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2.2 Haematology > Red Cell Membrane Disorders > Flashcards

Red Cell Membrane Disorders Flashcards

1
Q

Write about the structure of the red cell membrane
(5)

A

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

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2
Q

What are the four main proteins in the cytoskeleton

A

Spectrin
Actin
Protein 4.1
Ankyrin

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3
Q

What is the point of the cytoskeleton

A

Important for maintaining the normal biconcave shape of the red cell

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4
Q

What is the point of cholesterol

A

Adds rigidity to cell

Cholesterol content of the membrane also reflects the plasma concentration

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5
Q

Describe the chemical structure of the red cell
(4)

A

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

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6
Q

What are some characteristics of the red cell membrane?
(7)

A

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

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7
Q

List the characteristics of the red cell membrane, in your own words
(4)

A

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

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8
Q

Write about the permeability of the red cell

A

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

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9
Q

What anions is the red blood cell freely permeable to?

A

H2O
Chloride (Cl-)
Bicarbonate (HCO2-)

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10
Q

Where does rbc exchange for anions occur?

A

Through exchange channels formed by integral proteins

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11
Q

What cations is the red blood cell relatively impermeable to

A

Na+
K+

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12
Q

Why is control of the Na+ and K+ concentration in red blood cells important?

A

Na+ and K+ concentration gradient controls the red cell volume and water homeostasis

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13
Q

What controls red cell Na+/K+ concentration

A

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

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14
Q

How do red cells maintain their cell potential

A

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

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15
Q

What happens if the Na+/k+ regulation breaks down

A

Sodium will leak into the cell and bring H2O with it causing the cell to rupture

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16
Q

How is Ca++ regulated by the red blood cell

A

Ca++ is actively pumped from the interior of the cell, by a Ca++ ATP-ase pump

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17
Q

Write about the sodium potassium pump
(5)

A

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

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18
Q

What happens when the sodium potassium pump hydrolyses ATP
(4)

A

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

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19
Q

How does the pump become dephosphorylated?

A

The pump binds 2 extracellular K+ ions

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20
Q

What happens when the NA/K pump is dephosphorylated?

A

ATP binds, and the pump reorients to release potassium ions inside the cell so the pump is ready to go again

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21
Q

What is spectrin

A

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

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22
Q

What does protein 4.1 do

A

Enhances the binding between the tails of spectrin tetramers to a protein cluster of short actin microfilaments

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23
Q

What does protein 4.2 do

A

The protein forms a two dimensional web that is secured to the overlying lipid bilayer by means of ankyrin

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24
Q

What does ankyrin do

A

It anchors spectrin to the cytoplasmic domain of the anion transporter

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25
Q

What is the function of spectrin?
(3)

A

Composed of alpha and beta chains

Principle structural element of the cell membrane

Plays a major role in cytoskeleton membrane organisation

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26
Q

What is the function of ankyrin

A

Involved in the attachment of the lipid bilayer

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27
Q

What is the function of adducin

A

Promotes the association of spectrin with F actin

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28
Q

What is the function of band 3

A

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

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29
Q

What is the function of band 4.1

A

Consists of 5% of the rbc mass

Promotes affinity of spectrin to actin

Promotes the linkage of the skeleton to the membrane

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30
Q

What is the function of band 4.2

A

Associates with band 3

Deficiency of this protein is associated with haemolytic anaemia

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31
Q

What is a red cell membrane disorder

A

Absence/abnormality of the membrane proteins or lipids or defective protein interactions resulting in deformability and premature destruction of the red cell (haemolytic anaemia)

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32
Q

What causes anaemia

A

When the rate of destruction exceeds the capacity of the marrow to produce RBCs

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33
Q

What is normal RBC survival time

A

110-120 days

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34
Q

What are the two main red cell membrane defects

A

Hereditary spherocytosis (HS)

Hereditary elliptocytosis (HE)

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35
Q

What is hereditary spherocytosis
(6)

A

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

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36
Q

What are the main symptoms of HS

A

Anaemia
Jaundice
Splenectomy

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37
Q

Comment on the prevalence of HS

A

Most common hereditary haemolytic anaemia among Northern Europeans

Affects 1/2000-5000 Northern European

Found in most ethnic groups

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38
Q

Write about the pathology of hereditary spherocytosis
(4)

A

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

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39
Q

Write about the clinical finding of HS
(6)

A

Anaemia
Family history
Mild jaundice
Pallor
Splenomegaly

Haemoglobin fluctuates
10% of anaemia cases will have severe low haemoglobin and require blood transfusions

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40
Q

What causes hereditary spherocytosis and how does this happen

A

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

41
Q

What does defects in vertical stabilisation of the phospholipid bilayer do?
(4)

A

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

42
Q

Write about ankyrin-1 deficiency in HS
(3)

A

Mild to moderate severity

Seen in 50-67% of patients

Dominant and recessive inheritance

43
Q

Write about band 3 deficiency in HS

A

Mild to moderate severity

Seen in 15-20% of patients

Mostly dominant inheritance

44
Q

Write about B spectrin deficiency in HS

A

Mild to moderate severity

Seen in 15-20 % of patients

Dominant inheritance

45
Q

Write about alpha spectrin deficiency in HS

A

Severe disease

seen in less than 5% of patients

Recessive inheritance

46
Q

Write about protein 4.2 deficiency in HS

A

Mild to moderate severity
Less than 5% HS
Recessive inheritance

47
Q

What deficiencies can cause HS

A

Ankyrin-1
Band 3
B Spectrin
A spectrin
Protein 4.2

48
Q

Write about the relationship between parvovirus B19 and HS
(4)

