ABO Blood group system (and H) Flashcards
Who first described the ABO system?
-Karl Landsteiner
-collected samples from himself and 5 other associates
-separated cells and serum and mixed each cell sample with each serum
-he was the first individual to inadvertently perform forward and reverse grouping
-he discovered the A, B, and O blood groups
-this is the only blood group system in which individuals have antibodies in their serum to antigens absent from their RBCs
What happens if you fail to determine an accurate ABO group?
-mortality and morbidity
-transfusion of incompatible blood may result in an immediate lysis of donor RBCs producing a severe if not fatal reaction
What is the leading cause of death in hemolytic transfusion reactions today?
-transfusion of the wrong ABO group
What is the ABO group frequencies of B +
8.5%
What is the ABO group frequencies of B-
1.5%
What is the ABO group frequencies of A-
6.3%
What is the ABO group frequencies of O-
6.6%
What is the ABO group frequencies of AB+?
3.4%
What is the ABO group frequencies of AB-
.6%
-most rare
What is the ABO group frequencies of A+
35.7%
What is the ABO group frequencies of O+?
37.4%
-most common
ABO frequencies for white people
O - 45
A - 40
B - 11
AB - 4
*most common is O and A
ABO frequencies for black people
O - 50
A - 26
B - 20
AB - 4
*most common is O
ABO frequencies for Hispanic people
O - 56
A - 31
B - 10
AB - 3
* most common is O
ABO frequencies for Asian people
O - 40
A - 28
B - 25
AB - 7
*Most common is O
Forward grouping
-use known commercial antisera (anti-A, anti-B)
-use the patient’s red cells
-detect antigens on the patient’s red cells
AKA: Front type
Reverse grouping
-use known reagent RBCs (A1 cells and B cells)
-use the patient’s serum
-detects ABO antibodies in the patient’s serum
AKA: Back type
ABO grouping
-most frequently performed test in the blood bank
-front and back typing is done on all patients
-inverse reciprocal relationship between front and back type (serve as a check for each other)
Group A: antigens, antibodies
Antibodies in plasma: Anti-B
Antigens in red blood cells: A antigen
Group B: antigens, antibodies
Antibodies in plasma: Anti-A
Antigens in red blood cells: B antigen
Group AB: antigens, antibodies
Antibodies in plasma: None
Antigens in red blood cells: A and B antigens
Group O: antigens, antibodies
Antibodies in plasma: Anti-A, Anti-B
Antigens in red blood cells: None
-O phenotype is an autosomal recessive trait because 2 non-functional genes are inherited
Naturally occurring antibodies
-it has been postulated that bacteria/pollen are chemically similar to A and B antigens
-bacterial/pollen is widespread, and we are constantly exposed
–exposure serves as a source of stimulants of Anti-A and Anti-B
-antibody production in most other blood groups requires the introduction of foreign RBCs (via transfusion or pregnancy)
What does naturally occurring mean
-produced without any exposure to RBCs
ABO antibodies
-naturally occurring
-individuals normally produce antibodies directed against the A/B antigens absent from their RBCs
-predominantly IgM
-activate complement
-react at RT and colder
-produce strong direct agglutination reactions during ABO testing
-production begins at 3-6 months old, peaks at 5-10 years, and diminishes when elderly
Can you detect antibodies in babies just birthed?
-Even though the production of antibodies is initiated at birth, the titers are too low to detect until around 4 months
-this is why front types are only performed on babies
What happens to elderly people and their antibodies?
-elderly people might have lower levels of Anti-A and Anti-B and therefore their back type (reverse grouping) may be weak or missing, resulting in ABO discrepancy
What class is Anti-A and Anti-B predominantly?
-IgM but may have IgG component
What antibodies do group O individuals have?
-they produce Anti-A, Anti-B, and Anti-A, B
-Anti-A.B is not a mix of Anti-A and Anti-B
-it has a separate cross-reacting antibody
-it is IgG in nature
**This can be a problem for a group O mom having an A or B baby, which can cause hemolytic newborn disease. Often the cord samples for babies of type O mom are tested for possible ABO HDFN
When is the Anti-A, B reagent used?
