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4.1 Transfusion and Transplant Immunology > Antigen/Antibody Reactions > Flashcards

Antigen/Antibody Reactions Flashcards

1
Q

What six factors influence the antigenicity of red cell antigens?

A

Chemical properites - protein/lipid/polysaccharide - molecular weight etc

Size e.g. RhD= 36 amino acids compared to RhD-

Degree of foreigness e.g. only one amino acid difference between E and e

Degradability

Dosage - pregnancy versus transfusion

The individuals response

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2
Q

Why is antigen dosage important?

A

How much exposure to the antigen you get can influence how likely you are to become immunised

This is an important consideration in HDFN as there is nearly always some degree of exposure of foetal cells to mothers circulation -> always happens at birth due to micro tears etc

This can cause sensitisation to any blood group e.g. Fya -> Fya antibody often seen in Fya- mothers who have never been transfused but have Fya+ children

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3
Q

What three factors influence how a person will react to an antigen, what affects immunogenicity of a person?

A

Genetic factors
Age
Immune Status: Immunosuppressed, inflammatory SCD etc

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4
Q

What genetic factors affect the immunogencity of a person?

A

A persons HLA type determines wether they are responders or non-responders

Those with the HLA-DRB1*15 allele are much more likely to have multiple alloantibodies -> half of responders are this type

Other HLA types are associated with only an increased likelihood of producing certain antibodies e.g Fy i.e. some HLA types are better are displaying Fy antigens to T cells then others

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5
Q

What genetic factors affect the immunogencity of a person?

A

A persons HLA type determines wether they are responders or non-responders

Those with the HLA-DRB1*15 allele are much more likely to have multiple alloantibodies -> half of responders are this type

Other HLA types are associated with only an increased likelihood of producing certain antibodies e.g Fy i.e. some HLA types are better are displaying Fy antigens to T cells then others (increase of like 45% in some)

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6
Q

What HLA type is associated with being a responder?

A

HLA-DRB1*15

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7
Q

How does age affect immunogenicity of a person?

A

Usually the very young and very old have a dimished ability to produce antibodies

There is evidence to suggest that being transfused before the age of 1 (a highly transfused cohort of patients) actually shows tolerance to transfusions much later in life - a lot of these go on to not produce any antibodies

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8
Q

what are the two ways in which blood group antibodies develop?

A

T cell dependent

T cell independent

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9
Q

What are T cell dependent antibodies, give two examples?

A

‘immune-type’

IgM converted to IgG antibodies

e.g. Anti-D and anti-K

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10
Q

What are T cell independent antibodies?

A

IgM
No isotoype switching to IgG - ‘non immune’
e.g. anti-A and anti-B, Lw and P
Cold antibodies

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11
Q

What are the main steps in T cell dependent antibody formation?
(9)

A

Antigen is engulfed by APC

Proteins are degraded into small peptides

These peptides bind to MHC class II molecules

These are transported to cell surface

These are recognised by TCR on CD4+ cells

Results in cytokine production

Cytokines stimulare B cells to convert to plasma cells

Immunoglobulins produced

igM converts to IgG

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12
Q

What are the six steps to T cell independent antibody formation?

A

Antigen stimulates B cells directly
T cells do not influence this reaction
B cells are stimulated directly to produce plasma cells
Plasma cells secrete IgG for specific stimulating antigen (carbohydrate antigens only)
IgM only

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13
Q

WHat antigens can directly stimulate B cells?

A

Carbohydrate based antigens such as ABO

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14
Q

Talk about the antigen/antibody reaction in general

A

There are 5 classes of antibodies

They respond to blood group antigens

Primary response is usually low titre IgM which will disappear over time

Secondary response is usually rapid with aggressiv rebound with rising titre of antibody

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15
Q

When is IgM usually produced?

