ABO Blood Group System

Janet Vincent with thanks to

Bettina Turner, Camellia St. John
and Clare Wong
 Terms-Flashcards
 Forward  Type 1 versus type 2
 Reverse  Lectins
 Codominant  Phenotype
 Amount of H  Genotype
 A antigen  amorph
 B antigen
 H antigen
 General OBJECTIVES
 Describe the reciprocal relationship between
ABO antigens and antibodies
 Identify the frequencies of the four major blood
types
 Explain the effect of age and disease on the
production of ABO antigens and antibodies.
 Describe the characteristics of the Bombay
phenotypes
 Interpret the results for an ABO typing and
resolve any discrepancies.
 Blood Group Systems
 Hundreds of antigens on red cells
 ABO most important
 Reciprocal antibody consistently present
 History
 1900 Landsteiner
 A, B, O
 1902 von Descastello and
Sturli
 AB
 "Forward" Typing
 "Reverse" Typing
 Genetics of ABO system
 List the different phenotypes and
genotypes in the ABO System
 Genetics
 Antigens made on structurally related
carbohydrate molecules
 Genes at 3 separate loci control where
expressed
 ABO,
 Hh,
 Sese
 Glycosyltransferases and
immunodominant sugars
responsible
Gene Glycosyltran Immunodo Antigen
sferase minant
Sugar
H(FUT1) Alfa-2-l L-fucose H
fucosyltransfe
rasa
A ALFA-N- N-actyl-D- A
acetylgalactos GALACTOSAM
aminyltransfer INE
asa
B Alfa-3-D D-Galactose B
galactosyltran
sferase
 Genetics
 Four Major alleles
 A1, A2, B, O
 Chromosome 9
 Six Major Phenotypes
 A1, A2, B, O, A1B, A2B
 Genetics
Phenotype Genotype
A1 A1A1, A1A2, A1O
A2 A2A2, A2O
B BB, BO
A1 B A1 B
A2 B A2 B
O OO
 ABO Genes
 Codominant
 enzymes attach
sugars

Harmening – page 112

 H antigen
 H/h genes
 H common gene

 Fucosyl transferase

 h rare gene - amorph

 Homozygous hh

Bombay (Oh)
 H antigen

Harmening, page 112

 Precursor Substance 2
(RBC’s only have Type 2)

Gal Gluc RBC

Gal GlcNAc
H gene

Fucosyl Transferase Fuc H Antigen

 ABO Genes
 O gene
 Amorph
 No detectable product
 A antigen
A gene
 Enzyme

 N-acetyl galactosyl
transferase
 Sugar
N-acetyl galactosamine
A antigen

Harmening, page 113

 Precursor Type 2
H Antigen

A Gene
Gal Gluc RBC
Gal GlcNAc
GalNAc Transferase GalNAc

Fuc A Antigen
 B antigen

B gene
 D-galactosyl transferase
 Sugar

 D-galactose
B antigen

Harmening, page 113

 Precursor Type 2
H Antigen

B Gene
Gal Gluc RBC
Gal GlcNAc
Gal Transferase
Gal

Fuc B Antigen
 Precursor Type 2
H Antigen

A Gene Gal Gluc RBC

Gal GlcNAc
GalNAc Transferase GalNAc

Fuc A Antigen

B Gene RBC
Gal Gluc
Gal GlcNAc
Gal Transferase Gal

B Antigen
Fuc
Antigens in Secretions
 Secretor vs. Nonsecretor
 Se/se genes
 Closely linked to H
 Directly responsible for H substances in
secretions
 Indirectly responsible for A,B, H
substances in secretions
 ABO RBC & SECRETORY Ag’s

• RBC’s ANTIGENS can be glycolipids,

glycoproteins or glycosphingolipids
with a terminal (end of chain) sugar of
Galactose
VS.
• In Secreted substances are
glycoproteins with a terminal sugar of N-
acetyl-D-galactosamine
27
 TYPE 1 & 2 CHAINS