-routinely used in ABO confirmation of blood donors, it is more economical to use one reagent instead of two when verifying group O RBCs
Reagent Anti-A
-Monoclonal
-highly specific
-IgM
-clear blue reagent
-expect 3-4+ rxn
Reagent Anti-B
-monoclonal
-highly specific
-IgM
-clear yellow reagent (contains acriflavine dye)
-expect 3-4+ rxn
Reagent Anti-A.B
-more effective at detecting weak A/B antigens
-not as necessary since monoclonal Anti-A and Anti-B antisera were developed
-This reagent is more sensitive and can detect weaker expressions
-not routinely used for patient ABO grouping
-used for ABO confirmation of donor blood
-more economical, cheaper to use one reagent
Bernstein- Inheritance of ABO blood groups
-1924
-inherit one ABO gene from each parent
-these two genes determine which ABO antigens are on the RBC membrane
-Inheritance follows Mendelian genetics
-codominant expression
Which chromosome is the A, B, and O gene located on?
9
Is the O gene an amorph?
-Yes, no antigens are produced
Autosomal codominant inheritance
-blood group genes are not carried on sex genes, hence autosomal and not sex-linked. Whenever the gene is inherited the antigen is expressed on the RBCs, thus its codominant
Phenotype
-saying someone is group O or A
Genotype
- AA/OO/AO etc is referring to genotype
Can you determine genotype serologically?
-no you cannot, family studies or molecular assays are necessary to determine the exact genotype
-you can for O because it is an autosomal recessive and results from inheriting 2 O genes (OO)
-you can for AB because codominant, so A came from one parent and B from another
What are the 3 different genes that form the ABH antigen?
-ABO
-Hh
-Se
What is the precursor material A, B, and H antigens are made from?
-paragloboside or glycan
A, B, and H antigens
-ABH genes do NOT code for antigens, they produce (code for) glycosyltransferases (enzymes)
-these enzymes add sugars to precursor substances
Which antigen is found in the greatest concentration of a group O individual
H antigen
How come the H antigen may not be detected in group A1 individuals?
- The A1 gene is so good at converting H antigens into A
-the more A, the less H antigen available
-the H antigen on A1 and A1B RBCs are so well hidden that occasionally anti-H is found in the serum
What is anti-H?
-a naturally occurring IgM cold agglutinin that reacts best below room temp
-IgM can react at temperatures up at 37 C but will react better at cold
What is the precursor on which A and B antigens are made of?
H antigen
Are Hh and Se genes a part of the ABO system?
NO
-but their inheritance does influence A and B antigen expression (Chromosome 19)
Hh genes form ABO antigens where?
RBCs
Se gene form ABO antigens where?
Secretions
-you need to inherit this to form ABO antigens in secretions
Type 1 and Type 2
-type 1 and 2 refer to linkages found between the terminal sugars of D-galactose and N-acetylglucosamine
Type 1
-precursor substance in secretions
B1 –> 3 Linkage
-number 1 carbon of D-galactose –> number 3 carbon of N-acetylglucosamine
Type 2
-precursor substance on RBCs
-B1 –> 4 linkage
-the terminal galactose on the precursor substance is attached to the N-acetylglucosamine in a beta 1 –> 4 linkage
ABH antigen expression
-ABH antigen develops early in fetal life
-Newborn RBCs have 25-50% number of antigenic sites found on adult RBCs (newborns have weaker front-type reactions)
-A and B antigen expression is fully developed by 2-4 years of age
-ABH antigen phenotypic expression can vary with race, genetic interaction, disease states
H gene
-Group O individuals inherit at least one H gene (genotype = HH or Hh) and 2 O genes
-H gene produces enzyme a-2-L-fucosyltransferase
-Transfer sugars (L-fucose) to an oligosaccharide chain on terminal galactose of type 2 chain
-produce the H antigen
-O gene does NOT make catalytically active transferase
-H substance is unmodified in group O
Immunodominant sugars
-the sugars that occupy terminal positions on this precursor chain confer blood group specificity
ex: L-fucose confers H specificity, blood group O has the highest concentration of H antigens
-need L-fucose (H substance) for other sugars to attach in response to A and B genes
Bombay phenotype (Oh)
-lacks normal expression of ABH antigens, so do not react with anti-A, anti-B, or anti-H
-FUT1 (H gene) is silenced
-genotype is hh (extremely rare), is an autosomal recessive trait, and 99.99% of the population have H gene
-Does not produce a-2-L-fucosyltransferase
-L-fucose is NOT added to type 2 chains and H substance is not expressed on RBCs
-individuals may inherit ABO genes, but not express them because no H antigen to build upon
-ABH substance also absent from saliva, FUT2 (Se gene) is also silenced
What will a Bombay phenotype show as?
-As group O, but true O RBCs react with anti-H lectin and Bombay does not (because no H antigen)
Who can a Bombay patient receive blood from?
