ABO Blood Groups
Brian Poirier, MD
UCDavis Medical Center
1
Topics
•
•
•
•
•
Basic genetics of ABO blood groups
Formation of H, A, and B antigens
ABO antigens and antibodies
ABO testing
ABO discrepancies
2
Objectives
• Describe the inheritance of the ABO Blood
Groups and predict the ABO phenotypes and
genotypes of offspring from various ABO
matings
• Explain the roles of Secretor and H genes in the
formation of H, A, and B antigens on the red
cells
• Describe the reciprocal relationship between
ABO antigens and antibodies for blood types O,
A, B, and AB.
3
Objectives (Continued)
• Describe the procedures on ABO forward
and back typings; interpret the results; and
resolve any discrepancies if present.
• Describe the quantitative and qualitative
differences between the A1 and A2
antigens.
• Correctly identify all the ABO compatible
blood components for each blood type
4
5
Co-Dominance
1. A “Big” letter doesn’t mean dominant
2. A “small” letter doesn’t mean recessive
3. The exceptions to #1 and #2 are:
– The Secretor genes: Se and se
– The Lewis genes: Le and le
– The H genes: H and h
6
Inheritance of the ABO
Blood Groups
• First described by Bernstein in 1924.
• A, B, O genes on chromosome #9
• The expression of antigens are based on
the combination of three gene alleles: A,
B, and O.
7
Phenotypes vs Genotypes
Phenotypes
Group O
Group A
Group B
Group AB
Genotypes
OO
AA or AO
BB or BO
AB
8
ABO Phenotype
Frequencies in U.S.
Populations
Phenotype
O
A1
A2
B
A1B
A2B
White
45
33
8
10
3
1
Blk
49
12
8
19
3
1
Mexican Asian
56
43
22
27
6
Rare
13
25
4
5
Rare
Rare
9
B Allele World Map
10
Exercises
• Mother is type A and father is type O:
What are the possible blood types for their
offspring?
• Mother is type A and father is type B: What
are possible blood types for their
offspring?
11
12
Basic Biochemistry
•
•
•
•
•
Type I and Type II chains
Se gene
H gene
Formation of the H antigen
Formation of the A and B antigens
13
14
Type I and Type II chains
Type I: primarily glycoproteins in secretions
and plasma
• Saliva, clostrum, mothers’ milk, gastric fluid, bile,
urine, serum, plasma, ovarian cyst
Type II: primarily glycolipids on RBCs
• RBC, WBC, platelets, normoblast, sperm,
epidermal and epithelial cells
15
Type 1: Secretor ABH
Glycoprotein
Substances
16
Se Gene and Formation of
the H Antigen
• Secretor = SeSe, Sese; Nonsecretor =
sese
• 80% of random population is either SeSe
or Sese
• Secretor gene codes for fucosyl
transferase
• Enzyme (FUT2) adds fucose to type I
chains at terminal galactose; product is H
antigen.
17
18
H Gene and Formation of
the H Antigen
• Phenotypes: HH, Hh, hh
• Virtually 100% of random population is either
HH or Hh; hh genotype (lack of H =“Bombay
phenotype”) is rare.
• H gene also codes for fucosyl transferase
(FUT1)
• Enzyme (fucosyl transferase) adds fucose to
terminal galactose of type II chains
• Final product is H antigen
19
Formation of the H
Antigen
20
Type 2: H, A, and B Antigens
• H Ag:
Gal–GlcNAc–Gal-X
|
Fuc
• A Ag:
GalNAc–Gal–GlcNAc–Gal-X
|
Fuc
• B Ag:
Gal–Gal–GlcNAc–Gal-X
|
Fuc
21
“A” Gene and Formation of
the A Antigen
• H antigen is required for A antigen
formation on RBCs or in
secretions/plasma
• Formation of A antigen:
N-acetylgalactosamine is added to H
antigen to make A antigen.
