ABO blood group system

Daw Pyae Mon Kyaw
Department of Medical Laboratory Technology
University of Medical Technology, Mandalay
1

Discovery
 Karl Landsteiner(1990) discovered human A, B, O groups.
 Van Deacastello and Sturli (1902) discovered AB blood group.
 Von Sungern and Kirzsledl (1911) divided group A into 2 subgroups, A1 and A2.
ABO system is classified into 6 groups: A1, A2, A1B, A2B, B, AB and O.
3

Landsteiner’s Law
1. If an agglutinogen is present on red blood cell membrane, the corresponding agglutinin
must be absent in the plasma.
2. If an agglutinogen is absent on red blood cell membrane, the corresponding agglutinin
must be present in the plasma.
4

Antigen location
 Human antigens are found on cells, organs, and tissues as well as in plasma and
other body fluids.

protrude from cell surface
 Physical location - on cell membrane (eg. ABO ag)
integral part of the membrane
(eg. Rh Ag)
8

ABO antigen
 Blood group antigens are actually sugars attached to the red blood cell.
 Antigens are built onto the red cell.
 Individuals inherit a gene which codes for specific sugar to
be added to the red cell.
 The type of sugar added determines the blood group.
10

ABO Antigen
 Appear in the sixth week of fetal life and remain unchanged until death.
 Human red cells contain on their surface a series of glycoprotein and glycolipid 
blood group antigen
11

ABO Antibodies
Can be classified as
1. Naturally occurring and immune antibodies
Depending on presensitization
2. Complete and incomplete antibodies
Depends on aggulutination of saline suspended red cells
IgM is complete antibody, most naturally occurring antiboides are complete and of IgM class
Ig G is incomplete antibody
3. Cold and warm antibody
thermal range of antibodies
Most natural antibodies are cold and some e.g. wide thermal range like Anti A and Anti B
Most immune antibodies are warm and can destroy red cell in vivo
12

Naturally occurring antibodies
 Almost all normal healthy individuals above 3-6 months of age have “ Naturally
occurring antibodies” to the ABO antigens that they lack
 Peak at 5-10 yrs
 Decline progressively with advanced age.
 These Abs termed Naturally occurring because they were thought to arise without
antigenic stimulation.
 These naturally occurring Abs are mostly IgM class. That means that,they are Abs
capable of aggulutinating saline/low protein suspended red cell without
enhancement and may activate complement cascade.
13

Immune or acquired antibodies
 Produced as a result of stimulation by a red cell antigen which is not present on his
own red cell or in his body fluid.
 Arise from blood transfusion or the intravenous or intramuscular injection of blood
or injection of substances which are chemically closely related to a red cell antigen
or as a result of pregnancy.
 More often IgG
 Most importance in transfusion is IgG which can pass through the placenta during
pregnancy.
14

Complete antibody and incomplete antibody
Compete antibody
- produced without any antigenic stimulus
- most ofen IgM
- capable of aggulutinating red cells
suspended in normal saline at 20-25 C
Incomplete antibody
- requires a bridge like the Coomb’s sera
for bilnding to the antigenic site
- most IgG
15

Detection of antibodies
1. Saline aggulutination method
2. Albumin agglutination method
3. Test using enzyme treated cells
4. The indirect antiglobuliln (Coomb’s tsest)
17

Antigen-Antibody reaction
 Red cell Ag-Ab reaction can detected by a number of techniques
 Most frequently used
 Hemolysis
 Agglutination
 Used as indicator of Ag-Ab reaction
18

Agglutination Reactions
Two Stage Process:
Stage 1 Sensitization:
 attachment of Antibody to Antigen on the RBC membrane.
Stage 2 Lattice formation (agglutination):
 formation of bridges between the sensitized red cells to form the lattice that constitutes agglutination.
19

This represents what occurs during stage one of
agglutination.
• Antibody molecules attach to their corresponding
antigenic site (epitope) on the red blood cell membrane.
• There is no visible clumping.
• Red cells must be close enough for the Fab portion of
Ab to bind and make bridges between cells
Stage 1: Sensitization
20

Stage 2: Lattice Formation
This represents what occurs during stage 2 of agglutination:
Antibody molecules crosslink RBCs forming a lattice that
results in visible clumping or agglutination.
21

 Sensitization by IgG does not result in agglutination
 IgG is too small to span the distance between two red cells
 IgM can easily cause agglutination
 For agglutination to occur, the repulsive forces keeping red cells apart must be
overcome
22

The Zeta Potential
 The electric repulsion between cells
 This explains why cells do not agglutinate
 Red cells have negative charge due to
sialic acid molecules
 When red cells are in solution containing
free ions:
 Cations are attracted to the –vely charged red
cells
 This forms a repelling cloud around the cell
24

