W9TL2 - ABO Subgroups Flashcards
H Expression between ABO Groups
Presence of A and B antigen “masks” the H antigen
Relative amount of H antigen present in each group:
- O > A2 > B > A2B > A1 > A1B
Lectin
Plant or seed extracts equivalent to antibodies
Bind specific sugar residues
Agglutinate human RBCs
The lectin Ulex europeus binds exposed H antigen, causing agglutination
- reaction strength is proportional to amount of H Ag exposed
Results for Lectin Ulex Europaeus
- O: 4+
- A2: 3+
- B: 2+
- A2B: 1+
- A1 and A1B: neg
Secretion of ABO Antigens
Individuals expressing the FUT2 (Se) gene secrete ABO antigens
- expression is dominant (1 Se allele = expression)
- termed “ABO secretors”
- ~80% of the population
FUT2 gene encodes for α-1,2-L-fucosyltransferase
- adds L-fucose to terminal D-galactose of type 1 precursor substance
- type 1 precursor substance is produced by secretory tissues => production of the H antigen in secretions
Product of A or B genes can modify this secreted H antigen
→ A, B, and H antigens found in secretions
- saliva, tears, urine, milk
Bombay Blood Group (Oh)
Those with Bombay blood group lack FUT1 (H) gene
- H gene is required for production of H antigen
- inheritance of H antigen is dominant, so only 1 H allele (Hh) needs to be present for expression
- individuals with Bombay phenotype are hh
Non-secretors (sese)
Very rare (0.01%-0.0001% of the population)
Do not produce H, A, or B antigens
Have anti -A, -B, -A,B and anti-H in their plasma
Anti-H is an IgM antibody that reacts @ 37°C and binds complement and will lyse H antigen expressing RBCs
Bombay Blood Group Testing
Group as O
Agglutinate all screening cells in an antibody screen
- screening cells are group O
- anti-H in Bombay plasma binds H antigen on screening cells
Identify Bombay individuals using Ulex europeus
Won’t agglutinate Bombay RBCs
Must be transfused with blood from other Bombay individuals
Para-Bombay
Weak or no expression of H antigen on RBCs
Weak expression of A or B antigen on RBCs, if individual produces A or B transferase
One of two mechanisms:
1. Inactive FUT1 gene, active FUT2 (Se) gene
- => type 1 H substance produced
- H (A/B) antigens in secretions
- a small amount adsorbs onto RBC
2. Mutated FUT1 gene with decreased activity, +/- active FUT2 (Se) gene
- anti-H (weaker than Bombay), -A (? + -B), -B (? + -A1) in plasma
ABO Genetics
ABO gene on chromosome 9
Encodes the glycosyltransferase responsible for adding the immunodominant CHO residue to the H ag
The ABO gene contains 7 exons
- exons 6 and 7 encode the catalytic domain of the enzyme
- SNPs in this region → production of transferases with different specificities, including ↓ functional activity => ABO subgroups
A1 and A2 Subgroups
Major subgroups of A
A1 - 80% of all A and AB (A1 and A1B) individuals
A2 - 20% of all A and AB (A2 and A2B) individuals
1-8% of A2 individuals, 22-35% of A2B individuals produce an anti-A1
- directed against A type 4 structure
- cold reacting IgM antibody
- clinically significant ONLY if it reacts at 37°C
- agglutinates cells expressing A1 antigen in vitro
- causes ABO discrepancies; affects reverse grouping results (1+ for A cells and 4+ for B cells)
Identify A2 subgroup using the lectin Dolichos biflorus
- dolichos biflorus binds to A1 antigen
- therefore, no agglutination in A2 individuals (only in A1)
A Subgroups
Immunodominant sugar is N-acetyl-D-galactosamine
A2 individuals produce a mutant α-3-N acetylgalactosaminyltransferase
- less efficient at converting H Ag -> A Ag
- therefore A2 individuals have less A Ag on RBC surface
- A qualitative difference also exists
Weak A Subgroups
Rare
All contain anti-B in their plasma
How to Differentiate between A and B Subgroups
The presence/absence of:
- ABO antigens in saliva
- The corresponding transferase in plasma
Absorption and Elution
Adsorption
- the binding of aby’s in solution (plasma) to the corresponding ag on the RBC surface
Elution
- technique used to dissociate aby’s from RBC antigens
- eluate (supernatant) contains concentrated aby
- importantly, the aby is not degraded, which means it can be used in subsequent testing
Absorption/Elution for ABO Subgroups
Take patient cells, mix with reagent anti-A (for suspected A subgroup) or anti-B (for suspected B subgroup)
If the corresponding ag is present, aby will bind
Wash cells multiple times to remove excess aby, ensuring that you keep the last wash
Perform elution, using heat or Lui freeze-thaw (these work for ABO abys, or any IgM aby)
Perform a standard forward grouping reaction, testing the eluate and last wash against known A and O cells (or B and O cells)
If there is agglutination against A (or B) cells only, and only with the eluate, then we can conclude that the patient expresses the corresponding antigen
