ABO and Rh Blood

Group Systems
 Teaching Aim

 Understanding inheritance, synthesis, various

antigens and antibodies and their clinical
significance in ABO & Rh blood group systems
 Understanding practical aspects of ABO & Rh blood
grouping
 Human Blood Groups
 Red cell membranes have antigens (protein /
glycoprotein) on their external surfaces
 These antigens are
o unique to the individual
o recognized as foreign if transfused into another individual
o promote agglutination of red cells if combine with antibody
o more than 30 such antigen systems discovered
 Presence or absence of these antigens is used to
classify blood groups
 Major blood groups – ABO & Rh
 Minor blood groups – Kell, Kidd, Duffy etc
 ABO Blood Groups
 Most well known & clinically important blood group system.
 Discovered by Karl Landsteiner in 1900
 It was the first to be identified and is the most significant for
transfusion practice
 It is the ONLY system that the reciprocal antibodies are
consistently and predictably present in the sera of people
who have had no exposure to human red cells
 ABO blood group consist of
o two antigens (A & B) on the surface of the RBCs
o two antibodies in the plasma (anti-A & anti-B)
Reciprocal relationship between ABO
antigens and antibodies

Antigens on Antibody in plasma / Blood

RBCs serum group
A Anti-B A
B Anti-A B
AB None AB
None Anti-A, Anti-B O
 All the ABH antigens develop as early as day 37 of fetal
life but do not increase very much in strength during
gestational period

 Red cell of newborn carry 25-50 % of number of antigenic

sites found on adult RBC

 Although cord red cells can be ABO grouped, the

reactions may be a bit weaker than expected

 A or B antigen expression fully developed at 2-4 yrs of age

and remain constant throughout life
 Although the ABO blood group antigens are regarded as
RBC antigens, they are actually expressed on a wide variety
of human tissues and are present on most epithelial and
endothelial cells

 ABH antigens are not only found in humans, but also in

various organisms such as bacteria, plants, and animals

 Present both on red blood cells and in secretions only in

humans and some of the apes (chimpanzee, gorilla)

 In all other mammalian species these substances are found

only in secretions
 Not present in the newborn, appear in the first years of life (4-6
months usually), reach adult level at 5-10 years of age, decreases in
elderly
 Naturally occurring as they do not need any antigenic stimulus
 However, some food & environmental antigens (bacterial, viral or
plant antigens) are similar enough to A and B glycoprotein antigens
and may stimulate antibody development
 Immunocompetent person react to these antigens by producing
antibodies to those absent from their own system
 Usually IgM, which are not able to pass through the placenta to the
fetal blood circulation
 Anti-A titer from group O > Anti-A titer from group B
 Anti-A titer from group B > Anti-B titer from group A
ABO antigens & corresponding
antibodies
 'Landsteiner's law :-
the plasma contains natural antibodies
to A or B, if these antigens are absent
from the red cells of that person
Inheritance of ABO Blood Groups
 Follows Mendelian principles
 Blood group antigens are “codominant”- if the gene
is inherited, it will be expressed.
 There are three allelic genes -A, B & O
 Some aberrant genotypes do occur but they are
very rare.
 Understanding of basic inheritance important.
 Inheritance of ABO Blood Groups
 Two genes inherited, one from each parent.
 Individual who is A or B may be homozygous or
heterozygous for the antigen.
o Heterozygous: AO or BO
o Homozygous: AA or BB
 Phenotype is the actual expression of the genotype, ie,
group A
 Genotype are the actual inherited genes which can only
be determined by family studies, ie, AO.
 Example of Determining Genotype

 Mother’s phenotype is group A, genotype AO

 Father’s phenotype is group B, genotype BO

B O
A AB 25% (Group AB) AO 25% (Group A)
O BO 25% (Group B) OO 25% (Group O)
 Other Examples

Mother Father Offspring Blood Group

AA BB 100% AB

BO OO 50% each of B or O

OO OO 100% O

OO AO 50% each of A or O
 ABO Antigen Synthesis
 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(s) to be added to the red cell.
 The type of sugar added determines the blood
group.
 ABO and H Antigen Genetics
 Genes at three separate loci control the occurrence
and location of ABO antigens
 Presence or absence of the ABH antigens on the
red cell membrane is controlled by the H gene
 Presence or absence of the ABH antigens in
secretions is indirectly controlled by the Se gene
o H gene – H and h alleles (h is an amorph)
o Se gene – Se and se alleles (se is an amorph)
o ABO genes – A, B and O alleles
 H Antigen
 The H gene codes for an enzyme (fucosyltransferase) that
adds the 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)
 The H antigen is the foundation upon which A and B
antigens are built
 A and B genes code for enzymes that add an
immunodominant sugar to the H antigen
 Immunodominant sugars are present at the terminal ends of the
chains and confer the ABO antigen specificity
RBC precursor substance

