PREPARED BY:
JOSEPHINE C. MILAN, RMT, MSMT
WAGGAS AHMEDELAAS; M.Sc, MLT
(Revised, Feb 2014)
Objective and Focus
 To recall basic concepts of Immunology and Antigen-




Antibody reactions as they apply to Blood Banking
practice, such as:
Immune response arising from blood group antigen
stimulation
Characteristics of antigens
Describing clinically significant antibodies
Enhancing Ag-Ab reactions
Topic Outline
 Definition and properties of Antigen, immunogen
 Definition and properties, types of antibodies
 The immune system response in relation to
Immunohematology or blood banking practice
 Nature/ principle of Ag-Ab reactions
ANTIGEN, IMMUNOGEN
 ANTIGEN (Ag)– substance recognized as foreign by the
body, which when introduced to a host , will stimulate an
immune response.
 Immune response 
1. activation of the cellular components of immune
system
2. the production of specific antibodies
 Properties of antigen:
1. Specific reactivity – react with specific antibody
2. Immunogenicity- stimulate immune response
Blood Group Antigens
 Biochemically : Protein, large polysaccharide,
glycoproteins or glycolipids
 Immunogenic, antigenic
 Represent the blood group phenotype
 Determined by blood group genes
 Antigens are integral structural component of the RBC
membrane
BLOOD GROUP ANTIBODIES
 Gamma globulins / immunogloblins
 Produced in response to antigenic
stimulation. How?
 Exposure through pregnancy
 Exposure through transfusion
 Exposure through transplant
 Essentially of Ig M and IgG classes; some IgA
 Characterized with specific reaction with
blood group antigen epitopes/ determinants
Antibodies:
 Produced and secreted by activated B lymphocytes
(plasma cells)
 Types:
1. Heteroantibodies (xenoantibodies) – produced in
response to antigens from another species.
2. Alloantibodies / isoantibodies – produced in response
to antigens from individuals of the same species;
such as those involved in transfusion reactions
3. Autoantibodies – made in response to the body’s own
antigens.
Ig CLASSES OF SOME
BLOOD GROUP ANTIBODIES

IgM antibodies important in Blood
banking includes those against :
ABH, Ii, MN, Lewis, Lutheran, and P.

IgG antibodies important in Blood
banking includes those against:
Rh(D), Ss, Kell, Duffy, and Kidd.

