Antigen/Antibody Reactions Flashcards
What six factors influence the antigenicity of red cell antigens?
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
Why is antigen dosage important?
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
What three factors influence how a person will react to an antigen, what affects immunogenicity of a person?
Genetic factors
Age
Immune Status: Immunosuppressed, inflammatory SCD etc
What genetic factors affect the immunogencity of a person?
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
What genetic factors affect the immunogencity of a person?
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)
What HLA type is associated with being a responder?
HLA-DRB1*15
How does age affect immunogenicity of a person?
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
what are the two ways in which blood group antibodies develop?
T cell dependent
T cell independent
What are T cell dependent antibodies, give two examples?
‘immune-type’
IgM converted to IgG antibodies
e.g. Anti-D and anti-K
What are T cell independent antibodies?
IgM
No isotoype switching to IgG - ‘non immune’
e.g. anti-A and anti-B, Lw and P
Cold antibodies
What are the main steps in T cell dependent antibody formation?
(9)
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
What are the six steps to T cell independent antibody formation?
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
WHat antigens can directly stimulate B cells?
Carbohydrate based antigens such as ABO
Talk about the antigen/antibody reaction in general
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
When is IgM usually produced?
In the first 3 to 7 days
What happens if a B cell doesnt convert to a plasma cell
They retain a memory of the stimulated antigen i.e. they become memory B cells
This is known as the humeral response
What is the significance of the humeral response?
Has relevance in delayed transfusion reactions
Can result in a failure to detect an extremely weak positive antibody
What happens to a red cell when it is coated in antibody in vivo?
Direct lysis of rbcs resulting in intravascular haemolysis
Rbcs coated in Ig sequested in the liver or spleen for extravascular haemolysis
Describe the process of intravascular haemolysis
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
Describe the process of extravascular haemolysis
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
On a cell basis, explain how red cell lysis occurs
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
What are the two pathways of cell lysis
Classic pathway
Alternative pathway
*both result in MAC formation and cell lysis
What is considered the standard for detecting antibodies?
3 cell screen
- positive => antibody panel + enzyme panel
What are the two stages of red cell agglutination?
Stage one: sensitisation
Stage two: agglutination
Give two examples of where in practice we would manipulate the antigen-antibody bond
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
What are the four types of bonds that occur between antigens and antibodies?
Hydrophobic
Hydrogen
Van der Walls forces
Electrostatic forces or Ionic bonds
What kind of bonds d carbohydrate antigens favour?
They favour exothermic bonds with their corresponding antibody i.e. low temperature will have highest reactivity
The formation of these bonds let energy out
What kind of bonds do proteins antibodies prefer?
Endothermic bonds
They take in heat so they prefer to react at higher temperatures
What is the main/primary bonding in antibody/antigen reactions?
Main bond is electrostatic
-> charges/ionic bonds
-> corresponding positives and negatives
Bonding such as van der wals forces are considered secondary bonds, what does this mean?
This means the antibody and antigens have to be very close together fo these bonds to occur
What kind of antibodies prefer immediate spin/cold temps and what kind prefer antiglobulin phase/37degrees?
IgM antibodies prefer immediate spin phase
IgG antibodies prefer 37 degrees
What blood group system antibodies are IgM?
ABO and H
I
M and N
Lea and Leb (can also be IgG)
P1
What blood group systems are IgG?
D, Cc, Ee
K
Fy
Jk
S, s
Lea, Leb (also IgM)
What do hydrophobic interactions do?
They make the antibody and antigen want to stick together - think of two drops of oil
What kind of bonding occurs in stage one/sensitisation?
Chemical bonding
Explain what is meant by “goodness of fit” in terms of sensitisation
In order for maximum complementarity both structual fit and complementary distribution of chemical groups must be achieved
What would an antigen-antibody complex with good structural fit with complementary chemical attraction result in?
If opposite charges and they perfectly fit each other then this is ideal
This would have a high KA/affinity
What would an Ab/Ag complex with complementary chemical groups but poor structural fit result in?
This would still work
Much lower affinity for antigen
Lower KA
What would an Ab/Ag complex with complementary chemical groups but poor structural fit result in?
This would still work
Much lower affinity for antigen
Lower KA
What would an Ab/Ag complex with good structural fit but non corresponding chemical groups result in?