RBC

Glucose

Galactose
Precursor
Substance
(stays the N-acetylglucosamine
same)
Galactose
 Formation of the H antigen

RBC

Glucose

H antigen Galactose

N-acetylglucosamine

Fucose Galactose
 A and B Antigen
 The “A” gene codes for an enzyme (transferase)
that adds N-acetylgalactosamine to the terminal
sugar of the H antigen
o N-acetylgalactosaminyltransferase

 The “B” gene codes for an enzyme that adds D-

galactose to the terminal sugar of the H antigen
o D-galactosyltransferase
 Formation of the A antigen

RBC

Glucose

Galactose

N-acetylglucosamine

Fucose Galactose

N-acetylgalactosamine
 Formation of the B antigen

RBC

Glucose

Galactose

N-acetylglucosamine

Fucose Galactose

D-Galactose
 Immunodominant sugars responsible for
antigen specificity

Gene Glycosyltransferase Immunodominant Antigen

sugar
H L-fucosyltransferase L-fucose H
A N-acetylgalactosaminyltransferase N-acetyl-D- A
galactosamine
B D-galactosyltransferase D-galactose B
 Secretor Status
 A, B, H substances are found in all body secretions
(except CSF) in 80% of individuals
 Ability to secrete these substances is determined by the
presence of secretor gene (Se) in either homozygous
(SeSe) or heterozygous (Sese) state.

Blood Group Substances Secreted

O H
A A&H
B B&H
AB A, B, & H
Oh Nil
 Characteristics of Bombay Phenotype
 First reported by Bhende et al in Bombay in 1952.
 Frequency estimated to be about 1 in 7600 in Bombay.
 Absence of H, A & B antigens. No agglutination with anti-A,
anti-B or anti-H
 Presence of anti-H, anti-A and anti-B in the serum
 No A, B or H substances present in saliva
 Incompatible with any ABO blood groups, compatible with
Bombay phenotype only
 A recessive mode of inheritance (identical phenotypes in
children but not in parents)
 ABO Subgroups
 ABO subgroups differ in the amount of antigen present
on the red blood cell membrane
o Subgroups have less antigen

 Subgroups are the result of less effective enzymes.

They are not as efficient in converting H antigens to A or
B antigens (fewer antigens are present on the RBC)

 Subgroups of A are more common than subgroups of B

 Subgroups of A
 Two principle subgroups of A are: A1 and A2
 Both react strongly with reagent anti-A
 To distinguish A1 from A2 red cells, the lectin Dolichos
biflorus is used (anti-A1)
 80% of group A or AB individuals are A1 and A1B
 20% are A2 and A2B
 A2 phenotype
 Clinical significance of A2 phenotype
o 8% of A2 and 25% of A2B individuals may produce anti-A1 in the
serum
o This may result in discrepancy in blood grouping or
incompatibility in cross match
o However, these anti-A1 antibodies are cold reacting & therefore
may not cause problems routinely.
 Difference between A1 and A2
o It is quantitative
o The A2 gene doesn’t convert the H to A very well resulting in
fewer A2 antigen sites compared to the many A1 antigen sites
 A1 and A2 Phenotypes

Anti-A Anti-A1 Anti-H Antibody Antigens /

in serum RBC
A1 4+ 4+ 0 Anti-B 9 x 105

A2 4+ 0 3+ Anti-B & 2.5 x 105

Anti-A1
 Subgroups of A
Reaction with antisera Antibodies Substance
Pheno in Saliva
type AntiA Anti B Anti AB Anti A1 Anti H Common Unexpected (secretors)

