Module 3 - 2nd half

Question 1

Where are embryonic haemoglobins produced? Which ones are the embryonic Hb and when are they produced?

Answer 1

Embryonic haemoglobins are produced in the erythroblasts of the human yolk sac:
Hb Gower 1 (zeta2 epsilon2) at 2-4 weeks
Hb Gower 2 (alpha 2 episilon 2)
Hb Portland (alpha 2 gamma 2) from 4 weeks
HbF (alpha 2 delta 2) from 4 weeks

Question 2

When is adult hb produced?

Answer 2

Hb a from 6-8 weeks

HbA2 from 30 weeks

Question 3

When do Beta globin and gamma globin cross over in terms of production?

Answer 3

~3 weeks, before 6 weeks.

Beta globin is pretty high at 12 weeks

Question 4

Which globinopathies are present in the newborn?

Answer 4

a - evident
B- only found incidentally
gamma - may cause transient haemolysis, confined to the neonatal period

Question 5

What Hb can alpha thal major babies produce?

Answer 5

Gower 1, Portland

Remember almost all is Hb barts - gamma 4. The above two are the only NORMAL two that can be produced

Question 6

Splenic sequestration

Answer 6

Reversible
Cells temporarily trapped by adhesion to reticular meshwork of cords
Can result in acute severe drop in Hb
May require transfusion (taking care not to over-transfuse)

Question 7

Malaria impact and protective diseases against malaria

Answer 7

Impact of malaria:
o Incidence 350-500million; 41% of world population lives in endemic areas
o Mortality up to 25-30%; 1.5mill deaths/yr mostly in children
Red cell disorders that are protective against malaria include:
oBlood group Ag - Duffy/ABO (O is most protected due to inhibition of rosetting)
oRed cell membrane – Elliptocytosis/ovalocytosis
oHb disorders-Haemoglobinopathies/variants/Thalassaemias
oEnzyme disorders - G6PD deficiency (decr risk of cerebral malaria)

Question 8

Protective mechanisms in HbAS/C against malaria

Answer 8

Innate:
1.Infected RBC have lower O2 tension due to parasite. Causes sickling and is removed via phagocytes or reticuloendothelial system.
2.Sickle trait RBC produce higher levels of the superoxide anion and hydrogen peroxide than normal RBC do, both are toxic to malarial parasites.
Structural:
3.RBC reduce display of PfEMP1 on the surface of infected cells preventing pathology of infection e.g. endothelial wall adhesion and rosette formation
4.Cytoskeletal network is disrupted stopping parasite from using it to traffic essential parasite proteins to RBC membrane
5. Prolonged mild parasitaemia is seen in HbAS with P. falciparum giving host opportunity to develop humoral immunity
6. In HbC marked resistance to osmotic lysis so cells cannot burst and release merozoites.
7. Increased crystallization of Hb C induced by the presence of Hb S, creating an inadequate substrate for the parasite’s proteases.

Question 9

Protective mechanisms in thalassemia against malaria

Answer 9

• Substantial oxidant damage in RBCs due to excess globin chains; oxidant injury enhanced by the release of superoxide anions by effector T cells upon binding to infected red cells. Infected cells more likely to be destroyed
Innate:
• Enhanced phagocytosis of infected cells has been seen which may be antibody and complement mediated.
• Reduced expression of red cell complement receptor 1 (CR1), responsible for red cell rosetting of infected and non-infected red cells
• increased erythrocyte count and microcytosis, allowing a greater reduction in erythrocyte count than children of normal genotype during malaria infection before the haemoglobin level falls to cause severe anaemia
• increased susceptibility to infection with the nonlethal P. vivax, particularly in young children, thereby inducing limited cross-species protection against subsequent severe P. falciparum infection

Rings developing in beta-thal, sickle cell trait RBCs, and HbH RBCs were phagocytosed more intensely than ring-parasitized normal RBCs. Ayi et al proposed that iwas because the oxidative events leading to enhanced phagocytosis are in sequence: increased denaturation of Hb, membrane binding of hemichromes (a form of metHb) and free iron; aggregation of band 3; and deposition of antibodies and complement C3c fragments. Nonoxidative aggregation of band 3 was also found to enhance opsonin deposition and phagocytosis without hemichrome deposition. This explain why r

Question 10

Malaria life cycle

Answer 10

1 Injection of sporozoites into the human host by an infected female Anopheles mosquito
2 Sporozoites infect liver cells where they mature into schizonts. The schizonts rupture and release merozoites 
NB: In P. vivax and P. ovale a dormant stage [hypnozoites] can persist in the liver and cause relapses by invading the bloodstream weeks, or even years later.
3 Merozoites infect red blood cells where they reproduce asexually and the RBC ruptures.
4 Some merozoites differentiate into gametocytes
5 The gametocytes are ingested by an Anopheles mosquito, while in the mosquito’s stomach, they form into ookinetes (fertilized motile zygotes) which invade the midgut wall where they develop into oocysts
6 The oocysts grow, rupture, and release sporozoites, which make their way to the mosquito’s salivary glands