• Type 2 Chains (only) on RBC’s where

– #1 carbon of galactose at end of chain is
attached to
– #4 carbon of the N-acetyl-D- glucosamine in the
precursor substance
• Type 1 & 2 Chains in the secretions.
Type 1 predominates where
– #1 carbon of D-galactose is attached to
– #3 carbon of the sub- terminal N-acetyl-D-
glucosamine
28
 Rbc antigens vs secretors
• Enzyme produced from by the H(FUT 1)
gene (alfa-2-L- fucosyltransferase) acts
primariy on type 2 chains,which are
prevalent on the RBC membrane

• Enzyme produced by the se(FUT 1)

gene( alfa-2-L fucosyltransferase)
preferentially acts on type 1 chains in
secretory tissues
Type 1 and Type 2 Chains
Precursor Substance 1
(found mainly in secretions)

Gal NAcGal Secrete

GlcNAc
Gal
Se gene

Fucosyl Transferase H Antigen in Secretions

Fuc
Precursor Substance 2
(RBC’s only have Type 2)
Gal Gluc RBC
Gal GlcNAc
H gene

Fucosyl Transferase Fuc H Antigen on RBC

Precursor Substance 1
(found mainly in secretions)
Gal NAcGal Secrete
GlcNAc
Se gene Gal

Fucosyl Transferase Fuc H Antigen in Secretions

 ABH Substance in the saliva of
secretores(SeSe or Sese)
ABO GROUP A B H

O None None 2+

A 2+ None 1+

B None 2+ 1+

AB 2+ 2+ 1+
 Expected Findings
Blood Antigen(s) Expected Serum Genotype(s)
Group Present Antibody
(Forward) (Reverse)
A A Anti-B AA or AO

B B Anti-A BB or BO

AB A,B AB

O Anti-A,-B,-A,B OO
 What are the percentages in
Guyana?
Race
Blood Group Whites African Americans
O 45% 49%
A 40% 27%
B 11% 19%
AB 4% 4%

Harmening, Fifth Edition . Table 6 – 5, p111

 ABH on Red Cells
 Type 2 chains
 Highly branched
 Infants – linear structures
 Detect some antigen at 5 weeks gestation
 Less than 50% adult antigens at birth
 Cannot determine subgroups until around
3 years of age
 Blood Group A
 Normally detectable anti-B
 Subgroups of A
A1 and A2


Genotype Phenotype Genotype Phenotype

A1 O A2 O
A1 A1 A1 A2 A2 A2
A1 A2
 Distinguishing A subgroups
 A1 subgroup reacts with Anti-A1 lectin
 Dolichos biflorus
 Subgroups other than A1 do not react with
Anti-A1 lectin
 A1 and A2 most common subgroups
 A3 gives a mixed field reaction with anti-A
and anti-A,B
 A1 Versus A2 phenotypes
Reaction of patients RBCs
Bld group ANTI-A Reagent Anti Anti1 lectin Reagent
A plus Anti-A1

A1 + +

A2 + 0

A1 + +

A2 + 0
• Anti- A1 formed by
– about 25% of the Gr. A2B’s and
– less commonly by Gr. A2 people.
• Suggests that Anti-A1
– is more highly specific
– (or a weaker Ab with a narrower specificity)
• Use Lectin as Reagent Anti-A1
– Dolicos biflorus is
– a plant lectin containing a substance that
– behaves “as if” it were Anti-A1
 Distinguishing Subgroups of A
 Degree of agglutination by anti-A,-A1
 Degree of agglutination by anti-A,B
 Degree of agglutination by anti-H
 Presence/absence of anti-A1 in serum
 Presence A/H substances in saliva of
secretors
 CELL REACTIONS OF SUBGROUPS OF A

A SUBGROUP ANTI-A ANTI-A,B ANTI-H ANTI-A1 LECTIN

A1 ++++ ++++ ++ ++++

A2 +++ +++ +++ neg

A3 MF MF +++ neg

Aint +++ +++ +++ ++

Ax neg + +++ neg

Am +/- +/- +++ neg

 Group B

 Detectable anti-A expected

 Anti-A and anti-A1
 Subgroups of B
 Infrequent
 Distinguish similarly to A subgroups
 B3 mixed field
 Bx, Bm, Bel
 Anti-A & Anti-A1