IgA antibodies – Le, ABH subs
II. Antibody Classes
IgM
IgG
IgA
IgD
IgE
Other names
Macroglobulin
-
Secretory Ig
-
Reagin
%
5-10
75 – 85
5-15
0.001
0.0003
MW
900,000
150,000
160,000 & 400,000
180,000
190,000
Half-life
9 - 11d
25 - 35d
6-8d
2-3d
2d
56 C stable
yes
yes
yes
no
no
Pass thru
placenta
no
yes
no
no
No
valence
10
2
2,4
2
2
C-fixing
yes
IgG3
No
no
no
subclass
2
4
2
-
-
form
pentamer
monomer
dimer
Monomer
Monomer
Ig G structure
Ig M structure
Secretory Ig A
Serologic properties of Abs:
IgM
 Strong agglutinins (3-4+ agglutination)
 Reactive at cold temperature (4 0C to room temp)
 React to cells suspended in saline
 Fix/ activate the complement
IgG
 Weak agglutinins, need AHG
 Reactive at warm temperature (37 oC)
 React to red cells in protein or potentiating medium
 Weakly activate the complement
ANTIBODY FUNCTIONS
 Primary function: to bind with antigen
 Secondary functions/ biological effector
functions:
 Complement fixation/ activation
 hemolysis of red cells
 Placental transfer ( Ig G)
 Bind on receptor sites on tissues and other
cells
The Complement in Immunohematology
 Proteins normally produced in the body.
 Unstable, heat labile (56 oC); serum must be fresh
 When activated, they cause lysis of cell membrane
of antibody-coated RBCs.
 Complement is activated by the classic pathway :
 IgM or IgG3 antibodies bound to red cell antigens 
C1 component binds to the Fc portion of the Ab 
activate C4, C2, C3, C5, C6 + C7 + C8 + C9
lysis.
 Reason why: hemolysis of RC is a positive result.
IMMUNE RESPONSE
 Immune system – activated by the presence of
foreign antigen, or abnormal autoantigen.
 Cells involved :
 APCs (antigen presenting cells)= macrophages,
dendritic cells, monocytes
 Lymphocytes = T helper, T cytotoxic, B/ plasma cells
 Natural killer, neutrophils, eosinophils, basophils,
platelets
Plasma cells; Monocytes; Neutrophols;
Platelets; Lymphocytes
IMMUNE RESPONSES in
Immunohematology
 1. antibody production  IgM and IgG
 2. hemolytic reaction  mediated by Ab-Ag and
complement (cascade pathway)
 3. inflammatory reaction  tissue necrosis
(organ transplantation)
 4. allergic reaction  mediated by IgE with
basophils and mast cells
IMMUNE RESPONSE BY BLOOD
GROUP ANTIGEN STIMULATION
 PREGNANCY – incompatible blood type of fetus
to that of mother (baby’s antigen foreign to the
mother) E.g. Baby is D(+), mother is D(-)
 TRANSFUSION- incompatible blood type
between patient (recipient) and donor blood
 Tissue/ organ transplantation – incompatible
type between patient (recipient) and donor
tissue
PRINCIPLES OF
ANTIGEN- ANTIBODY
REACTION
MANIFESTATIONS OF
ANTIGEN – ANTIBODY REACTION
 1. Red cell agglutination
 Clumping of red cells
 Various grades of strength of reaction
 (4+, 3+, 2+, 1+, +/-)
 2. Hemolysis (red cell destructionHb)
 Red tinge of supernatant
 Minimal red cell button or none at all
 May be misread as negative
Grades of agglutination reaction
NON- SPECIFIC AGGREGATION OF
RED CELLS; NOT (+) RESULT
Rouleaux Formation

Stacked-coin formation
 Found in patients with
Multiple Myeloma,
Walderstrom’s
Macroglobulinemia
or Hyperviscosity Syndrome
 The red cells cluster
and resembles a
macroscopic agglutination)
NATURE OF Ag- Ab REACTIONS
A. Lock and Key principle
specific reaction of antigen and
antibody
the antigenic determinant fit in a
cleft / space formed by the
combining site of the antibody (at
the Fab region, formed by variable
regions of light and heavy chains)
Ag-Ab binding
NATURE OF Ag – Ab REACTIONS
B. Non-covalent Bonds



That hold the Ag in the antibody
combining site
Include hydrogen bonds, electrostatic
bonds, Van der Waals forces and
hydrophobic bonds
Multiple bonding ensures the Ag bound
to the Ab is tight and stable.
STAGES OF RED CELL AGGLUTINATION
1. PRIMARY STAGE ( ANTIBODY SENSITIZATION)
Involves binding of the
paratope /combining site
of the Ab and the
epitope/ determinant
of the Ag
in a reversible reaction).

2. SECONDARY STAGE OF RED CELL AGGLUTINATION :
LATTICE FORMATION
Multiple erythrocytes with
bound antibodies form a
latticework through
Ag--Ab bridges
formed between adjacent
erythrocytes
 The lattice formed is the
basis of all visible
agglutination reactions.

FACTORS AFFECTING THE
PRIMARY STAGE OF REACTION
1. Temperature
The nature of the bonds determines if the
reaction occurs better at colder or warmer
temperature.
 With

hydrogen bonds- they are exothermic,
reaction occurs better at colder temperature
Associated with carbohydrates antigen  e.g.
ABH, Lewis, P1, I)
FACTORS AFFECTING THE
PRIMARY STAGE OF REACTION
 If
with hydrophobic bonding, the
reaction is optimally reactive at
body temperature, 37 OC
 This type of bond is normally
associated with protein antigen 
e.g. Rh, Kell, Duffy
FACTORS AFFECTING THE
PRIMARY STAGE OF REACTION
2. Effect Of pH
 Optimum
pH for most antigen-antibody
reactions = 6.5 to 7.5
 Some antigens show stronger reactivity at
lower pH
 e.g. anti-M, and anti-D at pH 6.5 – 7.0
FACTORS AFFECTING THE
PRIMARY STAGE OF REACTION
3. Ionic Strength
refers to charge concentration of the
suspending (reaction) medium.

decreasing the ionic strength of the
suspending medium reduces the
interfering effects of the electrostatic
barrier…
 THUS, better attraction between antigen
and antibody.

