibitors (CD55 and CD59) on red blood cells
Red blood cells become susceptible to complement attack
What are the main symptoms of PHN
Fatigue
Bone marrow failure
Thrombosis
Anaemia
Haemoglobinuria
Smooth muscle dystonia: dysphagia, abdominal pain, male ED
