An introduction to Blood Group Serology Flashcards
Blood Group Antigens are?
- Glycoproteins and glycolipids present on the surface of red cells
- Some (eg ABO) may be present more widely on endothelial surfaces
- Genetically determined
- Generally autosomal and co-dominant
- Xg system sex linked
- Limited understanding of biological function of the antigens
Describe the Genetic control of Blood groups
- Protein Determinants
- Gene codes for the antigenic determinant itself
- Rh, Kell, Duffy and Kidd systems
- Glycolipid Determinants
- Gene codes for production of enzymes that add or remove carbohydrate or lipids, through this you get definition of an antigen!
- ABO, Lewis group systems
Duffy Blood Group System and Malaria?
- The Duffy antigen on the RBCacts as the entry point to the red cell for the malarial parasite
- in caucasian populations the Fya-Fyb negativephenotype is rare
- In individuals with a black african ethnic backgroup up to 40% are Fya-Fyb- negative this represents the impact of natural selection for malarial resistance
The McLeod Phenotype
- Kx null phenotype associated with Chronic Granulomatous Disease and acanthocytosis!
Blood Groups Variation with Populations
- Varies significantly in different population types
- Most likely due to Genetic drift
- Usually no biological advantage apparent
- May have a clinical impact on the provision of compatible blood!!
Blood group antigens/systems are clinically important because they have the ability to
generate Blood Group Antibodies
Antibodies rcognise ‘foreign’ antigens
- May be IgM, IgG or occasionally IgA
- May be naturally occurring or immune stimulated
Difference between Naturally occuring and Immune stimulated Antibodies
Naturally Occurring:
- No exposure to foreign red cells but exposure to bacteria containing, for example, ‘A-like Antigens’.
- At birth baby has no ABs againsts ABO antigens, but by 6 months almost all babies will have formed some form of anti-A or Anti-B. This is because A or B antigens are very close to the antigens formed on the surface of bacteria
- Can Activate the Complement Cascade > intravascular destruction
- Anti-A produced
Immune Stimulated:
- exposure to foreign red cells (for example Rh+) by transfusion or pregnancy (Rh+ fetus)
- Cannot fully activate Complement Cascade > extravascular destruction
- can activate early phases, and coat early proteins
- Anti-Rh produced
Naturally Occurring Red Cell Antibodies
- Antigens that develop in the absence of exposure to the red cell antigen
- Most likely stimulated by cross reacing antigens derived from bacteria (as they’re very similar)
- Not present at birth but develop during 1st year of life
- Usually related to lipid antigens
- Significant IgM component to antibody, but IgG may also be present
- ABO and Lewis antigens fall in this category
Immune Stimulated Red Cell Antibodies
- Develop only following exposure to specific antigen
- May be produced following
- transfusion
- pregnancy
- injection (eg IVDU)
- Normally IgG in nature
A brief overview of the history of Transfusion.
Just for extra info
- Earliest record of transfusion in the early 19th century, for women bleeding heavily following childbirth. Would use husbands blood via cannuler. “Blundell Transfusion”
- Some died immediately, and some survived.
- Later on Dr Karl Landsteiner discovered the ABO blood group system
- the ABO blood group system is the most likely to kill you, and the best to know about
Where are ABO antigens found?
Present on the surface of
- Blood cells
- Epithelial cells
- Body fluids
What determines the ABO phenotype?
The phenotype is determined by a series of glycosyltransferase enzymes. These are resposible for addition of CHO molecules to the basic membrane structure.
The H antigen is neccessary for the ABO phenotype to be expressed
this is present in (almost) everyone
The ABO groups all have an H group attached, but whats the molecular difference in this!
H:** base structure with a D-galactose and fucose sugar. **This alone is known as the O blood group!!
A: N-acetylgalactosamine added to the surface of the red cell
B: Galactose added to the surface of the red
Therefore your blood group is determined by the presence of a sugar molecule
What does it mean by the ABO blood group being ‘co-dominant’
You inherited one gene from your mum and one from dad, and this COLLECTIVELY determines phenotype.