FACTORS AFFECTING THE
PRIMARY STAGE OF REACTION
Low Ionic Strength Saline (LISS)
Consists of 0.2 % NaCl
 Bring about increased rate of antibody
uptake for the Ag during sensitization
shortens incubation period of 15 to 5
minutes.

FACTORS AFFECTING THE
PRIMARY STAGE OF REACTION
4. Time of Incubation
 To
allow antigen/antibody reactions
reach equilibrium
 15 to 60 minutes
FACTORS AFFECTING THE
PRIMARY STAGE OF REACTION
5. Effect Of Antigen-antibody Ratio
 Amounts
of antigen and antibody should
be in optimal proportions
 Optimum ratio is 80 parts antibody to 1
part antigen
 2 drops serum : 1 drop RCS
 Excess in either antigen or antibody
results to non occurrence of lattice
formation leading to a false-negative
result.
FACTORS AFFECTING THE
PRIMARY STAGE OF REACTION
FACTORS AFFECTING THE
SECONDARY STAGE
1. ZETA POTANTIAL
 The surface of red cells carry a negative charge .
 In saline, red cells will attract positively charged
Na+, and an ionic cloud will form around each cell.
 Thus the cells repel each other and stay a certain
distance apart.
ZETA POTENTIAL
• IgG antibodies
cannot cause
agglutination
when zeta
potential exists.
• To overcome
zeta potential
techniques are
needed to
neutralize these
charges.
FACTORS AFFECTING THE
SECONDARY STAGE
2. Effect of Centrifugation
 The antibody - sensitized red cells are
subjected to high gravitational force to
overcome the natural repulsive effect of
the red cells to one another
 Closer
physical proximity (nearness)
increases antigen-antibody bridging
FACTORS AFFECTING THE
SECONDARY STAGE
3. Effect of Immunoglobulin Type
 Most efficient for agglutination reactions.
 The
 The
physical size of IgM
number of antigen binding sites of IgM
molecules (valence 10) compared with IgG
(valence 2), increases the chances of
random antibody-antigen collisions,
leading to a greater chance of effective
red cell cross-linking.
FACTORS AFFECTING THE
SECONDARY STAGE
ENHANCEMENT MEDIA FOR
Ag- Ab REACTIONS
1. ALBUMIN:



1. Reduce the charge density (dielectric
constant) of the red cell suspending medium.
2. Reduce net repulsive force between red cells
3. It replaces the water hydration surrounding the
red cell
Therefore: IgG antibody able to span the
gap between individual positive red cells
to produce agglutination.
ENHANCEMENT MEDIA
FOR Ag- Ab REACTIONS
2. ENZYMES
The proteolytic enzymes:
1) papain (papaya)
2) ficin (figs)
3) bromelin (pineapples)
4) trypsin (lining of a hog's stomach)
Enzyme treatment is known to increase the avidity of both IgM &
IgG Abs. They reduce Zeta potential allowing cells to come closer.
They also remove proteins adjacent to antigens, allowing Abs to
have better access to Ags.

3. ANTIHUMAN GLOBULIN (AHG)
 Produces
a
“bridging effect” by
cross linking the
antibody-sensitized
red cells.
4. POLYETHYLENE GLYCOL:
 Water- soluble polymer used with AHG to bring
sensitized cells close together and facilitate crosslinking and enhancement of agglutination reaction.
Does not produce non-specific reactions.
5. POLYBRENE (hexadimethrine bromide)

Positively charged macromolecule which cause
non-specific aggregation of sensitized red cells
with IgG antibody after incubation with LISS.
Thanks