 The Zeta Potential can
be varied by altering the
charge on red cells
 This can affect both
sensitization and
agglutination
 Reducing the cloud
density allow Abs to
approach the cells,
sensitize and then
agglutinate them
25

3. Increase the ionic strength of the medium
 Increasing conc. of cations in medium cause
 Increase in the density of ions around the red cell which cause
 Size of cloud of cations is decreased
 Zeta potential decreases
 Red cell approach each other easily
 Agglutination is facilitated
27

4. Decreasing the ionic strength of medium by using low
ionic strength saline (LISS)
 Decreasing conc. of cations in medium
 Leads to decrease in density of ions around red cells
 This increases sensitization
 But decreases agglutination
28

Factors affecting Red Cell Sensitization
1. Ratio of Ab to Ag
 Sensitization occurs easily when at higher conc. of Ab
 This can be done by increasing conc. of serum containing
the Ab to conc. of cells
29

Factors affecting Red Cell Sensitization
2- The pH of reaction mixture
 At a pH below the pI, Abs have
+ve charges
 This makes it easier for the Ab
to bind to the –vely charged
red cells
 Optional pH for sensitization is
6.5 to 7.5 (Ab +vely charged)
pH 8 pH 7
30

3- Temperature
Ag- Ab reactions are exothermic
Therefore, Abs bind to a greater degree at lower temperature
But at lower temperatures, rate of reaction is reduced
To speed up reaction, tests are done at 37oC
31

 Temperature can also affect Ag accessibility on red cells
 Some IgM Abs bind best at 4oC (cold Abs)
 Temperature can make conformational changes in the Ag
 More Ag sites are exposed as the temperature is lowered allowing
increased binding of Ab
 Most naturally occurring cold Abs are of no clinical
significance
 Compatibility testing is done at 37C
4oC 37oC
32

4. Ionic strength of the medium
When RBCs are suspended in LISS the cloud of ions around
the cell is less dense than in isotonic saline
Reduced conc. of cations surrounding RBCs allow +vely
charged Abs easier to access Ag sites
Rate of sensitization increases
33

5- Antigen Density
 The greater the number of Ags on red cell, the greater the sensitization
 Binding of +vely charged Abs to red cells lower the zeta potential
 And therefore enhances agglutination
 Increased Ag density also increases chance of bridging
34

Factors Influencing RBCs Agglutination
6- Ag Clustering and Mobility
 Clustering facilitates agglutination by increasing likelihood of Ab binding at that
site
 Cluster of some Ags can occur after enzyme treatment of cells
Clustering of Ags
35

Factors Influencing RBCs Agglutination
7- Antibody Characteristics
 Ability of Ab to agglutinate cells depend on the Ig
class
 IgM has a wider span than IgG, and therefore more
effective agglutination
 IgG can be chemically modified to increase its span
IgMA
g
300 Ao
IgG IgG
150 Ao250-300 Ao
36

 8. Specificity
 9. Bonding
 10. Physical fit
 11. Incubation time
37

Genetics of ABO blood group system
38

Basic genetics of Blood group
39

ABH antigen
 Express on RBC, platelet, epithelial cells are mainly glycospingolipids
 Also present in body fluid (saliva, serum) are glycoproteins
 Genes ABO,Hh, Se control the expressions of antigens.
40

Location
 The presence or absence of the
ABH antigens on the red blood
cell membrane is controlled by
the H gene.
 The presence or absence of the
ABH antigens in secretions is
indirectly controlled by the Se
gene.
41

H antigen
 The H gene codes for an enzyme that adds sugar fucose to the terminal sugar of a
precursor substance
 The precursor substance (proteins and lipids) is formed on an oligosaccharide chain
(the basic structure)
42

H antigen
 Is the foundation upon which A and B
antigens are built.
 A and B genes code for enzyme that add
an immunodominant sugars to the H
antigen
 Immunodominant sugars are present at the
terminal ends of the chains and confer the ABO
antigen specificity
44

A antigen
 The A gene codes for an enzyme
(transferase) that adds N-
acetylgalactosamine to the terminal sugar of
the H antigen
 N-acetylgalactosaminyltransferase
45

B antigen
 The B gene codes for an enzyme that
adds D-galactose to the terminal sugar
of the H antigen
 D-galactosyltransferase
46

 Certain blood types possess more H antigen than others.
 Most H O>A2>B>A2B>A1>A1B
Least H

49

 Why do Group O individuals have more H
antigen than the other groups?
 Group O individuals have no A or B genes
to convert the H antigen that means more
H antigen sites
50