• A Ag
22
Formation of the A Antigen
23
“B” Gene and Formation
of the B Antigen
• H antigen is required for B antigen
formation on RBCs or in
secretions/plasma
• Formation of B antigen: D-galactose is
added to H antigen to make B antigen.
24
Formation of the B
Antigen
25
The Residual H Antigen
• The more A or B antigen is made, the less
H remains
• Relative amounts of H by blood group
O>A2>B>A2B>A1>A1B
26
The Use of Lectins for
Antigen Confirmation
• Dolichos biflorus = anti-A1
• Ulex europaeus = anti-H
27
Questions?
• How is H antigen formed?
– Relationship of Type I chain and Se gene
– Relationship of Type II chain and H gene
• How are A and B antigens formed?
• What blood type has the highest amount
of H antigen? What blood type has the
least amount of H antigen? How would
you determine that?
3/16/2016
28
29
ABO Antigens and
Antibodies
• ABO antigens based on combinations of
three genes: A, B, and O
• Antibodies are clinically significant and
“naturally occurring”
– causing most fatal acute HTRs
– some causing HDFN
• ABO antibodies neutralized with secretor
saliva.
30
Group O
• Generally the most common blood
group
• Genotype: OO
• Antigen: H
• Antibodies: anti-A, anti-B, and anti-A,B
– Antibodies are naturally occurring and very
strong
– Anti-A,B (mostly IgG) may cross placenta
to cause HDFN
31
Group A
•
•
•
•
Genotype: AA, AO
Antigen:
A, H
Antibodies: anti-B (primarily IgM)
A subgroups
– A1 (80%) and A2 (20%) most important
– A1 has more A than A2 (quantitative
difference); qualitative differences, too.
– ~25% of A2B’s form anti-A1
– 1-8% of A2’s form anti-A1
– Lectin of Dolichos biflorus agglutinates A1
32
Group B
•
•
•
•
Genotype: BB, BO
Antigen:
B, H
Antibodies: anti-A (primarily IgM)
B subgroups:
Not important
33
Group AB
•
•
•
•
•
Genotype: AB
Antigen:
A, B, very little H
Antibodies: None
B subgroups:
Not important
A2B:
25% of A2B individuals
produce anti-A1
3/16/2016
34
35
ABO Testing
• Cell typing (forward grouping) to
determine antigen types on RBCs
• Serum/plasma typing (reverse grouping
or backtyping) to determine type of
antibody in serum:
• Note the opposite reactions
– If the forward reactions are opposite of
reverse, an ABO discrepancy is not
present.
36
ABO Grouping Reagents
• Forward Grouping Reagent
• Reverse or Back Tying Cells
37
Forward Grouping Reagent
38
Forward Grouping
• Reagent: Monoclonal antibody
– Highly specific
– IgM
– Expected 3+- to 4+ reaction
– 1 drop
– Anti-A=Blue; anti-B=Yellow (Acroflavin dye)
• A and B antigens on patient red cells are
agglutinated by known sera (anti-A, anti-B)
39
Reverse or Back Tying
Reagent Cells
40
Reverse or Back Typing
• Reagent Cells: Human Source
– Expected 2+ to 4+ reaction
– 4-5% cell suspension
– 1 drop
• Anti-A or anti-B antibodies in patient serum
(or plasma) agglutinate with A1 and B
antigens on Reagent cells
41
Forward Typing
Procedures
• To determine what antigens are present on
RBCs.
42
Step 1. Label test tubes.
43
Step 2: Make a 2-5%
patient red cell suspension.
44
Step 3: Add reagent
antisera (1 drop).
45
Step 3A: Add reagent Anti-A
antisera
(1 drop).
46
Step 3B: Add Anti-B
reagent antisera (1 drop).
47
Step 4: Add one drop of 2-5%
suspension of patient RBC to each
tube.
48
Step 5: Mix and centrifuge
(approximately 20 seconds).