-A Bombay person can only receive blood from another Bombay because anti-H can be potent and react at 37 C
-normal group O RBCs have many H antigens and Bombays anti-H would cause immediate lysis
The A gene codes for what enzyme?
a-3-N-acetylgalactosaminyltransferase
What does a-3-N-acetylgalactosaminyltransferase do?
transfer N-acetyl-D-galactosamine (GalNAc) sugar to the H substance
What is GalNAc sugar responsible for?
-A specificity
A gene
-has more transferase than B gene, thus producing more antigen sites
-810,000 to 1,170,000 A antigen sites on adult A1 RBC
The B gene codes for what enzyme?
a-3-D-galactosyltransferase
What is the job of a-3-D-galactosyltransferase?
-transfers D-galactose (Gal) sugar to the H substance
What sugar is responsible for B specificity?
D-galactose
When both A and B genes are inherited, which one competes more efficiently?
-the B enzyme seems to compete more efficiently for the H substance than the A enzyme
-so on an AB person’s RBC there are approximately 600,000 A antigens and 720,000 B antigens
What are the three immunodominant sugars?
Fucose - Group H
N-acetyl-D-galactosamine - Group A
Galactose - Group B
*** As more A or B antigen is made, less H antigen remains
H antigen amount
O >A2>B>A2B>A1>A1B
ABH soluble antigens
-found in all body secretions
-presence depends on ABO genes inherited AND inheritance of secretor genes (Sese)
-solubles are only on glycoproteins
Secretors
SeSe or Sese
-80% of the US population are secretors
Non-secretors
sese
What does the Se gene code for?
a-2-L-fucosyltransferase
What does a-2-L-fucosyltransferase do?
-modifies type 1 precursor substance in secretions to form H substance
-that H substance can then be modified to express A or B substance if the corresponding ABO gene is present
Where can RBC antigens be found on?
-glycolipids, glycoproteins, and glycosphingolipids
secretor studies
-80% of the population has the Se gene
-these people secrete water-soluble blood group substances in their saliva and body fluids
Group A = secrete A substance and some H
Group B = secrete B substance and some H
Group O = secretes H only
Group AB = secretes A and B substances and a little H
Agglutination inhibition
-is used to determine the secretor status
-the presence of agglutination = negative test
-no agglutination = positive test
* for valid tests the control needs to agglutinate
*2 steps to this process
1. antibody neutralization
2. agglutination inhibition
Antibody neutralization
-saliva is mixed with commercial antisera and incubated
-if the patient is a secretor, soluble blood group antigens in the saliva will react with the antibodies in the commercial antisera
Agglutination inhibition
-commercial RBCs of appropriate blood groups are added to the test mixture
-if the patient is a secretor, there is no free antibody left for the commercial RBCs to react with
-thus if a patient is a secretor there will be NO agglutination
ABO subgroups
-subgroups show weaker variable serologic reactivity with polyclonal anti-A, anti-B, and anti-A, B reagents
-due to decreased # of antigen sites
-not as often seen now due to the use of monoclonal reagents
A subgroups
-more common than B subgroups
-99% of group A people are A1 or A2
What is the percentage of A1?
80%
What is the percentage of A2 or other A subgroups?
20%
What are the immunodominant sugars on the A1 and A2?
-N-acetyl-D-galactosamine
A1 gene
-higher concentrations of a-3-N-acetylgalactosaminyltransferase than A2
-A1 enzyme is 5-10 times more active than A2
-A1 is more effective at converting H antigen to A antigen
-A1 cells have 1,000,000 A antigens
A2 gene
-A2 individuals have increased levels of H antigen, second only to group O
-A2 cells have 250,000 A antigens
Weaker A subgroups
-subgroups weaker than A2 are rare, usually found through ABO discrepancies
-Subdivide individuals into A3, Ax, Aend, Am, Ay, Ael, etc
* secretor studies
*adsorption-elution tests
*molecular testing
-have less antigen, varying degrees of agglutination with anti- A, B variability in detectability of H antigen (anti-H)
-A3 RBC characteristically demonstrates a mixed-field pattern of agglutinations with anti-A
-if a weak A subgroup is suspected make sure the patient doesn’t have a disease that makes altered ABH antigens
Potential problems: Weaker A subgroups
-if an Ax donor is mistyped as O type and transfused to O recipient, the recipient’s anti-A, B will agglutination/ lyse Ax RBCs –> rapid intravascular hemolysis
A3 RBCs
-looks mixed field with anti-A and anti-A, B
-antigen sites: 35,000
-Anti -A1 may be present in the serum of A3 people and A substance is detected in saliva if secretor
Ax RBCs
-not agglutinated by anti-A, but do agglutinate with anti-A, B
-Antigen sites: 4,000