Bombay Phenotype (Oh)
 Inheritance of hh
 The h gene is an amorph and results in little or no production of L-fucosyltransferse
 Orginally found in Bombay
 Very rare (130 worldwide)
 The hh causes NO H antigen to be produces
 Results in RBCs with no H, A, or B antigen (patient type as O)
 Bombay RBC are NOT agglutinated with anti A, anti B,or anti H(no antigens present)
 Bombay serum has strong anti A, anti B and anti H, agglutinating ALL ABO blood
groups
54

 What blood ABO blood group would you use to transfuse this patient?
 Another Bombay
 Group O RBC cannot be given because they still have H antigen
 You have to transfuse the patient with blood that contains NO H antigen
55

Blood group Antigens on cell Antibodies in plasma
A A Anti-B
B B Anti-A
AB A and B Anti-AB
O None Anti A and B
56

Methods of Blood Grouping
 Several methods can be done for ABO grouping.
 Serology - direct detection of the ABO antigens. It is the main method
used in blood transfusion centre and hospital blood banks.
 This form of testing involves two componenets.
 1 Antibodies that are specific at detecting a particular ABO antigen on RBCs.
 2 Cells that are of a known ABO group that are agglutinated by the naturally occurring antibodies in
the person’s serum
58

Blood grouping
Blood grouping
Forward or cell
grouping or front typing
or forward typing
Reverse or serum
grouping or confirming
grouping or back
typing
59

Forward grouping
Test for antigens
 Patient’s cells containing unknown antigens tested with known antisera.
 Reaction of patient red blood cells tested with reagent anti A and anti B antisera
Forward
grouping
Slide
method
20-40% Patient’s RBC
suspension + known antiserum
Tube
method
2-5% Patient’s RBC suspension+
known antiserum(centrifuge before
read)
60

Reverse grouping
 Serum is combined with cells having known red cell content in a 2:1 ratio
Reverse
grouping
Slide
Method
20-40% Known red cell
suspension + patient’s serum
Tube
method
2-5% Known red cell suspension +
patient’s serum
62

Reverse grouping
 Reverse Group ; reaction pattern
63

Use of Group O cells when carrying out a serum group
 Some donors have antibodies in their serum other than anti A or anti B
 These antibodies are not naturally expected to be present
 These are called irregular antibodies
 As a result of earlier immunization , during pregnancy in the case of a woman,
previous transfusion
 The presence of irregular antibodies may be demonstrated by using group O cell in
serum grouping
Viva,
MSQ
67

Subgroups of A (A1 and A2)
They both react strongly with reagent anti-A.
80% of group A individuals phenotype as A1
20% phenotype as A2
Reagent anti-A is a mixture of two Abs ;
anti-A which react with both A1 and A2 cells.
anti-A1 which reacts with A cells but not with A2 cells in simple testing .
68

Difference Between A1 and A2
 A1 has more A and less H antigen on the cell.
 A2 has less A and more H antigen
 Cannot be detected serologically
 A2 can produce anti- A1 – qualitative difference
69

Differentiation between the A blood subgroups
 Reagent anti-A is a mixture of two Abs
Anti-A1-lectin: is another source of anti-A1.
lectins are seed extracts that agglutinate human cells with some degree of specificity.
The seeds of the plant Dolichos biflorus serve as the source of the anti-A1 lectin this
reagent agglutinate A1 or A1B cells but does not agglutinate A2 or A2B cells.
70

Anti-A1
 1-8% of A2 and 22-35% of A2B people will have anti-A1
 Causes ABO discrepancy – reverse type
 Incompatible crossmatch if donor A1
 NOT clinically significant unless reactive at 37C or AHG.
 Clinically significant – ability to cause red cell destruction – donor blood or
hemolytic disease of the fetus and newborn.
71

Subgroup A Subgroup frequency Antibodies always
present
Antibodies sometimes
present
A1 80% Anti B None
A2 20% Anti B Anti A1 in 2%
A1B 80% None None
A2B 20% none Anti A1 in 25%
72

Anti AB
 Used anti AB as a part of standard blood grouping to ensure weak A and B antigen
 This mixture has a much higher affinity for weaker antigens
 Not required for testing patient’s red cells
 Recommended for donor blood grouping
73

Anti A
 Consist of a mixture of two antibodies.
 Anti A  agglutinates A1, A2, A1B, A2B cells
 Anti A1 agglutinates only A1 and A1B cells
74

Secretor Genes
 A, B and H antigens may be present in fluids.
 Controlled by Se and se, secretor genes.
 Need only one copy of the Se gene.
 The gene se is an amorph.
 Not linked to ABO locus, inherited independently
75

Secretor Genes
 Persons who have A, B and/or H in secretions are called “secretors”
Blood Group Substance in Secretions
A A and H
B B and H
AB A, B and H
O H
77

