Ag-Ab reaction

1. Basic Principles of
Immunohematology

2. • Antigen:
A substance that on introduction give rise to a
formation of antibody that reacts specifically with
the same antigenic substance.
Substance(usuallyforeign) that binds specifically
to an antibodyor a T-cell receptor
Antibody:
Protein(immunoglobulin) that recognizes a
particular epitope on anantigen and facilitates
clearance of that antigen.
Antigen and AntibodyAntigen and Antibody

3. Epitope
• Single antigenic determinants; functionally
they are the parts of the antigen that combine
with the antibody.

4. Characteristics of antigenCharacteristics of antigen
• Factors Contributing to Immunogenicity:
• Chemical structure of antigen
• Protein is the most immunogenic, followed by
carbohydrates, and then lipids
– Antigens composed of oligosaccharides tend to stimulate
IgM type of antibody production
– Antigens which are primarily protein in nature, produce
IgG antibody
• Degree of foreignness
• The greater the difference from self, the Greater likelihood
of eliciting an immune response

5. • Number of antigens introduced
• Number of red blood cells introduced and the
amount of antigen that they carry contribute
tot he likelihood of animmune response
• Route of administration
–Intramuscular or subcutaneous route are
generally better routes for eliciting an
immune response
• Size
• Molecules with a molecular weight greater
thran10,000 daltons are better immunogen

6. ImmunogenicityImmunogenicity
• Ability of an antigen to stimulate the
production of its corresponding
antibody in a person who lacks the
antigen
• Rh D most potent antigenic
• Fya
is poor antigenic

7. Immunogenicity of antigensImmunogenicity of antigens
Antigens System Immunogenicity (%)
D
Rh
70
C 0.2
c 04
E 3.3
e 1.1
K
Kell
10
k 1.5
Fya Duffy 0.6
Jka
Kidd
0.1
Jkb 0.03
S
MNS
0.08
s 0.06

8. ImmunoglobulinsImmunoglobulins
 Five types, viz. IgG, IgM, IgA, IgD & IgE.
 Blood group antibodies are mainly confined to IgG &
IgM.
 IgG is a smaller molecule, with a pair each of heavy
& light chains of amino acids.
 IgG antibody can just coat but not agglutinate the
cells.
 IgM has 5 such pairs joined together by the J chain.
 IgM antibody agglutinate the cells bearing
corresponding antigen.

10. IgM Antibodies (Complete)
• Agglutinate in saline phase
• Pentavalent contains 10 potential
antigen-combining sites
• Usually naturally occurring
• Do not cross placenta
• React at temperature varying
from 4 – 20o
C( some with wide
thermal range)
• Example: ABO antibodies

12. IgG Antibodies (Incomplete)
• Agglutinate in IAT phase
• However, may cause
agglutination in saline phase
using albumin / enzymes
• Monovalent
• Usually immune in nature
• Can cross placenta
• React at 37o
C
• Example: Rh antibodies

13. 14 nm 35 nm
24 nm
IgG IgM
Intracellular distance

14. Complete and Incomplete antibody
Complete antibody -IgM
Incomplete antibody -IgG

15. Naturally occurring Vs ImmuneNaturally occurring Vs Immune
AntibodiesAntibodies
Feature Naturally occurring Immune
Antigen stimulus Obscure, possibly
from microbial origin
Human red cell
antigens
Type of Immunoglobulin IgM IgG
Optimum temperature < 22o
C at 37o
C
Clinical significance Acute HTR HDN, DHTR
Examples ABO antibodies Rh, Kell, Kidd,
Duffy antibodies

16. Clinical Significance of AntibodyClinical Significance of Antibody
Clinically significant
• ABO
• Rh
• Kell
• Duffy
• Kidd
• Ss
Clinically
insignificant
• Lewis
• M,N
• P1
• Lutheran
• A1

17. Types of Antigen-Antibody Reactions
 Agglutination
 Sensitization
 Haemolysis
 Neutralization (inhibition)
 Precipitation

20. Antigen-Antibody Reaction

21. Stages of ag-ab reactionStages of ag-ab reaction
Stage of sensitizationStage of sensitization
• The antibody binds to an
antigen on the red blood
cell membrane. (coating
of red cells with antibody)
• This stage requires an
immunologic recognition
• between the antigen and
antibody.
Stage of agglutination cellStage of agglutination cell
interactioninteraction
• Characterized by
formation of bridges
between sensitized red
cells resulting in visible
aggregate of red cells

22. SensitizatioSensitizatio
nn• The sensitization is defined as binding of
antigen and antibody, in vitro or in vivo with or
without agglutination.

23. AgglutinationAgglutination
• Whenever the sensitized cells
come into contact of each other
the end result is clumping of red
cells known as agglutination.