49
Group A: 4+ Agglutination with
Anti-A
0 Agglutination with Anti-B
50
Group B: 4+ Agglutination with
Anti-B
0 Agglutination with Anti-A
51
Group AB: 4+ Agglutination
with Anti-A and Anti-B
52
Group O:
No Agglutination with Anti-A or
Anti-B
53
Back Typing
• To determine what antibodies are present
in patient’s plasma.
54
Step 1: Label Test Tubes
55
Step 2: Add two drops of
patient serum to each tube
56
Step 3: Add one drop of
reagent cells to each test tube
57
Step 3A: Add one drop of
Reagent A1 cells
58
Step 3B: Add one drop of
Reagent B cells
59
Step 4: Mix and centrifuge
(approximately 20 seconds)
60
Group A: 4+ Agglutination with
B Cells
0 Agglutination with A1 Cells
61
Group B: 4+ Agglutination with
A1 Cells
0 Agglutination with B Cells
62
Group O: 4+ Agglutination with
A1 Cells
3+ Agglutination with B Cells
63
Group AB:
No Agglutination with A1 and B
Cells
64
Exercises
• Interpretation of test results
65
Exercises: Interpretation of
ABO Testing Results
Forward
Reverse
Interpretation
anti-A Anti-B
A1 cells B cells
Group
4+
0
4+
0
0
4+
4+
0
0
4+
0
4+
4+
0
0
4+
ABO
??
??
??
??
66
Exercises: Interpretation of
ABO Testing Results
Forward
Reverse
Interpretation
anti-A Anti-B
A1 cells B cells
Group
4+
0
4+
0
0
4+
4+
0
0
4+
0
4+
4+
0
0
4+
ABO
A
B
AB
O
67
68
What can Cause ABO
Discrepancies?
• Disagreement between the
interpretations of forward and
reverse grouping
• Antigen problems
• Antibody problems
69
Antigen Problems
• Lack of expected antigens
– A subgroup
– B subgroup
– Bombay
• Presence of unexpected antigens
– Acquired B phenotype
– Polyagglutinable RBCs, recent marrow
transplant, nonspecific agglutination
70
Antibody problems
• Lack of expected antibodies
– Immunodeficiency, neonates, abnormally high
concentrations of Ab (prozone)
• Presence of unexpected antibodies
– Anti-A1, cold auto-or alloantibodies, rouleaux
(false positive)
71
A Subgroups
• A1
• A2
• A3
• Ax
• Aend
• Am
• etc
72
A1 vs A2 Phenotypes
Blood Group
A1 (80%)
A2 (20%)
Anti-A
+
+
Anti-A1 lectin
+
0
• A1 & A2 account for 99% of A group
73
A1vs A2 Phenotypes
• Quantitative differences: More antigenic
sites on A1 than A2.
• Qualitative differences between A1 and
A2 antigens:
– 1-8% of A2 individuals produce anti-A1
– 25% of A2B individuals produce anti-A1.
74
B Subgroups
• Very rare and are less frequent than A
subgroups.
• B subgroups demonstrate variations in
the strength of the reaction using antiB and anti-A,B
• Examples are: B3, Bx, Bm, Bel
75
Acquired B phenotype
• Occurs in type A individuals with:
–Colon cancer, intestinal
obstruction, gram negative sepsis
• Bacteria deacetylate group A sugar
(GalNAc); remaining galactosamine
crossreacts with reagent anti-B.