Question 11

Sickle cell pathogenesis

Answer 11

codon 6 = change from GAG to GTG = swapping glutamic acid for valine (uncharged)
Valine fits into a hydrophobic pocket of neighbouring deoxyHbS, which triggers polymerisation of deoxyHbS and formation of 7 twisted double fibres of HbS
o HbA aren’t as able to form polymers because normal beta chains can’t engage with the hydrophobic pocket
•Polymerisation deform the cell into a sickle shape. Polymerisation is triggered by
ohypoxia
oinfection (low pH, increased temp, increased OSM)
•Sickling is initially reversible (‘boat cell’), but ultimately becomes irreversible (we know it is a reversible process because veins have more sickled cells than arteries)
o sickling manifests in organs where there is a long time for cells to travel from the arterial bed to the venous bed. this allows time for sickling. the spleen is an example
o secondary events makes cells irreversibly sickled

Question 12

Mechanisms of irreversible sickling

Answer 12

HbS has further roles in oxidation
Cytoskeletal damage
CSM damage
Cellular dehydration
NO
Other cells

Question 13

Explain HbS role in further oxidation

Answer 13

• metHbS is formed via oxidation. it breaks down into hemichromes and releases haem and Fe3+
• Haem and Fe3+ oxidise membrane lipids, cytoskeleton and other HbS molecules
• oxidised HbS precipitates as Heinz bodies which bind to ankyrin band 3 on inner CSM
o this is seen and removed by the spleen; extravascular haemolysis

Question 14

How does cytoskeletal damage occur

Answer 14

Oxidation causes a loss of ability of the cytoskeleton to tether to CSM. This results in a loss of CSM vesicles. A decreased CSM:cell content ratio means that cell becomes more rigid.

Question 15

CSM Damage/loss

Answer 15

Polymers of HbS can pierce the membrane/ produce a spicule which is then lost
•oxidation of lipids causes rearrangement of inner and outer CSM lipids and loss of lipids. This results in a relocation of PPDserine to outer CSM =
• increased adhesion to endothelium & macrophages (this happens before changes in cell shape and rigidity)
• phagocytosis by macrophages
• complement activation = intravascular haemolysis
• prothrombotic effect

•increased IgG binding = intravascular haemolysis

Question 16

Cellular dehydation mechanism

Answer 16

Cellular dehydration (high MCHC)
•	Oxidative damage to CSM = activation of ion transport channels = loss of K + Cl = loss of water
•	Mg usually inhibits KCl cotransport; SCD patients usually have low Mg
•	Polymerisation increases CSM permeability = Ca influx & activation of Gardos channels

Question 17

Role of other cells

Answer 17

Roles of other cells
•Endothelial dysfunction = prothrombotic, increased adhesion, and production of pro inflammatory cytokines
o pro inflammatory cytokines also activate Gardos channels
•Macrophages bind to Ig and complement on RBCs, particularly those with altered CSMs, and phagocytose them
•Neutrophils are activated, particularly during infection, and this increases their adhesive properties
o adhesion of neutrophils to endothelium helps to trap RBCs in venules = increased transit time = promoted sickling

Question 18

Role of NO in SCA

Answer 18

Role of NO
• IV haemolysis = free Hb in circulation = breakdown of NO = vasoconstriction and more platelet activation
o haptoblobin and haemopexin, two mechanisms by which Free Hb is removed from the blood, are both reduced in SCD
o IV haemolysis = release of arginase = breakdown of arginine = less NO synthesis
• activated neutrophils (infection) also inhibit eNOS = decreased synthesis
• Low NO correlates with high LDH (infarcts)

Question 19

Why does vasoocclusion occur in SCD?