• Gr. B’s produce a mixture of

– Anti-A & Anti-A1
• Anti-A appears to be a broader
specificity Ab (or stronger Ab)
– reacts with A1, A2, & A3 cells
• Anti-A1 ONLY reacts with A1 cells,
– not with A2, or A3 cells
– Produced by some A2B’s
 Group AB
 A gene
 B gene
 Cis AB
 Rare
 Unequal crossing-over
 Inheritance of both A
and B gene from one
parent
 Offspring receives 3
ABO genes
 Anti-H

• Produced by Bombay individuals who

– lack the H, A, & B Ag’s
• Can also be produced by A1B
individuals
– who have converted almost all their
H Ag to A & B Ag’s
• Ulex europeaus is
– a plant lectin containing a substance that
– acts as if it were Anti-H
 Amount of H

O>A2>B>A2B>A1>A1B
 Naturally occurring Antibodies
 A antigens: ? Antibody expected
 B antigens: ? Antibody expected
 No A or B antigens: ? Antibody expected
 Both A and B antigens: ? Antibody
expected
 Unexpected ABO Antibodies
 Anti-A1
 1-8% A2
 22-35% A2B
 Anti-H
 “Naturally Occurring”
 Testing for ABO blood group
 Outline the procedure for Forward and
Reverse testing
 Recognize the expected reactions for the 4
blood groups
 List the reagents and samples needed for
ABO typing.
 Reagents
 Common ABO Antisera
 Anti-A
 Anti-B
 Anti-A,B
 Reagents-Lectins

Anti-H
 Anti- A1 lectin
(Dolichos biflorus) (Ulex europaeus)
Monoclonal
Antibodies
 Mouse immunized, spleen removed, antibody-
producing B cells harvested.
 Fusion with myeloma cells
 Selection/testing process
 The hybridoma cell is patented and frozen
 Advantages
 Very specific; very homogeneous
 Initially expensive, eventually cheaper
 Disadvantages
 Too specific – antibody to a single antibody to a
single epitope
 Some Monoclonal antibodies are pH dependent
 Some will cross react with other antigens; must
dilute the antibody.
 RULE
 KNOWN + UNKNOWN = Reaction
 X+Y=Z
 What is known
 What is unknown
 ABO GROUPING

• Forward • Reverse Grouping

Grouping – What Ab(s)
– What Ag(s) Present
Present – What’s Unknown?
– What’s Unknown? – What is Known
– What is known?
 Antigen Typing
“Forward”
 1 drop of antibody (anti-A or anti-B) and
 1 drop of 3-5% suspension of rbcs
 Mix and centrifuge

 Agglutination = presence of antigen

 No agglutination = absence of antigen
 Gel Typing example
 Negative
 1+
 2+
 3+
 4+
 Antibody Typing
“Reverse”
 Two drops of plasma or serum and
 One drop of reagent A1 rbcs or B rbcs
 Mix and centrifuge

 Agglutination = presence of antibody

 No agglutination = absence of antibody
Reagent Cells
 ABO Reagent Cells
 A1 cells
 A2 cells
 B cells
 Expected ABO Reactions

Anti-A Anti-B Forward A1 cells B cells Reverse Group

Interp Interp
4+ - A - 4+ A A
- 4+ B 4+ - B B
- - 0 4+ 4+ O O
4+ 4+ AB - - AB AB
 RULES
 Each test result must be recorded as
observations are made.
 Must always do QC on reagents each day
of use.
 Must always have a negative reaction on
Forward grouping
 Others?
 Review
 ABO antibodies are not present at birth
 “Naturally occurring”
 Most ABO antibodies are IgM
 Group O is more likely to have an IgG
component
ABO Discrepancies
 Objectives
Recognize ABO discrepancies and list some possible
causes.
 Discuss the initial steps to resolve an ABO

discrepancy.
 Identify the steps to resolve the following ABO

discrepancies:
- Asubgroup with anti-A1
- Cold alloantibody such as anti-M, -P1
- Cold autoagglutinin such as autoanti-I
What is an ABO Discrepancy?

