Blood Grouping

NAEEM ULLAH
Lecturer MLT (pathology)
NWIHS
  Discovered the ABO Blood Group
System in 1901

History - Karl
 He and his five co-workers began
Landsteiner
mixing each others red cells and
serum together and inadvertently
performed the first forward and
reverse ABO groupings
  Karl Landsteiner’s law :

 If an antigen is present in the RBC’s of an

Landsteiners
individual, the corresponding antibody
Rule
must be absent from the plasma

 If an antigen is absent in the RBC’s of an

individual, the corresponding antibody
must be present from the plasma
 ABO Antigen Antigen Antibody
Group Present Missing Present
Major ABO A A B Anti-B
Blood Group B B A Anti-A
O None A and B Anti-A&B
AB A and B None None
  Blood group antigens are actually sugars
attached to the red blood cell.

 Antigens are “built” onto the red cell.

ABO Basics
 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
 There are two principles

1-almost all normal healthy individuals above 3-6

Principle of months of age have “ naturally occurring Abs” to

blood the ABO Ags that they lack
grouping
These Abs termed naturally occurring because they
were thought to arise without antigenic stimulation
 2- These “naturally occurring” Abs are mostly IgM

Principle of class. That means that, they are Abs capable of

blood agglutinating saline/ low protein suspended red
grouping cell without enhancement and may activate
complement cascade.
  Ags belonging to ABH blood group system are present on
RBCs and other body cells and body fluids.
ABO and H
Antigen
Genetics  The presence of A,B, and O Ags on RBCs depends upon the

ABO chromo 9 allelic genes, A,B, and O

O gene on  An H genes at a separate locus codes for the precursor

chrom 19 substance on which the A and B gene products act

 The products of the A and B genes are enzymes that act as a

specific transferases
  The ABO genes do not code for the production of
ABO antigens, BUT rather produce specific glycosyl
transferases

Genetics
 ABO produces a specific glycosyl transferases that
add sugars to a basic precursor substance on the
RBCs
 RBC

RBC Precursor
Structure Glucose

Galactose
Precursor
Substance
(stays the N-acetylglucosamine
same)
Galactose
 There are two potential precursors substance (PS) both are
comprised of identical sugar (galactos-N- acetyl
Precursor gluctosamin - galactose -glucose) but different in linkage.
Substance  Type I PS has a terminal galactose (Gal) linked to a
subterminal N acetylgucoseamine (GlcNAc) in 1-3 linkage

 Type II PS, has the same sugar combine in 1-4 linkage

 ABH Ags on RBCs are derived from Type II chains

  The inheritance of at least one H gene (HH or Hh)
elicits (obtain) the production of an enzyme called,
α-2-L-Fucosyl transferase, which transfers the
sugar from the Guanosine diphosphate L-fucose
(GDP-Fuc) donor nucleotide to the terminal
H Antigen galactose of the precursor chain.

 The H substance must be formed for the other

sugars to be attached in response to an inherited A
and /or B genes
 RBC

Formation of
the H antigen Glucose

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

Fucose
  The “A” gene codes for an enzyme (transferase) that
adds N-acetylgalactosamine to the terminal sugar of
the H antigen

A and B  N-acetylgalactosaminyltransferase

Antigen
 The “B” gene codes for an enzyme that adds D-
galactose to the terminal sugar of the H antigen
 D-galactosyltransferase
 RBC

Formation of
the A antigen Glucose

Galactose

N-acetylglucosamine

Galactose
A antigen

N-acetylgalactosamine
Fucose
 RBC

Formation of
the B antigen Glucose

Galactose

N-acetylglucosamine

Galactose

Galactose B antigen
Fucose
 RBC

Formation of
the AB Glucose

antigen Galactose

N-acetylglucosamine

Galactose

Galactose B antigen

N-acetylgalactosamine A antigen
Fucose
 RBC

Formation of
the H antigen Glucose

Galactose
Precursor
Substance
(stays the N-acetylglucosamine
same)
Galactose
H antigen
O antigen
Fucose
  Certain blood types possess more H antigen than
others:

H antigen
O>A2>B>A2B>A1>A1B
  The H antigen is found on the RBC when you have the
Hh or HH genotype, but NOT from the hh genotype

 The A antigen is found on the RBC when you have the

Genetics
Hh, HH, and A/A, A/O, or A/B genotypes

 The B antigen is found on the RBC when you have the

Hh, HH, and B/B, B/O, or A/B genotypes
  Inheritance of hh

Bombay  The h gene is an amorph and results in little or no

Phenotype production of L-fucosyltransferase
(Oh)
 Originally found in Bombay

 Very rare (130 worldwide)