A

In the first 3 to 7 days

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16
Q

What happens if a B cell doesnt convert to a plasma cell

A

They retain a memory of the stimulated antigen i.e. they become memory B cells

This is known as the humeral response

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17
Q

What is the significance of the humeral response?

A

Has relevance in delayed transfusion reactions

Can result in a failure to detect an extremely weak positive antibody

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18
Q

What happens to a red cell when it is coated in antibody in vivo?

A

Direct lysis of rbcs resulting in intravascular haemolysis

Rbcs coated in Ig sequested in the liver or spleen for extravascular haemolysis

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19
Q

Describe the process of intravascular haemolysis

A

Direct rbc lysis -> a totally unnatural process i.e. should never happen in vivo

Rupture of red cell membrane and release of ‘free’ haemoglobin which activates other responses:
- uncontrolled clotting
- hypotension
- organ failure due to poor perfusion

Can cause death in very ill patients, remeber one is never getting a transfusion when they are healthy

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20
Q

Describe the process of extravascular haemolysis

A

Red cells coated with antibody (and C3) are sequested in spleen or liver

These cells are damaged or released as spherocytes

These spherocytes have a much slower release of Hb

This results in a raised bilirubin, fever and a failure to oxygenate tissue as a result of persisting anaemia -> not as dangerous as Intravascular haemolysis

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21
Q

On a cell basis, explain how red cell lysis occurs

A

Red cells coated with igG antibody

Monocytes/any effector cell has an Fc receptor on its cell surface to detect Fc region of already bound anitbodies

IgG binding to Fc receptor occurs -> cell signalling -> target cell lysed

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22
Q

What are the two pathways of cell lysis

A

Classic pathway
Alternative pathway

*both result in MAC formation and cell lysis

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23
Q

What is considered the standard for detecting antibodies?

A

3 cell screen
- positive => antibody panel + enzyme panel

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24
Q

What are the two stages of red cell agglutination?

A

Stage one: sensitisation
Stage two: agglutination

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25
Q

Give two examples of where in practice we would manipulate the antigen-antibody bond

A

If we have a weak reverse group we would go to tubes at 4 degrees to increase sensitisation

We can elute of antibodies e.g. in positive DATs

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26
Q

What are the four types of bonds that occur between antigens and antibodies?

A

Hydrophobic
Hydrogen
Van der Walls forces
Electrostatic forces or Ionic bonds

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27
Q

What kind of bonds d carbohydrate antigens favour?

A

They favour exothermic bonds with their corresponding antibody i.e. low temperature will have highest reactivity

The formation of these bonds let energy out

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28
Q

What kind of bonds do proteins antibodies prefer?

A

Endothermic bonds

They take in heat so they prefer to react at higher temperatures

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29
Q

What is the main/primary bonding in antibody/antigen reactions?

A

Main bond is electrostatic
-> charges/ionic bonds
-> corresponding positives and negatives

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30
Q

Bonding such as van der wals forces are considered secondary bonds, what does this mean?

A

This means the antibody and antigens have to be very close together fo these bonds to occur

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31
Q

What kind of antibodies prefer immediate spin/cold temps and what kind prefer antiglobulin phase/37degrees?

A

IgM antibodies prefer immediate spin phase

IgG antibodies prefer 37 degrees

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32
Q

What blood group system antibodies are IgM?

A

ABO and H
I
M and N
Lea and Leb (can also be IgG)
P1

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33
Q

What blood group systems are IgG?

A

D, Cc, Ee
K
Fy
Jk
S, s
Lea, Leb (also IgM)

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34
Q

What do hydrophobic interactions do?

A

They make the antibody and antigen want to stick together - think of two drops of oil

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35
Q

What kind of bonding occurs in stage one/sensitisation?

A

Chemical bonding

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36
Q

Explain what is meant by “goodness of fit” in terms of sensitisation

A

In order for maximum complementarity both structual fit and complementary distribution of chemical groups must be achieved

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37
Q

What would an antigen-antibody complex with good structural fit with complementary chemical attraction result in?