Results of red cell tests

do not agree with the serum tests
 Unexpected positive or negative results
 Weaker or mixed-field results as defined by
the laboratory’s SOP
 Technical Problems
 First step in resolving any ABO
Discrepancy – Repeat the test on
the same sample.
 Common Technical Errors
 Improper identification of samples, tubes
 Sample mix-up
 Clotting Deficiency
 Cell suspension too heavy or too light
 Clerical errors
 Reagent not added
 Manufacturer’s directions not followed
 Hemolysis overlooked
 Reagent contamination
 Improper temperature used
 Improper centrifugation
 False Negatives
 Failure to add serum or antibody
 Inappropriate ratio
 Under centrifugation
 Incubation above RT
 Inactive reagents
 Improper interpretation/recording results
 False Positives
 Over centrifugation
 Contaminated antisera, red cells, saline
 Dirty glassware
 Incorrect recording/Interpretation of
results
 Categories of ABO
descrepancies
 Group I-IV
 Descrepancy I
 Unexpected reaction in the reverse grouping
 Due to weak or missing antibodies in the
reverse
 Normal RBC and serum are usually strong
reaction(4+)
 Dpressed antibody production or cannot
produce the ABO Antibodies
 Descripancy 1 example
Anti A Anti B A1 cells B cells
0 0 0 0
 Descrepency II
 Unexpected reactions in the forward
grouping
 Due to weakly or missing antigens
 Least frequent
 Example of the acquired B
ANTI-A Anti-B A1 CELLS B CELLS
4+ 2+ O 0
 Descrepancies III
 Between forward and reverse
 Cause by protein and plasma
abnormalities result in puedoagglutination
 Descrepancies IV
 DESCREPANCY BETWEEN FORWARD AND
REVERSE GROUPINGS
 DUE TO MISCELLANEOUS PROBLEMS
 ABO Discrepancies
 Red cell Mediated
 Subgroups
 Chimera
 Polyagglutination
 Substances in serum or plasma
 Reagents
Red Cell mediated ABO Discrepancy

 Chimera
 Two cell populations
 Polyaggultination
 exposure of “Crypt”/buried Ag’s
 Weakened ABO antigens

 Disease:
 Leukemia
 stomach or pancreatic carcinoma
 Subgroup
 ABO Discrepancies
 Serum Mediated
 Subgroups
 Alloantibodies
 Autoantibodies
 Rouleaux
 Transfusion of Non –ABO Identical Plasma Products
(Plts)
 Age
 Disease
 Reagents
 UNEXPECTED Ab’S

• Cold Agglutinins - commonly Anti-I

• Unexpected Ab’s (non-RBC immune):
– anti-H
– anti-I, anti-P, anti-H, anti-M, anti-N,
anti-Lea, anti-Leb
• Unexpected Ab’s (strong RBC
immune):
– strong/high titered IgG class Ab
– for example - a Rh Ab
83
 ROULEAUX

• Cells aggregate & mimic’s

agglutination
• volume expanders (Dextran, PVP)
• Hypergammaglobulinemia
• Confirm by “Saline Replacement”
– gently removing serum without
disturbing RBC button &
– gently replace with saline - then re-
suspend
84
 Examples of ABO
Discrepant Results
Anti-A Anti-B Forward A1 B Reverse Blood
interp cells cells interp Group

= = O = 4+ A

4+ = A 2+ 4+ O

= 4+ B = = AB

4+ 4+ AB 4+ 4+ O
 ABO Discrepancies
General Indicators
 Strength of reactions

 Previous records
 Controls Necessary
 Autologous (Auto)  Group O Cells (Screening
 Patient plasma (serum) Cells)
and patient red blood  Supplied by manufacturer
cells with a list of antigens
 If positive: present mixed with
- ? Auto antibody patient’s plasma or serum
 If positive = possible Allo
enhanced
antibody
- cells coated with
(Ab other than anti-A or
allo antibody
anti-B)
 Examples
Anti-A Anti-B Forward A1 B Reverse
interp cells cells Interp

Patient 1 4+ = A = = AB

Patient 2 = = O = = AB
 To resolve:

1. Repeat testing

2. Incubate reverse group at Room Temp or

at 4C for up to 30 minutes with group O
cells and Auto Control

(Incubation may enhance other common cold antibodies such as anti-I)