By 6 months of life you’ve developed antibodies against the A or/and B antigens which you lack
O is the most common phenotype!
Clinical Relevance of ABO systems
- ABO antibodies: naturally occuring and appear ~3-6months old
- Most important blood group as → to fatal transfusion errors
- ABO incompatible transfusion results in complement activation leading to…
- intravascular haemorrage
- renal failure
- DIC
Therefore for a tranfusion we select antigens that don’t have ___________
Therefore for a tranfusion we select antigens that don’t have corresponding antibodies present in the recepiants plasma
eg; blood group O (has therefore anti-A and Anti-B present) cannot be transfused with A or B groups. Can only be transfused with O
O is therefore the universal Donor but as a small population they can’t sustain everyone!
The Rh Blood Group System
- Second most important blood group system
- Protein antigen
- Expressed only on RBC
- antibodies only produced post exposure to the D+ red cells ‘immune stimulation’
- Rh(D) is highly immunogenic,
- if you expose a group to D+ red cells 80-90% will have a reaction
What is Rh(D)?
- Most important antigen of the Rh system
- Everyone is either Rh(D) positive or Rh(D) negaticed antigen is amorph
Rh(D) negative: dd
Rh(D) positive: DD or Dd
population frequency of Rh(D) in NZ?
Rh(D) and transfusion
- 90% of Rh(D) negative individuals transfused with a unit of Rh(D) positive red cells will produce anti-D strong immune reaction
- Anti-D is an IgG antibody that is unable to bind complement, Red cell destruction is extravascular
- Anti-D is the most common cause of Haemolytic Disease of the newborn: fetus RBC cross into mum, if fetus is D positive from Dad and mum is negative > anti-D formation > problems for mum in subsequent pregnancies
- Normally transfuse red cells of the same Rh(D) type as the recipient
- Never transfuse Rh(D) positive red cells to an Rh(D) negative female of child bearing age; male would only require D negative cells, not a biggie
Describe what the Rh antigen is??
Describe the Expanded Rh system
- THe Rh system is the most complex of known blood group systems
- Rh antigen: product of 3 genetically closely linked alleles; which make allelic pairs
- C and c
- D and d (d is an amorph)
- E and e
- 3 allelic antigens behave as a single entitiy
- a number of common alleles can therefore be defined
Minor Blood Group Systems
- Over 400 blood group systems have been defined
- Majority not of clinical interest, but some relevance to
- frequency of antigen in pop
- frequency of antibody production following transfusion
- ability of antibody to destroy transfused red cells
- Relevance is higher if a small amount of the population will react
What are the 3 blood group systems of common clinical interest?
- Kell: K (Kell) and k (cellano)
- Kidd: Jka and Jkb
- Duffy: Fya and Fyb
Don’t need to know gene frequencies
Laboratory Techniques for detecting antigens and antibodies
- Often involves an agglutination technique; when the antibody + antigen put together they CLUMP
- Antigen testing usually uses commercially sourced monoclonal reagents
- these have high specificity and sensitivity
- are IgM in nature
- produce direct agglutination
- Molecular typing methods increasingly available
Laboratory Techniques; Red cells and zeta potential
- Red cells are negatively charged, so called zeta potential
- IgM molecule is larger then the ZP so can produce cross-linking → agglutination
-
IgG molecule is too small to produce cross-linking; therefore a potentiator (enables red cells to get closer) is need to have agglutination
- usually Anti-human Globulin
- also enzymes, albumin
Immunoglobulin Structural differences of the IgG and IgM?
IgG: dimer with 2 antigen-binding sites that are very close together, soif a red cell is bound to one site the other is blocked
IgM: Pentamer, with 5 binding site, therefore you can get several RBCs around to form an agglutination via crossinglinking (see pic)
Describe what would happen via IgG and RBC, and what you need to do in the lab to fix this
As the IgG is too small, you get it coating the cell, but no cross link reaction occuring, and the IgG can’t overcome the pushing apart of the RBC (zeta potential) and you can’t see anything.