24. Factors affecting stage of sensitizationFactors affecting stage of sensitization
• Antigen – Antibody ratioAntigen – Antibody ratio
• Ratio of antigen on the red blood cell to antibody in the
serum.
– Two volume of serum and one volume of 5% red cells
– Sensitivity of test depends upon number of antibody molecules
bound per red cells
• Increasing the amount of serum placed in the test
tube increases the concentration of antibodies
available for binding to red blood cell

26. • PHPH
• The ideal pH of a test system for antigen-
antibody reactions is a range between 6.5 and
7.5
• TemperatureTemperature
• Different isotypes of antibodies may exhibit
optimal reactivity at different temperatures
– IgM antibodies react optimally ambient temperatures
while IgG at 37o
C
Factors affecting stage of sensitizationFactors affecting stage of sensitization

27. • Incubation timeIncubation time
– Time needed to reach ag-ab reaction at equilibrium
The length of time recommended for optimal antigen-antibody
reactivity varies with the test procedure and the reagents used
in testing.
Factors affecting stage of sensitizationFactors affecting stage of sensitization

28. • Low Ionic Strength Solution (LISS) MediaLow Ionic Strength Solution (LISS) Media
• They decrease the ionic strength of a reaction medium, which
reduces the zeta potential and therefore allows antibodies to
react more efficiently with RBC membrane antigens.
• LISS media are often used because they result in an increased
rate of antibody uptake during sensitization and a decreased
reaction incubation time
Factors affecting stage of sensitizationFactors affecting stage of sensitization

29. Factors affecting stage of agglutinationFactors affecting stage of agglutination

30. DISTANCE BETWEEN RED BLOOD CELLS.DISTANCE BETWEEN RED BLOOD CELLS.
• The zeta potential, or the force of repulsion
between red blood cells in a physiologic saline
solution, exerts an influence upon the
agglutination reaction
• Red blood cells possess a net negative charge
on the cell surface in a saline suspension.

31. • Cations from the saline environment are
attracted to these negative charges.
• Stable cationic cloud surrounds each cell and
contributes a force of repulsion between
molecules of similar charge
DISTANCE BETWEEN RED BLOOD CELLS.DISTANCE BETWEEN RED BLOOD CELLS.

33. OPTIMAL CONCENTRATIONS OF ANTIGENOPTIMAL CONCENTRATIONS OF ANTIGEN
AND ANTIBODYAND ANTIBODY
• Maximum amounts of agglutination are observed
when the concentrations of antigens (red blood
cells) and antibody (serum) fall within the zone of
equivalence
• Zone of equivalence: maximum agglutination
or precipitation; equilibrium between antigen
and antibody binding.
• Prozone: excess antibody causing a false
negative reaction.

34. 34
Equivalence point:
(suitable proportion between the virus particles
and RBCs)
12/10/17

35. Effect of centrifugationEffect of centrifugation
• The time and speed of centrifugation are
important factors for the detection of
agglutinated red blood cells.
• Centrifugation helps to facilitate the formation of a
latticed network by forcing the red blood cells closer
together in the test environment.

36. • Size & Class of antibodySize & Class of antibody
– IgM antibody, being a pentamer can bind antigen sites
up to 35 nm apart
– IgG antibody, being a monomer can bind antigen sites
up to 14 nm apart
• Antigenic sitesAntigenic sites
– Antigens located on surface of red cell membrane (ABO)
result in strong agglutination
– Antigens embedded in membrane (Rh antiges) result in
weaker agglutination

37. PRINCIPLES OF ANTIBODY POTENTIATOR
• Antibody potentiators, or enhancement
media, are reagents selected to adjust the in
vitro test environment to promote
agglutination.
• Theyare added to blood bank tests to enhance
the detection of antigen-antibody complex
formation.

38. Role of Poteniators
• Enhance antibody uptake (first stage of
agglutination).
• Promote direct agglutination (second stage of
agglutination).
• Or serve both functions

39. Types of potentiators
• Low-ionic strength solution (LISS).
• Bovine serum albumin (BSA).
• Polyethylene glycol (PEG).
• Proteolytic enzymes

40. POTENTIATOR MECHANISMOFACTION
Low-ionic strength solution Increases rate of antibody uptake
Bovine serum albumin Affects the second stage of agglutination
Polyethylene glycol Concentrates the antibody in the test
environment in a low-ionic strength
solution
Proteolytic enzymes Removes negative charges from the red
blood cell membrane, which reduces the

41. Polyethylene Glycol (PEG)
• PEG and polybrene are macromolecule
additives used with LISS to bring sensitized
RBCs closer to each other to facilitate
antibody cross-linking and agglutination
reactions.
• They are often used in place of albumin and
have some advantages and possible
drawbacks.

42. Enzymes
• Proteolytic enzymes (e.g., ficin, papain) cleave
RBC surface glycoproteins and can strengthen
reactions by enhancing antigen expression or
allowing antibodies to bind better to
previously shielded antigens
• Enzymes may also directly destroy other
antigens 3. Useful in antibody identification to
confirm or refute a particular antigen as target
of an antibody

51. Reading/Grading Agglutination ReactionsReading/Grading Agglutination Reactions
• Done by gently shaking the tubes containing
the serum and cells, and observing the cell
button as it is dispersed
• Hard shaking must be avoided because this
may yield to false result
• Attention should also be given to whether
discoloration of the supernatant is present
(Hemolysis).
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52. Reading/Grading Agglutination ReactionsReading/Grading Agglutination Reactions
• Pseudo agglutination or the Rouleaux
Formation also occurs
– Red blood cells appear as stacks of coins.
• Addition of physiologic Nacl will disperse
pseudo agglutination
• Saline Replacement is done after pseudo
agglutination is observed so that it may not
give false negative result due to the dilution
effect of the saline

53. GRADING AGGLUTINATION REACTIONS
GRADE DESCRIPTION Appearance
Negative (-) No aggregates
Weak (+/-) Tiny aggregates that are
barely visible
macroscopically; turbid
and reddish supernatant
1+ A few small aggregates
just visible
macroscopically; turbid
and reddish supernatant
53

54. 2+ Medium-sized aggregates;
clear supernatant
3+ Several large aggregates; clear
supernatant
4+ One solid aggregate; clear
supernatant