 Resolved
Anti-A Anti-B A1 B O Auto
cells cells cells Cont
Pt. 1 4+ = = =

Pt.1 4+ = = 2+ = =
15’ 4C
Pt. 2 = = = =
Pt. 2 = = 2+ 2+ = =
15’ 4C
 NOT Resolved
Anti-A Anti-B A1 B O Auto
cells cells cells
Pt. 1 4+ = = =

Pt.1 4+ = 2+ 2+ 1+ 1+
15’ 4C
Pt. 2 = = = =
Pt. 2 = = = = = =
15’ 4C
 Examples
Anti-A Anti-B A1 cells B cells

Pt. 1 = = O = 4+ A

Pt. 2 = = O = 4+ A
 To resolve
 If due to weak reacting antigen
 Incubate washed red cells with Anti-A, Anti-B
and Anti-A,B for up to 30 min at RT or 4C.
 If due to excess soluble blood group
substance
 Wash patient red cells and repeat forward
typing.
 Resolution
Anti-A Anti-B A1 cells B cells O cells Auto

Pt. 1 = = = 4+

Pt. 1 2+ = = 4+ = =
15’ 4C
Pt. 2 = = = 4+

Pt. 2 4+ = = 4+
wash
 Unexpected Forward Reactions
 Antibodies (other than anti-A or anti-B) reacting
 Polyclonal antisera contains
 Repeat with a different lot number or monoclonal
 Bystander Agglutination due to antigen-antibody
complexes
 Acriflavin/Sodium caprylate
 Reaction between antibody to additive and additive
 Acquired B

Example:
Anti-A Anti-B A1 cells B cells
4+ 1+ - 4+

To Resolve:
Repeat with monoclonal anti-B
Acidify polyclonal anti-B
 Wharton’s Jelly
 Contamination of cord blood sample
 Gelatinous intercellular substance
consisting of primitive connective tissue of
the umbilical cord
 Rich in hyaluronic acid
 Wash sample multiple times and repeat
 Obtain a heel stick sample
 Unexpected Reverse Reactions
(Antibody)
 Anti-A1
 Found in subgroups of A other than A1
 Most commonly A2 subgroup
Example 1:
Anti-A Anti-B A1 cells B cells
4+ - 1+ 4+
Example 2:
Anti-A Anti-B A1 cells B cells
4+ 4+ 1+ -
 Anti-A1

Example :
Anti-A Anti-B A1 cells B cells
4+ - 1+ 4+

 Could this be Anti-A1 reacting with A1 cells?

 Can this person make anti-A1?

 Anti-A1
Example :
Anti-A Anti-B A1 cells B cells
4+ - 1+ 4+

Test red cells with anti-A1 lectin.

 If positive the red cells have A1 antigen and

the patient cannot make anti-A1.
 If negative, the red cells are a subgroup of A
(A2) and the patient could make anti-A1.
 Anti-A1
Example :
Anti-A Anti-B A1 cells B cells
4+ - 1+ 4+

Anti-A1 lectin
-

The red cells lack the A1 antigen so this person

could make anti-A1.Must prove statistically.
 Anti-A1
Example :

Anti-A Anti-B A1 cells B cells

4+ - 1+ 4+

Anti-A1 lectin
-

Must test the SERUM/PLASMA with additional

lot #’s of red cells to confirm that the
antibody present is anti-A1.
Test with additional A1 cells, A2 cells, O cells
and an auto control.
 Anti-A1
Example :

Anti-A Anti-B A1 cells B cells

4+ - 1+ 4+

Anti-A1 lectin
-

A1 cells A2 cells O cells auto control

1+ - - -

CONCLUSION: ONLY the A1 cells reacted, so the

antibody is confirmed to be anti-A1.
 Unexpected Reverse Reactions
 Alloantibodies
 Autoantibodies

Example:
Anti-A Anti-B A1 cells B cells
- 4+ 3+ 2+

Again, note strength of reactivity

 Unexpected Reverse Reactions
Example:
Anti-A Anti-B A1 cells B cells
- 4+ 3+ 2+

Test with O cells and auto control.