A

If opposite charges and they perfectly fit each other then this is ideal

This would have a high KA/affinity

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38
Q

What would an Ab/Ag complex with complementary chemical groups but poor structural fit result in?

A

This would still work

Much lower affinity for antigen

Lower KA

39
Q

What would an Ab/Ag complex with complementary chemical groups but poor structural fit result in?

A

This would still work

Much lower affinity for antigen

Lower KA

40
Q

What would an Ab/Ag complex with good structural fit but non corresponding chemical groups result in?

A

This would not work
The chemical groups wouldnt be attractive and may even repel each other
Bonding wouldnt occur

41
Q

What is the chemistry behind elution?

A

Its the addition of hydrogen ions -> in the form of an acid

This will interfere with the corresponding charges in the antigen/antibody bond

H+ binds to any negatives

This forces the bond to separate -> antibodies released from surface of red blood cells i.e. are eluted into plasma

42
Q

How does antibody cross-reactivity occur?

A

This occurs when a determinant of the immunising antigen is shared by another antigen

43
Q

What is affinity

A

Essentially a measure of how well an antibody binds its antigen

Based on the law that all chemical reactions are reversible

=> Ag/Ab has a varying degree of affinity

44
Q

What is Ka, what does it mean cliically?

A

A measure of AgAb affinity

The likelihood that Ag+Ab = AgAb

A high Ka means a strong associated between the Ag and Ab which implies a sensitive test and complementary ‘fit’

45
Q

What is Kd, what does it mean clinically?

A

Kd is a measure of dissociation - how little affinity an AbAg bond has

The likelihood of AgAb -> Ab + Ag

The higher a KDA the more likely a bond is to dissociate

Lower affinity reactions will take longer to come up and can imply a flase negative finding

46
Q

What is avidity?
(3)

A

The strength of multivalent attachments of Ab populations to antigenic determinants on the Ag

Avidity is the combination of the strength of all Ab/Ag complexes against a particular antigen

e.g. there are 30 antigens on RhD, the combination of the affinity of all of these Ab/Ags is the avidity of RhD

47
Q

What is meant by high avidity low affintiy antibodies?

A

The patient has a large amount of antibodies to multiple different epitopes

But these antibodies are of low affinity i.e. they dont react well

You wont be able to dilute these out

High avidity can compensate for low affinity -> low reaction but cannot dilute them out

48
Q

How does IgM use avidity to make up for its low affinity?

A

IgM is 10 valent (actually 5 as only 5 sites can bind at a single time)

Compare to IgG which is 2 valent

49
Q

How does IgM use avidity to make up for its low affinity?

A

IgM is 10 valent (actually 5 as only 5 sites can bind at a single time)

Compare to IgG which is 2 valent

50
Q

What are five factors that can alter sensitisation?

A

Temperature e.g. decrease temp if weak reverse

pH optimised for each antisera

Incubation time -> 1 hour or 15 mins with LISS

LISS -> ionic strength -> increases Ab/Ag binding

Antigen-Antibody proportions/concentration -> can increase plasma if weak reverse etc etc

51
Q

How does LISS work?

A

Low ionic strength saline

It reduces incbation periods from 1 hour to 15 minutes

It increases antigen-antibody binding

Has to be low ionic strength as we can only slightly reduce ionic strength before bursting the red blood cells i.e. can only stretch the cells so far

52
Q

What four factors affect haemagglutination?

A

Overcoming the repulsive forces between RBCs

Ig Class

Use of potentiators and additives

Haemolysis:
- can be an endpoint caused by potent complement binding antibodies particularly if using 37 degrees incubation or enzymes

53
Q

What is the wingspan of IgG compared to IgM?

A

IgG has a wingspan of 12 to 14 while IgM has a wingspan of 20nm

54
Q

What is the wingspan of IgG compared to IgM?