-almost always produced anti-A1, but only H substance in saliva if secretor
-anti-A can be adsorbed and eluted from Ax cells without difficulty
Anti-A1
- 1-8% of A2 individuals make anti-A1 in their serum
- 22-35% of A2B individuals make anti-A1
- reagent anti-A agglutinates both A1 and A2 RBCs
-usually first detected in the BackType, anti-A1 will react with the A1 cells used in the reverse typing
-naturally occurring IgM cold reacting antibody and is unlikely to cause transfusion reaction b/c it only usually reacts below body temp
-Both A1 and A2 strongly react with Anti-A in routine testing, but A1 can be distinguished through A1 lectin
Anti-A1 lectin
name: dolichos biflorus
- use this to differentiate A1 and A2
-Anti A1 lectin will agglutinate A1 but NOT A2 cells
Lectins
-lectin are seed extracts that agglutinate human cells with some degree of specificity
Dolichos biflorus
-agglutinates A1 cells
Bandeiraea simplicifolia
agglutinates B cells
Ulex europaeus
-agglutinates O cells (is anti-H lectin)
-since the A2 phenotype reflects the inefficient conversion of H antigen to A antigen, A2 shows increased reactivity with anti-H lectin
Adsorption/eluate studies for identification of A subgroups
-commercial anti-A is adsorbed onto red cells believed to be A subgroup
-eluate is prepared from the RBCs and then tested for anti-A
-if anti-A is recovered, presenced of A antigens is confirmed
Subgroups of A
Aint, A3, Ax, Am, Aend, Ael, etc.
B subgroup
-very rare
-like A subgroups, result from alternate alleles at the B gene locus
-usually have variations in reaction strength with anti-B and anti-A, B
-Includes B3, Bx, Bm, and Bel
-can do adsorption/eluate studies with anti-B
-presence/absence of ABO isoagglutinin in serum
-may initial front type as group O
B3
-mixed field with anti-B and A,B (like A3), and B substances in secretions
* the most frequent B subgroup
Bx
weak agglutination with anti-B and anti-A, B, readily adsorb and eluate anti-B
-secretors have lots of H substance and some B substance that is hard to detect
Bm
-doesn’t agglutinate anti-A or anti-A, B
How can disease states alter RBC antigens?
-can weaken reactions
-can acquire pseudoantigens (these would be seen during forward grouping)
-can cause forward grouping issues
Weaker front-type reactions
-depressed antigen strength
-can look like mixed field
-can be due to leukemia, chromosome 9 translocations, thalassemia (stress hematopoiesis), Hodgkin’s disease
-antigen strength increases when in remission
-antigen strength increases when in remission
Weaker- Back type reactions
-isoagglutinin (anti-A, anti-B, anti-A, B) may be weak/absent
-chronic lymphocytic leukemia (CLL)
*leukemia with hypogammaglobulinemia
-malignant lymphomas (non-Hodgkin’s)
*decreases in gamma globulin fraction
-agammaglobulinemia
Acquired B phenomenon
-due to increased permeability of the intestinal wall (due to obstruction, colon/rectal cancer)
-allows passage of bacterial polysaccharides from E.coli serotype O86 into the patient’s circulation
-the patient’s group A RBCs adsorb the B-like polysaccharide and react with some anti-B reagents
-their forward grouping may appear as AB but with a weaker reaction with the anti-B antisera
-their reverse type would like group A
What does bacterial deacetylase do?
-works on the A antigen by converting N-acetylgalactosamine (the terminal sugar for A antigen) to N-galactosamine (very similar to the terminal sugar for the B antigen- D galactose)
When should you suspect acquired B phenomenon?
Acquired B is a transient serologic discrepancy seen in group A individuals. Acquired B should be suspected when a historical group A patient now has weak B expression in the front type (anti-B = 2+ or less)
How would you resolve a patient with acquired B?
-retype using a different monoclonal anti-B or acidified human anti-B, acidified human anti-B will not react with the acquired B antigen
What two mechanisms have been postulated for acquired B?
- In vivo: bacterial enzyme de-acetylase modifies the terminal a-N-acetyl-D-galactosamine into a D-galactosamine which looks like a D-galactose (B-antigen) and can react with anti-B
- In vitro: blood group B activity can be conferred onto A/O RBCs adsorbing B-active bacterial polysaccharides
Undetectable ABO antigens
-can occur with carcinoma of the stomach or pancreas
-patient’s RBCs are unchanged, serum contains increased concentrations of blood-group specific soluble substances (BGSS)
*may neutralize antisera used in forward grouping
ABO discrepancies
-forward and reverse grouping disagree
-must be resolved prior to transfusion
-can be due to problems with patient RBCs (forward group) or patient serum (reverse group)
-can be due to technical errors, labeling, missing reagents, contamination, etc.