ABO & Rh D Grouping using Tile Technique
Anti A Anti B Anti AB A cell B cell Ocell Anti D
Patient ID
Patient ID
Patient ID
The blood group reagents should be dispesne according to the
labeling on the tile.
Reagents
 Place one drop of anti A reagent in each of the wells in the
anti A columm.
 Place one drop of anti B reagent in each of the wells in the
anti B columm. 78

 Place one drop of anti AB reagent in each of the wells in the anti AB columm.
 Place one drop of A cells (15-20% reverse cells) in each of the wells in the anti A cells column.
 Place one drop of B cells (15-20% reverse cells) in each of the wells in the anti B cells
column.
 Place one drop of O cells (15-20% reverse cells) in each of the wells in the anti O cells
column.
 Place one drop of anti D reagent in each of the wells in the anti D column.
79

 Samples
 For each row:
 Place one drop of 15-20% patient cells in each of the anti A, anti B, anti AB and anti D wells.
 Place two drops of patient plasma (or serum) in each of the A cells, B cells and O cells wells.

 Procedure
 Mix the contents of all wells using the wooden mixing "sticks".
 Gently rock the tile and then place at room temperature.
 Examine for agglutination after 5 minutes.
 If a weak agglutination or negative reaction is obtained, examine again after 15 minutes.
 Using the Agglutination Grading chart as a guide, score the results for each well from 0-4+.
 Determine the group for the control and patient samples using the Tile Group Interpretation Chart.
80

ABO & RhD Grouping Using Tube Technique
 Principle
 The ABO forward group identifies the presence or absence of ABO antigens on the red cell
surface using antibodies to those antigens. The ABO reverse group identifies the presence
or absence of naturally occurring plasma (or serum) antibodies to ABO groups must
correlate.
 The Rh system is the second most important blood group system. Blood is classified as Rh
D positive or RhD negative depending on the presence or absence of the Rh D blood group
antigen.
 A weakened expression of the Rh D antigen may occasionally be present on red cells.
 Approximately 82% of the Caucasian populations are Rh D positive.
 The ABO and Rh D blood groups of blood donors and recipients are routinely determined
to ensure that ABO and Rh D compatible blood is used for transfusion.
 All blood group discrepancies must be resolved.
 This procedure describes ABO and Rh D grouping tests, using test tube based techniques.

81

 Specimen preparation
 A 3-5% red cell suspension of test cells and a separated sample of patient serum (or) plasma are
required.
 Materials
 Patient 3-5% red cell suspension
 Patient plasma or serum
 ABO forward Group Typing Reagents
 ABO Reverse Grouping Reagents Red Cells
 Reverse cell 3% cells
 Rh D Blood Grouping Reagents
 Anti D
 Buffered saline or Normal saline
 Plastic Pipettes
82

 suitable centrifuge
 Light Box
 Grading Chart
83

 Procedure
 The ABO cell (forward) and plasma or serum (reverse) groups and Rh D group are performed simultaneously.
 Prepare a 3-5% suspension of patient red cells as per SOP (1).
 Label 7 glass test tubes (12x75 mm) with a unique patient identifer and anti A, anti B, anti AB and anti D for
cell grouping (forward group) and A cells, B cells and O cells for plasma grouping (reverse group).
 Place two drops of patient plasma into the two tubes labeled A cells, B cells and O cells.
 Place one drop of each grouping reagent into the appropriate tubes.
 Add one drop of 3-5% test red cell suspension to each of the four tubes containing the forward grouping
reagents and add one drop of A1 and B reagent red cells (Revercells) to the 3 tubes labeled A cells, B cells
and O cells.
 Gently shake all tubes to allow mixing and incubate at room temperature for 5 minutes.
 Centrifuge tubes at 2000 rpm for 15 seconds or 1000 rpm for 1 minute using Immufuge or another suitable
centrifuge.
 Examine for macroscopic agglutination over a light box using the roll and tip method.
 Score and record results and interpretation on supplied worksheet.
84

Causes of False Positive Reaction
 1. Rouleaux formation
-pseudo agg  pile of coins due to high protein concentration
- can be differentiated from true agglutination by saline dispensing technique
2. Cold agglutinin  cause auto agg, active at RT. If suspected,
- serum grouping  repeated at 37C
- cell grouping  repeated after washing several time in warm buffered
saline
3. Infected red cells
- poly agg – less obvious – does not occur at 37C- true group can be
done at 37C
85

Cause of False negative reaction
 1 Use of impotent sera
 Loss of potency result if sera are left at RT or stored frozen in large volume
 2. No recognition of lysis
 Inspected for lysis
86

ABO blood group system

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