A

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

49
Q

How does a neonate present with HS

A

Hydrops fetalis (rare)
Neonatal anaemia (rare)
Neonatal jaundice

50
Q

How does a young child present with HS

A

Severe haemolytic anaemia

51
Q

How does a child present with HS?
(4)

A

Anaemia
Jaundice
Parvovirus infection
Incidental finding on blood count

52
Q

How does an adult present with HS
(4)

A

Parvovirus infection
Incidental finding on blood count
Extra-medullary haemopoiesis
Anaemia unmasked by pregnancy

53
Q

What are the four characteristics of mild HS

A

Normal Hb
Little or no splenomegaly
Haemolytic episode triggered by infection
20-30% of cases, autosomal dominant

54
Q

What are the five characteristics of Moderate HS

A

Mild to moderate anaemia
Moderate splenomegaly
Episodes of jaundice
Increased occurrence of gallstones
60 to 75% of cases are autosomal dominant

55
Q

What are the four characteristics of severe HS

A

Chronic jaundice
Enlarged spleen
Serious haemolytic anaemia needing transfusion
5% of cases, autosomal recessive

56
Q

What are the main laboratory findings of HS
(6)

A

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

57
Q

When might you see increased reticulocytes in HS?

A

During the recovery phase

Following recovery from an aplastic crisis

Permanent immunity usually results

58
Q

What do we need to rule out in order to diagnose hereditary spherocytosis

A

Autoantibodies
Eosin 5-malemimide (EMA)
Autoimmune haemolytic anaemia (AIHA)
Haemolysis caused by irregular maternal igG antibodies

59
Q

How do we rule out autoantibodies

A

Direct antiglobulin test -> needs to be negative to rule out

60
Q

How do we rule out EMA

A

Flow cytometry

61
Q

What does EMA stand for

A

Eosin 5-maleimide

62
Q

How do we test for EMA?
(3)

A

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

63
Q

Explain in your own words how we carry out EMA testing in Hereditary Spherocytosis

A

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

64
Q

What is CDA-II

A

Congenital dyserythropoietic anaemia type II

65
Q

When is additional testing used in HS?
(3)

A

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

66
Q

What additional testing may be carried out for abnormal cases of HS

A

Quantitative protein analysis by SDS-PAGE may be undertaken in these atypical cases

Osmotic fragility test -> confirmatory test

67
Q

What is quantitative protein analysis by SDS -PAGE

A

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

68
Q

What is the osmotic fragility test

A

Test to measure the increased sensitivity of spherocytes to lysis in a gradient of sodium chloride concentrations compared with normal red cells

69
Q

Why is the osmotic fragility test not carried out anymore

A

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

70
Q

How is HS treated

A

Splenectomy is the treatment for symptomatic moderate or sever HS, this eliminates the primary location of haemolysis

71
Q

Why is splenectomy discouraged
(2)

A

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

72
Q

What is Hereditary Elliptocytosis

A

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

73
Q

What causes HE

A

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

74
Q

What are the three main types of eliptocytosis

A

Common HE
Spherocytic HE
Southeast Asian ovalocytosis

75
Q

What is common HE

A

Minimal to severe haemolysis
Eliptocytes

76
Q

What is spherocytic HE

A

Haemolysis present

Spherocytes and fat elliptocytes

77
Q

What is southeast asian ovalocytosis

A

Mild or absent haemolysis

Roundish elliptocytes that are also stomatocytic

78
Q

When is there haemolysis in HE

A

Severly dysfunctional proteins cause membrane fragmentation (haemolysis)

79
Q

What is notable about the permeability of HE

A

The cells are abnormally permeable to Na+ which demands an increase in ATP to run cation pump and maintain osmotic equilibrium

80
Q

What are the laboratory findings of HE
(5)

A

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

81
Q

How is HE diagnosed

A

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

82
Q

Comment on the severity of HE

A

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

83
Q

What are the laboratory findings of HE

A

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

84
Q

Write about PNH
(6)

A

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

85
Q

How is PNH characterised

A

Continuous destruction of PNH RBCs

Often occurs in bone marrow failures

86
Q

How is PNH characterised

A

Continuous destruction of PNH RBCs

Often occurs in bone marrow failures

87
Q

Write about the diagnosis of PNH

A

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

88
Q

What can PNH cause

A

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

89
Q

What are the symptoms of PNH

A

Haemoglobulinuria
Anaemia
Fatigue
Abdominal pain
Thrombocytopenia
Dysphagia
Dyspnoea

90
Q

What are the clinical signs of PHN

A

Acute renal failure
CKD
Stroke
Cardiac dysfunction
Pulmonary hypotension
Hepatic failure
DVT

91
Q

How is PNH diagnosed
(3)

A

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

92
Q

Explain what cause PNH

A

Lack of expression of two GPI-anchored proteins involved in the regulation of complement renders PNH erythrocytes susceptible to complement-mediated lysis

93
Q

How is PNH treated

A

Eculizumab
A humanised monoclonal antibody directed against the terminal complement protein C5
Has resulted in dramatic improvement of survival and reduction in complications

94
Q

What does FLAER stand for

A

Fluorescein-labelled proaerolysin

95
Q

How is FLAER used in diagnosing PNH

A

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

96
Q

What are some requirements for PHN testing

A

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

97
Q

What happens in PNH

A

Lack of complement inhibitors (CD55 and CD59) on red blood cells

Red blood cells become susceptible to complement attack

98
Q

What are the main symptoms of PHN

A

Fatigue
Bone marrow failure
Thrombosis
Anaemia
Haemoglobinuria
Smooth muscle dystonia: dysphagia, abdominal pain, male ED

2.2 Haematology (38 decks)

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