-4 groups of discrepancies
*should always add serum and antiserum first then red cells, should always record results to prevent transcription error
Group 1 discrepancies
-unexpected reactions in BACK TYPE due to weak/missing antibodies
-most common discrepancy group
Who is affected by group 1 discrepancies?
- newborns/elderly (have lower antibodies levels)
- immunosuppression (ex: agammaglobulinemia)
- BMTs
- Dilution
- Subgroups (ABO)
*patients with malignant lymphomas/CLL
*patients who have been diluted by FFP transfusions
How to resolve Group 1 discrepancies?
-get patient history
-increase serum to cell ratio
-increase the incubation time
-decrease temperature
* incubate at 4C with auto control and group O control cells (additional cells needed because decreased temp increases chance of detecting cold agglutinin reactivity)
Group 2 discrepancies
-least frequent discrepancy
-unexpected reactions in FRONT TYPE due to weak/antigens
-blood group specific soluble (BGSS) substances
-chimerism
How to resolve Group 2 discrepancies?
-increase incubation time by up to 30 minutes
-can decrease the temperature to 4C and run with auto control and group O cells
*If acquired B:
secretor studies: if the patient is a secretor, only A substance will be present
-can also treat cells with acetic anhydride-acquired B cells lose reactivity
Who is affected by group 2 discrepancies?
*subgroups of A/B
*leukemias
*acquired B phenomenon
BGSS Substances
-can occur due to carcinoma of the stomach/pancreas
*excess amounts of BGSS substances are present in plasma
*neutralize reagent anti-A or anti-B
*leaves no unbound antibody to react with patient cells
*cause a false negative or weak reaction in front type
-resolve by washing patient cells several times with saline
chimerism
-presence of 2 cell populations in 1 person
-true chimerism is very rare and occurs in twins
-blood exchange occurs in utero
*2 cell population emerge
*both recognize as self, so the body doesn’t make anti-A or anti-B
-if no history of twins, could be due to dispermy (2 sperm + 1 egg) and maybe mosaic
Artificial chimerism
-is more frequent than true chimerism and can be due to non-ABO matched blood transfusions, BMTs, exchange transfusions, FMH
Group 3 discrepancies
-caused by protein or plasma abnormalities (in vitro problems like rouleaux)
Who causes group 3 discrepancies?
-waldenstrom’s macroglobulinemia
-multiple myeloma
-some advanced Hodgkins lymphomas
-increased levels of fibrinogen
-plasma expanders (dextran)
-wharton’s jelly (cord blood only)
Rouleaux
-RBCs stacked like coins
How to resolve group 3 discrepancies?
-resolve rouleaux with saline replacement
*remove patient serum and replace with equal volume saline
*true agglutination remains
-Resolve Wharton’s jelly by washing cord cells up to 6 times with saline
Group 4 discrepancies
-cold reactive autoantibodies
*RBCs so heavily coated that they spontaneously agglutinate
-mixed field
*more than one ABO group type RBCs circulating (ex: non-ABO identical RBC transfusion, BMT, stem cell transplant, ABO subgroup (A3))
-unexpected ABO isoagglutination or non-ABO alloantibody
-acriflavine antibody
How to resolve group 4 discrepancies? due to cold autoantibodies
-if due to cold autoantibody-incubate patient RBCs at 37C and wash with 37C saline 3 times (get valid front type)
-can also treat RBCs with DTT to break up IgM agglutination
-can pre-warm
-can perform auto adsorption (get valid back type)
How to resolve group 4 discrepancies? due to unexpected ABO isoagglutinin
-may be due to anti-A1
-identify anti-A1 using 3 examples of A1 cells, A2 cells, B cells, O cells, and auto control
-pattern determines the specificity
-test patient RBCs with dolichos biflorus (Anti-A1 lectin-agglutinates A1 cells)
How to resolve group 4 discrepancies? due to unexpected alloantibodies
ex: anti-M
-perform antibody identification process
-find reagent A1 and B cells negative for antigen to correct reverse typing
acriflavine antibody
-forms a complex that attaches to patients’ RBCs and causes agglutination in the forward type
-resolve by washing the patient’s cells with saline 3 times
What is acriflavine?
-is the yellow dye in some anti-B antisera reagents, when someone has an antibody to this dye, it forms an acriflavine-anti acriflavine complex that attaches to patient RBCs and causes agglutination in the front type