A1 +4 0 +4 +4 0 Anti-B - A and H
Sometimes
A2 +4 0 +4 0 +2-3 Anti-B A and H
Anti-A1
Sometimes
A3 +2 mf 0 +2 mf 0 +3 Anti-B A and H
Anti-A1
Weak Always
Ax 0 +2 0 +4 Anti-B H
Or 0 Anti-A1
No
Am 0 0 0 0 +4 Anti-B A and H
Anti-A1
Sometimes
Ael 0 0 0 0 +4 Anti-B H
Anti-A1
Weak Weak Sometimes
Aend 0 0 +4 Anti-B H
Mf Mf Anti-A1
 B Subgroups
 B subgroups occur less than A subgroups
 B subgroups are differentiated by the type of
reaction with anti-B, anti-A,B, and anti-H
 B3, Bx, Bm, and Bel
Practical aspects of ABO grouping
 Routine ABO grouping must include both cell & serum testing as
each test serves as a check on the other
 Test should be done at room temperature or lower; testing at
37oC weakens the reactions
 Tubes, slides should be dry and labeled properly
 Serum should always be added before adding cells
 Results should be recorded immediately after observation
 Hemolysis is interpreted as positive result
 Blood Grouping

 There are 2 components to blood typing:

o Test unknown cells with known antibodies
o Test unknown serum/plasma with known red
cells
 The patterns are compared and the blood
group is determined.
 Blood Sample for Blood Grouping

Blood sample
 Clearly labeled blood samples in sterile tubes (plain & EDTA)
 Test should be performed on the fresh sample for best results. In
case the test can not be performed immediately, sample can be
stored at 4oC & should be tested with in 48 hours
 No signs of hemolysis should be there
 If serum is not completely separated, centrifuge tube at 1000-3000
rpm fro 3 min
 Preferably use saline washed red cells and make 2-5% suspension
Red Cell Suspensions for Blood Grouping

 50%: Slide Method

 5%: Test Tube Method
 1%: Gel technology
 1%: Microplate
 Slide Method for ABO Grouping
Not recommended as a routine method
 Very rudimentary method for determining blood groups.
 CANNOT be used for transfusion purposes as false
positives and negatives do occur. Drying of reaction
mixture can cause aggregation - false positive
 Less sensitive, not reliable for weakly reactive antigens
and antibodies
 Can only be used for emergency ABO grouping or for
selection of plateletpheresis donors
 Slide Method for ABO grouping

 Put 1 drop anti-A & anti- B

separately on slide
 Add 1 drop of 40-50%
suspension of test red cells to
each drop of typing antisera
 Mix & spread each mixture
evenly on the slide over an
area of about 15 mm diameter
 Leave the test for 2 min at
room temp (20-24oC)
 Record the results immediately
 0.8 % cell suspension
for
Gel card grouping

5 % cell suspension
for
Tube grouping
 Test Tube Method of ABO Grouping

Recommended method
 Allows longer incubation of antigen and antibody
mixture without drying
 Tubes can be centrifuged to enhance reaction
 Can detect weaker antigen / antibody

Two steps in ABO grouping

Cell grouping (Forward grouping)
 Tests the patients red cells with known Anti-A & Anti-
B to determine the antigen expressed
Serum grouping (Reverse grouping)
 Test the patients serum with known A & B cells to
determine the presence of antibody
Lay Out of Tubes for ABO & Rh grouping

Forward grouping Reverse grouping

Rh grouping
Cell grouping Sera grouping
 2 vol of anti- A / 1 vol of 2-5% red
anti-B/ Anti-AB cell suspension

Incubate at room temp

(20-24oC) for 5 min

Forward Centrifuge at 1000 rpm for

Grouping 1 min

Check for agglutination

against well lighted
background
 1 vol of 5%
2 vol of test
suspension of
serum/plasma
reagent red cells in
respective tubes

Shake & leave at room

temp (20-24oC) for 5 min

Reverse
Grouping Centrifuge at 1000 rpm for 1
min

Centrifuge & record the

results similarly as for
cell grouping
Recording results of ABO grouping

Reaction of red cells Reaction of serum Interpretation

with with pooled cells
Anti-A Anti-B Anti-AB Ac Bc Oc
+ + + 0 0 0 AB
+ 0 + 0 +/H 0 A
0 + + +/H 0 0 B
0 0 0 +/H +/H 0 O
0 0 0 +/H +/H + Oh

+ = agglutination, 0 = no agglutination H = hemolysis

 Microplate Method
 It is ideal for testing large number of blood samples.
 More sensitive to detect weaker antigen-antibody
reactions
 Results can be photographed for archival storage
 Microplate can be incubated & centrifuged
 There is significant saving in time and in the cost of
disposables and reagents.
 Microplates are intended to be disposable however
they can be reused after cleaning them properly
making sure that all foreign protein are removed.
 Microplates can be adapted for automation
 Microplate Method