Answer 19

• VCAM1 is upregulated by the dysfunctional endothelium. it binds to reticulocytes via VLA4
• reversibly sickled cells also bind to dysfunctional endothelium and exposed ECM
o young sickle cells bind to integrins via LW antigen. LW antigen is upregulated by adrenaline so stress/adrenaline = adhesion and may lead to sickling
• The majority of adhesion is believed to occur in post-capillary venules
o RBCs elongate in capillary bed (surface area) and there is slow blood flow in the venule
o adhesion leads to retrograde capillary obstruction = ischaemia/infarction
•Vaso-occlusive disease starts after 6 months of life – HbF protects upto this point
o Painful crises (limbs/chest/abdomen) occur in 40% of patients?
o hand foot syndrome (dactylitis) in the first two years of life leads to growth delay
o adolescent sequelae include delayed puberty and priapism

Question 20

Conditions associated with secondary HH

Answer 20

Inherited  
•          Thalassaemia major/intermedia 
•          Sickle cell disease 
•          Pyruvate kinase deficiency 
•          Hereditary spherocytosis 
•          Diamond Blackfan anaemia 
•          X-linked sideroblastic anaemia 
Acquired 
•          Myelodysplastic syndromes 
•          Aplastic anaemia 
•          Myelofibrosis

Question 21

Sundic et al. study

Answer 21

A study of 62 patients by Sundic et al. investigated the treatment of hereditary haemochromatosis with erythrocytapheresis instead of whole blood phlebotomy and demonstrated a more rapid initial decline in ferritin levels and a reduced number of procedures per patient, but not in earlier achievement of target ferritin level. The frequency of discomfort was equally low with the two methods. The costs and, probably, technician time consumption were higher in the apheresis group.

Question 22

Secondary HH Mx

Answer 22

Iron chelators (desferrioxamine-removes iron from liver and deferiprone-removes iron from heart)
Deferoxamine is the drug traditionally used for iron chelation therapy. It is given by a slow subcutaneous infusion overnight through a portable pump for 5 to 7 nights/wk or via 24-h IV infusion. Dose is 1 to 2 g in adults and 20 to 40 mg/kg in children.
Deferiprone, another oral iron chelator, is indicated for the treatment of patients with transfusional iron overload due to thalassemia syndromes when chelation therapy with deferasirox or deferoxamine is inadequate. Deferiprone can also be used in combination with deferasirox. Initial dose is 25 mg/kg po tid. Maximum dose is 33 mg/kg po tid
As well as as a  careful record of transfused blood should be maintained for each patient (stating volume and haemocrit of each unit)

Question 23

Classical and lectin pathway

Answer 23

Classical pathway:
•IgG antibody- antigen complexes enable C1q binding to the FC region of the antibody. (IgG3>IgG1>1gG2). Sometimes IgM.
•In binding of C1q, C1r and C1s are activated. 
•@ C1s cleaves C4 to C4a and C4b & C2 to C2a and C2b.
•C4bC2a = C3 convertase -> cleaves C3 into C3a (anaphylatoxin) and C3b 
•C4BC2aC3b = C5 convertase -> cleaves C5 
•C5b initiates MAC complex formation
Lectin:
•Proteins involved in this pathway recognise sugar molecules on bacterial cell surfaces Ie. Binding of mannose-binding lectin (protein that binds to carbs) or ficolins to bacterial carbohydrate moieties. 
• MBL binds to mannose binding groups on bacteria -> activates proteases analogous to C1q, C1r and C1s known as MASPS (mannose binding lectin associated proteins. 
• Cleavage of C4 & C2 occurs normally, like the classical pathway.

Question 24

Alternative pathway

Answer 24

Constitutively auto-activated
•C3 undergoes hydrolysis to C3(H20)
•This allows factor D to cleave Factor B into Bb and Ba
•C3(H2O)Bb is a C3 convertase cleaving more C3 –> C3a and C3b
•C3b in contact with a FOREIGN surface activates C3 amplification loop.
•C3b does two things:
1.Opsonin which surrounds the pathogenic surface enabling phagocytosis
2.C5 @ and formation of MAC

Question 25

Terminal pathway

Answer 25

All pathways converge at the TERMINAL pathway (C5 onwards) where C5b forms MAC. C5b-9 complex forms a split washer on the membrane of pathogen -> membrane destruction.

Question 26

Why don’t MACs attack our cells? Explain the mechanisms in place to avoid this

Answer 26

This process does not occur on own cells as our cells; FLUID PHASE and MEMBRANE bound regulators to control this process. Dysregulation of these mechanisms leads to pathogenesis:
•Own cell membranes contain sialic acid which is absent from pathogenic cell membranes.
•FACTOR H in the presence of Sialic acid on self-cells regulates C3b amplification. Absence on pathogenic surfaces fails to confer protection leading to C3b accumulation (and clearance of pathogen)
•C3b on self-cells can be inactivated by FACTOR I.

Question 27

Atypical HUS

Answer 27

• Deficiency or polymorphisms in Factor H (which regulates the alternative pathway) confer risk of HUS in response to risk such as infection/pregnancy
• Disorder of complement regulation
• Characterised by: Haemolysis, Thrombocytopenia and Renal failure
• Complex genetic trait – multiple mutation required for manifestation.
• Associated with mutations of Factor I and MCP