- Wash the solution 4x to remove any free antibody present
- Take those free cells and expose them to anti-human globulin; antibody directed at human IgG
- IgG of the AHG will react with the IgG bound to the RBC, and you will get a reaction
This is partiularly important in detecting IgG antibodies
How is Anti-human Globulin produced?
- Used to be in rabbits, inject with serum containing IgG as well as other proteins
- Rabbit reacts against IgG and forms Antihuman Globulin against this (also forms anticomplement antibodies))
- Select the AHG elements you require.
Nowadays mixtures of monoclonal reagants more comonly used; as easier to produce and standerdise
How do we form/detect Antibodys?
Tile Groups
- Have a line of Antibodies (eg; anti-A and Anti-B) and a line of blood cell types (A, B O cells)
- Note if you get agglutination
Test Tubes:
Postitive: Agglutination in bottom of testtube
Negative: Dispersed cells
Micro Tetre Plate
No reaction: cells sink to bottom
Reaction: red cells will coat the surface of the cell;carpet like phenomenon
Chromotographic Gel Cards
Reaction: red cells can’t pass through so float on top
Can also do this testing Antibodies
What causes Haemolytic Disease of the Newborn?
- When maternal antibody crosses placenta → destruction of fetal RBCs
- Always involves IgG antibody (IgM too large to cross)
- Most frequently from Anti-D
- Followed by Anti-c
- And Anti-Kell
- Frequency reduced by immunoprophylaxis
Describe the course of events of Haemoytic Disease of the Newborn
maternal antibody crosses the plancenta → destroys fetal red cells → baby becomes progressively anaemic → develops cardiac failure → develops Hydrops Fetalis
Hydrops Fetalis : severe conjestive cardiac failure. If untreated can cause death in utero. Usually in will survive.
Usually babies will haemolytic disease will survive to birth, but what happens to them then?
They will have the maternal antibody present in their system for a few weeks. (T1/2 of IgG is 3 weeks) , baby continues to produce red cells that continue to be destroyed by IgG and the baby becomes jaundiced.
This doesn’t happen in utero as the bilirubin is sucked out into the maternal circulation.
The infants blood-brain-barrier is poorly developed → bilirubin crosses to brain surface → CSF→ irritant to the neuro system → causes a range of neurological issues/abnormalities.
Can cause deafness or Kernicterus (spasm), which can cause cerebal palsy
What is happening here?
- D positive infant
- Cells cross into maternal circulation
- usually at childbirth, but can be due to a variety of other reasons during the pregnancys
- The D positive red cells lead to the formation of anti-D in the mother
- This Anti-D will then travel back into the fetus and destroy its RBCs
This occurs less where there’s an ABO incompatibility between mum and baby, because the ABO antibodies would remove the D + red cells before anti-D formation could occur.
What has cause the decline of Anti-D sensitisation in births, and how does in work?
Immunoprophylaxis. (combined with improved obstrectric antenatal care)
- The basis of it is to break the cycle; and stop mum forming anti-D when the babies cells enter the circulation
- As this occurs at childbirth (And you have a D - mum and D+ baby) we give the mum an injection of Anti-D
- This clears the D+ red cells and prevents the mums immune response from occuring “passive immunisation”
Prevention of Rh (D) HDN?
- Anti-D immunoglobulin given routinely following birth of an Rh (D) + baby to a Rh (D) - mother
- Standard postnatal dose in NZ is 625 IU destroys 5ml fetal blood
- Kliehauer test: used to detect women with larger foetomaternal bleeds (as they’ll need a larger dose)
When is Anti-D immunoglobulin given during pregancy?
Following a potentially sensitising event
- Abortion
- Termination
- Amniocentesis
As this is a point where theres more bleeds, give in the 3rd trimester as bleeds occur most here!
ABO Haemolytic Disease of the Newborn?
- We all have ABO antibodies
- ABO incompatibility between mum and baby is common
- but Haemolytic ABO disease is rare
- AB antigens weakly expressed in fetus/newborn
- AB antigens widely distributed in placental tissue and absorb antibody before it reaches fetus
- When ABO HDN occurs it’s usually mild and transfusion rarely required