Reaction in the microplate after 1 hour incubation at room temperature

 Column Agglutination Technology
 One card is basically a set of 6
microtubes
 Microtubes contain either Sephadex
Gel or glass microbeads
impregnated with antisera
 Antigen-antibody reaction takes
place in the reaction chamber of
microtube
 Gel matrix or glass beads act as
sieve and allow free cells (un-
agglutinated) to pass through and
settle at the bottom of microtube
while agglutinated cells are trapped
in the matrix
Grading Result
Sources of errors resulting in ABO
discrepancies
 Inadequate identification of sample, test tubes or
slides
 Cell suspension either too heavy or too light
 Clerical errors
 Missed observation of hemolysis
 Failure to add reagents
 Uncalibrated centrifuge
 Contaminated / expired reagents
 Failure to follow manufacturer’s instructions
 Technical problems
• Glassware, Reagents, Equipment
o Dirty glassware, contaminated or outdated reagent,
temperature not proper
• Cell concentrations
o Too high or too low concentration
• Centrifugation
• Carelessness –
o patient identification,
o sample identification,
o reading and recording results
Resolution of Blood Group Discrepancies
 Obtain fresh sample
 Rule out clerical error
 Rule out technical error
 Obtain clinical history
o age, diagnosis, pregnancy, drug & transfusion
 Repeat test using
o Washed RBC
o Incubate at different temperatures (4oC, RT, 37oC)
o Put up auto control
o Antisera from different lot no.
o Antisera from different manufacturer
Resolution of ABO group discrepancies
….contd.
Additional test
 A2, O or cord cells if required may be used
 Anti AB antisera
 Lectins (anti A1, anti H)
 Increasing serum : cell ratio
 Increasing incubation time
 Decreasing incubation temperature
 Including autocontrol
 Saliva secretor status
 Adsorption elution test
 Rh Blood
Group System
Rh system: Nomenclature
 Rh (D) Antigen
 Of next importance is the Rh type.
o Rh is a blood group system with many antigens, one of which is D.
 Rh refers to the presence or absence of the D antigen on the
red blood cell.
 Unlike the ABO system, individuals who lack the D antigen do
not naturally produce anti-D.
 Production of antibody to D requires exposure to the antigen.
 The D antigen is very immunogenic, ie, individuals exposed to it
will very likely make an antibody to it.
 For this reason all individuals are typed for D, if negative must
receive Rh (D) negative blood.
 Rh (D) Antigen (continued)

 Rh antigens are an integral part of the red cell

membrane.
 They are protein in nature with an active phospholipid
component
 Rh antigens do not exist in the soluble form and,
therefore are not excreted in body fluids.
 Unlike ABO antigens, Rh antigens are present only on
red blood cells. These antigens are not found on other
blood cells including platelets and leukocytes
 Rh (D) Antigen (continued)

 A very potent antigen (50% may form antibody to exposure)

 Frequency in Indian population
o 95% Rh positive
o 05% Rh negative
 The most important patient population to consider is females
of child-bearing age.
 If immunized to Rh (D) antigen the antibody can cross the
placenta and destroy Rh (D) positive fetal cells resulting in
death.
 This is why Rh negative women are given anti-D (Rhogam)
after birth of Rh positive baby.
Various antigens in Rh system

Rh antigens Frequency % in India

D 95
d (absence of D) 05
C 70
E 15
c 90
e 98
 Rh Antibodies
• All Rh antibodies are immune in nature, developed after
immunizing event
• React at 37oC and require anti globulin test to demonstrate
the reaction
• Generally do not react at room temperature in saline
• Most are IgG in nature and therefore can cross the
placenta
• Generally, do not fix complement and cause extravascular
hemolysis
• All are important in HDN (Hemolytic disease of the newborn)
and delayed HTR (Hemolytic Transfusion Reaction)
 Rh typing

 Normal typing for Rh antigens only includes typing

for Rh (D).
 The result of this typing determines the Rh status of
the cells (Rh - positive or Rh - negative).
 Some Rh typing sera is diluted in high protein
solutions and may require a negative control.
 It is recommended to use two monoclonal anti-D
sera from two different manufacturers labeled as D1
and D2, especially to confirm all Rh negatives