This would not work
The chemical groups wouldnt be attractive and may even repel each other
Bonding wouldnt occur
What is the chemistry behind elution?
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
How does antibody cross-reactivity occur?
This occurs when a determinant of the immunising antigen is shared by another antigen
What is affinity
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
What is Ka, what does it mean cliically?
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’
What is Kd, what does it mean clinically?
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
What is avidity?
(3)
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
What is meant by high avidity low affintiy antibodies?
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
How does IgM use avidity to make up for its low affinity?
IgM is 10 valent (actually 5 as only 5 sites can bind at a single time)
Compare to IgG which is 2 valent
How does IgM use avidity to make up for its low affinity?
IgM is 10 valent (actually 5 as only 5 sites can bind at a single time)
Compare to IgG which is 2 valent
What are five factors that can alter sensitisation?
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
How does LISS work?
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
What four factors affect haemagglutination?
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
What is the wingspan of IgG compared to IgM?
IgG has a wingspan of 12 to 14 while IgM has a wingspan of 20nm
What is the wingspan of IgG compared to IgM?
IgG has a wingspan of 12 to 14 while IgM has a wingspan of 20nm
Give some examples of potentiators
AHG in Ireland
Polyehtylene glycol/PEG in USA
Albumin in USA
LISS in Ireland
Enzymes
Give some ezamples of enzyme potentiators, how do they work
These have a positive charge and when they act on rbcs they push them together
Polybrene
Protamine sulfate
Poly-L-lysine (polycations)
Give some ezamples of enzyme potentiators, how do they work
These have a positive charge and when they act on rbcs they push them together
Polybrene
Protamine sulfate
Poly-L-lysine (polycations)
How does albumin work as a potentiator?
It works in the same way as LISS -> reduces ionic strength
How does PEG work as a potentiator?
PEG excludes water and reduces the charge on rbcs
How does PEG work as a potentiator?
PEG excludes water and reduces the charge on rbcs
What is prozone?
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
How does lowering ionic strength affect haemagllutination
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
How does lowering ionic strength affect haemagllutination
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
Explain zeta potential in vivo
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
Explain zeta potential in vivo
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
What gives rbcs their negative charge?
Glycophorins A, B and C
What is the rbc slipping plane
The furthest reach of the rbcs negative charge
What is the rbc slipping plane
The furthest reach of the rbcs negative charge
Defne zeta potential
The force of repulsion between red cells determined by the net negative charge at the slipping plane
Defne zeta potential
The force of repulsion between red cells determined by the net negative charge at the slipping plane
What is the zeta potential for rbcs in normal saline
18mv
What is the principle behind column agglutination technology in gels
- 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
What are the two types of antiglobulin tests?
Direct and indirect
What is the AHG used in Gels specific for?
Polyspecific/broad spectrum anti-IgG and anti-C3b or Csd
Other than AHG what is present in gel matrix
LISS
Other than AHG what is present in gel matrix
LISS
What kind of gell is used?
Sefadex gell
What is the gold standard, gels or tubes, why?
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
Why might it be suggested to not use gels to detect Duffy or Kidd?
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
Why might it be suggested to not use gels to detect Duffy or Kidd?
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
What is the principle of the DAT?
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
What might cause a positive DAT
AIHA
HDFN
Transfusion reaction
How do you carry out an IAT?
(3)
Serum with specific antibody mixed with reagent red cells
Wash cells x3 to remove unbound globulins
AHG added to promote agglutination on centrifugation
What is the principle of the IAT
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
Give four examples of proteolytic enzymes used in blood group serology
Papain
Bromelin
Ficin
Trypsin
comment on the use of enzyme treated rbcs
First described by Morton and Pickles in 1946
Widely used up to te mid-eighties
What are the four effects enzyme treatment has on rbcs
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
How can red cell enzyme treated increase enzyme reactoins
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
How can red cell enzyme treated increase enzyme reactoins
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
Why is there no need for AHG in enzyme gels
The enzyme treatment cleaves M and S responsible for giving rbc its neg charge
What blood group systems does enzyme treatment destroy?
Fya and Fyb
What blood group systems does enzyme treatment enhance?
Rh
Kidd
P
Lewis
I
What are the clinically significant Ab according to the BSH?
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
if you can exclude a non-clinically significant antibody, what are you to do?
Just give crossmatch compatible blood at 37 degrees