A

IgG has a wingspan of 12 to 14 while IgM has a wingspan of 20nm

55
Q

Give some examples of potentiators

A

AHG in Ireland

Polyehtylene glycol/PEG in USA

Albumin in USA

LISS in Ireland

Enzymes

56
Q

Give some ezamples of enzyme potentiators, how do they work

A

These have a positive charge and when they act on rbcs they push them together

Polybrene
Protamine sulfate
Poly-L-lysine (polycations)

57
Q

Give some ezamples of enzyme potentiators, how do they work

A

These have a positive charge and when they act on rbcs they push them together

Polybrene
Protamine sulfate
Poly-L-lysine (polycations)

58
Q

How does albumin work as a potentiator?

A

It works in the same way as LISS -> reduces ionic strength

59
Q

How does PEG work as a potentiator?

A

PEG excludes water and reduces the charge on rbcs

60
Q

How does PEG work as a potentiator?

A

PEG excludes water and reduces the charge on rbcs

61
Q

What is prozone?

A

This is very rare but occurs when there is excess antibody

Too much anibody to antigen ratio -> no where for crosslinking so haemagglutination cannot ocur

Causes a false negative

Will have to dilute it down until it goes back positive, continued dilutions will make it negative again

62
Q

How does lowering ionic strength affect haemagllutination

A

Decrease the Na+ and Cl- concentrations from 0.17M to 0.03M

This causes a reduced ‘neutralising’ effect of Na+ and Cl- on oppositely charged Ag and Ab

Result is increased Ab uptake with possible increased sensitivity

i.e. basically reduces the affects of the zeta potential

63
Q

How does lowering ionic strength affect haemagllutination

A

Decrease the Na+ and Cl- concentrations from 0.17M to 0.03M

This causes a reduced ‘neutralising’ effect of Na+ and Cl- on oppositely charged Ag and Ab

Result is increased Ab uptake with possible increased sensitivity

i.e. basically reduces the affects of the zeta potential

64
Q

Explain zeta potential in vivo

A

Red cells in normal saline attract a cation cloud around them

This reduces the negative charge at the limit of any given red cell slipping plane relative to the negative charge at the red cell surface

The formce of repulsion between rbcs is determined by the net negative charge at the slippingplane

This force of repulsion is known as the zeta potential

65
Q

Explain zeta potential in vivo

A

Red cells in normal saline attract a cation cloud around them

This reduces the negative charge at the limit of any given red cell slipping plane relative to the negative charge at the red cell surface

The formce of repulsion between rbcs is determined by the net negative charge at the slippingplane

This force of repulsion is known as the zeta potential

66
Q

What gives rbcs their negative charge?

A

Glycophorins A, B and C

67
Q

What is the rbc slipping plane

A

The furthest reach of the rbcs negative charge

68
Q

What is the rbc slipping plane

A

The furthest reach of the rbcs negative charge

69
Q

Defne zeta potential

A

The force of repulsion between red cells determined by the net negative charge at the slipping plane

70
Q

Defne zeta potential

A

The force of repulsion between red cells determined by the net negative charge at the slipping plane

71
Q

What is the zeta potential for rbcs in normal saline

A

18mv

72
Q

What is the principle behind column agglutination technology in gels

A
  • Gel column IAT incorporates AHG within the gel matrix
  • Sensitised cells react with the AHG on centrifugation, living the liquid reactant including any unbound globulins in the reaction chamber
  • cells free from IgG and or complement components are centrifuged to the bottom of the microtibe
    -No control of negative results is necessary and no washing required
73
Q

What are the two types of antiglobulin tests?

A

Direct and indirect

74
Q

What is the AHG used in Gels specific for?

A

Polyspecific/broad spectrum anti-IgG and anti-C3b or Csd

75
Q

Other than AHG what is present in gel matrix

A

LISS

76
Q

Other than AHG what is present in gel matrix

A

LISS

77
Q

What kind of gell is used?