  The hh causes NO H antigen to be produced
 Results in RBCs with no H, A, or B antigen (patient
types as O)
Bombay  Bombay RBCs are NOT agglutinated with anti-A, anti-
Phenotype B, or anti-H (no antigens present)
 Bombay serum has strong anti-A, anti-B and anti-H,
(Oh) agglutinating ALL ABO blood groups
 What blood ABO blood group would you use to
transfuse this patient??
Another Bombay
Group O RBCs cannot be given because they still
have the H antigen
You have to transfuse the patient with blood that
contains NO H antigen
  Group A serum contains anti-B
 Group B serum contains anti-A
ABO  Group AB serum contains no antibodies
antibodies  Group O serum contains anti-A, anti-B, and anti-A,B
  IgM is the predominant antibody in Group A and Group
B individuals
 Anti-A
 Anti-B
ABO
antibodies  IgG (with some IgM) is the predominant antibody in
Group O individuals
 Anti-A,B (with some anti-A and anti-B)
  Reactions phase: Room temperature
 Complement can be activated with ABO antibodies
(mostly IgM, some IgG)
 High titer: react strongly (4+)
ABO  Usually present within the first 3-6 months of life
antibodies  Stable by ages 5-6 years
 Decline in older age
 Newborns may passively acquire maternal antibodies
(IgG crosses placenta)
 Reverse grouping (with serum) should not be performed on
newborns or cord blood
  Several methods for testing the ABO group of an
individual exist. The most common method is:

 Serology: This is a direct detection of the ABO

ABO antigens. It is the main method used in blood
routine testing transfusion centres and hospital blood banks.
 This form of testing involves two components:
a) Antibodies that are specific at detecting a particular
ABO antigen on RBCs.
 b) Cells that are of a known ABO group that are
agglutinated by the naturally occurring antibodies in
the person's serum.
 DIRECT OR FORWARD GROUPING
Test for antigens
• Patient’s cells containing unknown antigens tested
ABO with known antisera
ROUTINE • Antisera manufactured from human sera
TESTING Aantisera used:
Antisera Color Source
Anti-A Blue Group B donor
Anti-B Yellow Group A donor
Anti-A,B Red Group O donor
  Reaction of patient red blood cells tested with
Reagent anti-A and anti-B antisera

Forward  Slide: 20-40% RBC suspension + anti-serum

Grouping
 Tube (12x75mm): 2-5% RBC suspension + anti-serum
(centrifuge before read)
 Reaction Patterns for ABO Groups

Blood group Agglutination with Agglutination with

Anti-A Anti-B
A + -
Forward
Grouping B - +

AB + +

O - -
 • serum is combined with cells having known Ag
Reverse content in a 2:1 ratio
grouping • uses commercially prepared reagents containing
saline-suspended A1 and B cells
 Reaction Patterns for ABO Groups

Blood Group Agglutination with Agglutination with

A cells B cells
Reverse A - +

grouping B + -

AB - -

O + +
  Grading of Agglutination:

Negative (0) No clumps or aggregates

Weak (+/-) Tiny clumps or aggregates barely
Grading of visible macroscopically or to the
naked eye
Agglutination 1+ Few small aggregates visible
macroscopically
2+ Medium-sized aggregates
3+ Several large aggregates
4+ One solid aggregate
 Patient Red Cells Tested With

ABO blood Interpretation Anti-B Anti-A Patient

group
(forward blood 0 0 1
grouping) 0 4+ 2

4+ 0 3

4+ 4+ 4
 Patient Red Cells Tested With

ABO blood Interpretation Anti-B Anti-A Patient

group
(forward blood O 0 0 1
grouping) A 0 4+ 2

B 4+ 0 3

AB 4+ 4+ 4
 Patient SERUM Tested With

Interpretation B Cells A1 Cells

Patient
Reverse
4+ 4+ 1
Grouping
(Confirmatory 4+ 0 2
grouping
0 4+ 3

0 0 4
 Patient SERUMTested With

Interpretation B Cells A1 Cells

Patient
Reverse
O 4+ 4+ 1
Grouping
(Confirmatory A 4+ 0 2
grouping
B 0 4+ 3

AB 0 0 4
 Reaction of Cells Tested With Reaction of Serum Tested
Against ABO
Forward & Group
Anti-A Anti-B A1 Cells B Cells
reverse
ABO blood 1 0 0 + + O

grouping 2 + 0 0 + A

3 0 + + 0 B

4 + + 0 0 AB
 Reaction of Cells Tested With Reaction of Serum Tested
Against ABO
Forward & Group
Anti-A Anti-B A1 Cells B Cells
reverse
ABO blood 1 0 0 + +
grouping 2 + 0 0 +
3 0 + + 0
4 + + 0 0
  This ID-Card contains a mixture of
human polyclonal and monoclonal anti-
A, human polyclonal anti-B and human
polyclonal anti-D antibodies.
ID card
system  The microtube ctl is the negative control.

 Two microtubes with neutral gel serve

for reverse grouping with A1 and B cells.
Figure: Results of a gel microcolumn test. The
subject is group B D-negative. Red cells
remaining at the top of the gel represent a
positive result; red cells collected at the bottom
of the tube represent a negative result.
Thank you….