O cell(I) O cell(II) O cell(III) auto

Allo ab - 2+ 2+ -
Auto ab 2+ 2+ 2+ 2+
 Unexpected Reverse Reactions
 Possible Allo antibodies
 Anti-M, Anti-P1, Anti-N
 Test reverse group cell for antigen
 Test serum against antigen negative cell
 Possible Auto antibodies
 Anti-I, Anti-H, Anti-IH
 Warm
 Cold autoadsorption/Rabbit erythrocyte stroma
 Unexpected Reverse Reactions
 Passively Acquired Antibodies
 IVIg (may contain ABO antibodies)
 Group O platelet concentrates

Example:
Anti-A Anti-B A1 cells B cells
3+ - 1+ 3+

Check transfusion history.

 Unexpected Reverse Reactions
 Rouleaux Formation
 Abnormal high concentration of proteins
 Altered serum protein ratio
 Recipient of plasma expanders of high
molecular weight (dextran)
 Elevated levels of fibrinogen
 Rouleaux

Example:

Anti-A Anti-B A1 cells B cells

- 4+ 3+ 1+
Rouleaux?
Check microscopically - “stack of coins”
Try saline replacement of reverse group.
 Rouleaux
Example:

Anti-A Anti-B A1 cells B cells

- 4+ 3+ 1+

Resolution: A1 cells B cells

Saline replacement: 3+ -
Rouleaux was present – Resolved
 Unexpected Reverse Reactions

Sample contains antibody to chemical

constituents of the diluents used to preserve
A or B red cells.

Wash reagent A or B red cells and retest.

 ABO DISCREPANCY
(FORWARD AND REVERSE DO
NOT AGREE)
 R e s o lv in g A B O D is c r e p a n c ie s

M is s in g R e a c t io n s
R e v e rs e
A n t ib o d y
C h e ck A ge

N e w b o rn E ld e r ly O th e r
E x p e c te d A g e r e la te d ? D ia g n o s is ?

In c u b a te In c u b a te
R e v e rs e G ro u p R e v e rs e G ro u p
w i t h C o n t r o ls w i t h C o n t r o ls
 R e s o l v in g A B O D i s c r e p a n c i e s

M is s in g R e a c t io n s
F o rw a rd
A n tig e n
C h eck A ge

N e w b o rn O th e rs
W e a k e r R e a c t io n s E x p e c t e d D ia g n o s is ?
N o fu rth e r w o rk u p
( U n le s s m ix e d f ie ld )

H e a lt h y C ancer

W e a k S u b g ro u p L e u k e m ia E xce ss G S S
In c u b a te W e a k e n e d A n tig e n W a s h c e l ls
w i t h c o n t r o ls In c u b a te R e ty p e
w i t h c o n t r o ls

N o t R e s o lv e d
In c u b a te
w i t h c o n t r o ls
 R e s o lv in g A B O D is c r e p a n c ie s
E x t r a R e a c t io n s
R e v e rs e

F o rw a rd s a s F o rw a rd s a s
A or AB A1, A 1B or B
R o u le a u x ?

T e s t w i t h A n t i - A 1 l e c t in S a lin e R e p la c e m e n t

A s u b g ro u p A1 or A1 B N o t R e s o lv e d R e s o lv e d
A s u b g o ru p B

C o n f ir m A n t i- A 1 A n t ib o d y S c r e e n
( A 1 c e lls , A 2 c e lls , O c e lls ) ( O c e lls + a u t o )
If not confirmed
 A B O D is c r e p a n c ie s
E x t r a R e a c t io n s
F o rw a rd
D ia g n o s is ?

A n e m ia C o lo n C a n c e r H e a lt h y
B a c ti I n f e c t io n

A u to a n t ib o d y ? P o ly a g g lu t in a t io n A n tib o d y to E x t r a A n tib o d y N e w b o rn
A d d it iv e in S e r a W h a r t o n 's J e l ly

W a r m s a lin e w a s h M o n o c lo n a l T r y N e w L o t# T ry N e w L o t # W a s h C e ll s a n d
T r e a t w it h D T T / 2 M E o r M o n o c lo n a l o r M o n o c lo n a l R e ty p e
H e e ls t ic k

R e pe a t T ype
 Terms-Flashcards
 Forward  Type 1 versus type 2
 Reverse  Lectins
 Codominant
 Amount of H
 A antigen
 B antigen
 H antigen