76
Acquired B phenotype
77
Acquired B phenotype
• AB forward (with weak reactions with
reagent anti-B)
• A reverse
• Reaction with anti-B is negative, if:
– Acidify serum
– Acetic anhydride treatment
– Auto incubation
78
Acquired B typing result
Forward
Anti-A
Interp
Anti-B
4+
1-2+
A”B”
Reverse
Interp
AB
A1 cells B cells
0
4+
79
Bombay (Oh) Phenotype
• Total Lack of H, A, and B antigens
• Develop strong anti-H, anti-A, and
anti-B
• “O” forward, “O” reverse; with positive
antibody screen
• Require other Bombay donors for
blood transfusion
• (“Para-Bombay” = H antigen in
secretions)
80
Reactivity of Anti-H with
ABO Blood Groups
O>A2>B>A2B>A1>A1B
81
Blood Type:
Antigens vs Antibodies
Blood Type
A
B
AB
O
Antigens
on rbcs
A
B
A,B
None
Antibodies
in Plasma
Anti-B
Anti-A
None
Anti-A, Anti-B
82
Exercise
Blood
Compatible Compatible
Compatible
Type
RBCs
FFPs
Whole
Blood
A
B
AB
O
___
___
___
___
___
___
___
___
___
___
___
___
83
ABO Compatible
Blood Components
Blood
Type
Compatible
RBCs
Compatible
FFPs
A
B
AB
O
A, O
B, O
AB, A, B, O
O
A, AB
B, AB
AB
A, B, AB, O
84
ABO Compatible Whole
Blood
Blood type
A
B
AB
O
Compatible WB
A
B
AB
O
85
Consequences of
ABO incompatibility
• Severe acute hemolytic transfusion
reactions
– One of the most frequent causes of blood
bank fatalities
– Clerical errors
• Most frequent HDFN; usually mild.
86
Sources of Technical Errors
Resulting in ABO Discrepancies
•
•
•
•
•
•
•
•
•
•
Inadequate identification of blood samples
Cell suspension too heavy or too light
Clerical errors
A mix-up in samples
Missed observation of hemolysis
Failure to add reagents
Failure to follow manufacturer’s instructions
Uncalibrated centrifuge
Contaminated reagents
Warming during centrifugation
3/16/2016
87
Resolving ABO Discrepancies
Problems with RBCs
Resolution
Techniques
Rouleaux
wash RBCs 4X
MF agglutination
check tx hx
Unusual phenotype (hh) Test with anti-H
Disease processes (Acq. B)
check patient
diagnosis
88
Resolving ABO
Discrepancies (Cont’d)
Problems with serum
Resolution
Techniques
Rouleaux
Saline replacement
Presence of unexpected Ab
Do panel to ID
Absence of expected Ab Increase incubation time
89
Objectives
• Describe the inheritance of the ABO Blood
Groups and predict the ABO phenotypes and
genotypes of offspring from various ABO
matings
• Explain the roles of Secretor and H genes in the
formation of H, A, and B antigens on the red
cells
• Describe the reciprocal relationship between
ABO antigens and antibodies for blood types O,
A, B, and AB.
90
Objectives (Continued)
• Describe the procedures on ABO forward
and back typings; interpret the results; and
resolve any discrepancies if present.
• Describe the quantitative and qualitative
differences between the A1 and A2
antigens.
• Correctly identify all the ABO compatible
blood components for each blood type
91
Reference Materials:
1.
2.
3.
4.
5.
6.
Modern Blood Banking And Transfusion Practices, or 5th Edition
Denise M. Harmening. March 2005 . F.A. Davis. Philadelphia PA.
Textbook of Blood Banking and Transfusion Medicine, Sally V.
Rudman. February 2005. W.B Saunders. Philadelphia PA.
Transfusion Medicine Interactive: A Case Study Approach .
Marian Petrides MD, Nora Ratcliffe MD, and Roby Rogers MD.
2004. AABB Press Bethesda, Maryland.
Transfusion Reactions, 2nd Edition. Mark A. Popovsky (Editor).
AABB Press 2001. Bethesda, Maryland.
American Association of Blood Banks Technical Manual (AABB)
14th Edition , 2003. American Association of Blood Banks, 8101
Glenbrook Road, Bethesda, Maryland.
Standards for Blood Banks and Transfusion Services 23rd
Edition, 2004. Standards Committee, AABB. Bethesda, Maryland.
92