A

Sefadex gell

78
Q

What is the gold standard, gels or tubes, why?

A

Tubes are gold standard -> much clearer reactions and less contamination

Gels:
- Can miss Kid and Duffy reactions
- Lots of interference and false positiivty e.g. fibrin and drying

79
Q

Why might it be suggested to not use gels to detect Duffy or Kidd?

A

Gels are used on automated machines

These automated machines required anti-coagulated blood -> EDTA is used

EDTA chelates calcium which prevents complement activation (think of cascade from haem)

We loose a lot of Duffy and Kidd reactivity using gels -> serum sample ideal for their detection

80
Q

Why might it be suggested to not use gels to detect Duffy or Kidd?

A

Gels are used on automated machines

These automated machines required anti-coagulated blood -> EDTA is used

EDTA chelates calcium which prevents complement activation (think of cascade from haem)

We loose a lot of Duffy and Kidd reactivity using gels -> serum sample ideal for their detection

81
Q

What is the principle of the DAT?

A

Cells are coated in vivo, cells washed to remove unbound globulins, addition of AHG promotes agglutination after centrifugation

Demonstrates in vivo sensitisation of rbcs with Ig or complement

82
Q

What might cause a positive DAT

A

AIHA
HDFN
Transfusion reaction

83
Q

How do you carry out an IAT?
(3)

A

Serum with specific antibody mixed with reagent red cells

Wash cells x3 to remove unbound globulins

AHG added to promote agglutination on centrifugation

84
Q

What is the principle of the IAT

A

Detection of IgG antibodies

In tubes: unbound IgG is washed from sample and AHG is added to sensitised cells, AHG promotes agglutination and thus visible haemagglutination

It is used for the identification of antibodies in serum/plasma -> its what we always do

85
Q

Give four examples of proteolytic enzymes used in blood group serology

A

Papain
Bromelin
Ficin
Trypsin

86
Q

comment on the use of enzyme treated rbcs

A

First described by Morton and Pickles in 1946

Widely used up to te mid-eighties

87
Q

What are the four effects enzyme treatment has on rbcs

A

Allows cell to be in closer contact for antibody molecules to bridge the gap - increases some antibody reactions

Makes red cells come into closer contact with each other

Certain antigens become more readily accessible to antibodies

Certain antigens are cleaved from the rbc surface

88
Q

How can red cell enzyme treated increase enzyme reactoins

A

It cleaves sialic acid (NANA) a major contributor to red cell negative charge - reduces - charge

It exposes glycoproteins which are hydrophilic i.e. attract water molecules and since water molecules need to be shared between rbcs this forces them closer together

It cleaves glycoproteins that protrude from red cell reducing steric hindrance and thus making antigens more readily accessible

89
Q

How can red cell enzyme treated increase enzyme reactoins

A

It cleaves sialic acid (NANA) a major contributor to red cell negative charge - reduces - charge

It exposes glycoproteins which are hydrophilic i.e. attract water molecules and since water molecules need to be shared between rbcs this forces them closer together

It cleaves glycoproteins that protrude from red cell reducing steric hindrance and thus making antigens more readily accessible

90
Q

Why is there no need for AHG in enzyme gels

A

The enzyme treatment cleaves M and S responsible for giving rbc its neg charge

91
Q

What blood group systems does enzyme treatment destroy?

A

Fya and Fyb

92
Q

What blood group systems does enzyme treatment enhance?

A

Rh
Kidd
P
Lewis
I

93
Q

What are the clinically significant Ab according to the BSH?

A

Anti-D, C,c, E, e

Anti K and k

Anti Jka and Jkb

Anti M (active at 37)

Anti S, s, U

Anti Fya and Fyb

94
Q

if you can exclude a non-clinically significant antibody, what are you to do?

A

Just give crossmatch compatible blood at 37 degrees

4.1 Transfusion and Transplant Immunology (24 decks)

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