The document describes the procedure for performing an antigen-antibody reaction test. It involves collecting whole blood from patients, preparing red blood cell suspensions, and diluting the suspensions in normal saline solution. The diluted suspensions are then added to tubes containing different antisera and incubated. Initial and final grading of agglutination is observed after shaking and further incubation. Anti-human globulin is added to some tubes to differentiate naturally occurring from immune antibodies.

1. Antigen-Antibody Reaction
BSMT- 4C
Group 10
Bacalla, faith
David, Trisha Kamille
Pingul, Anabelle
Sotto, Jonell

2. Procedure

3. Specimen Collection and
Preparation
prepare 2-5% RCS
Collect whole blood
Label 12 tubes (1 through
12)

4. Dilute each tube to 15
drops using NSS
Add a drop of anti-sera
to each tube

5. Initial grading of
agglutination
Shake for 60 seconds

6. Final grading of agglutination
Incubate tubes @ 37 Degrees
Celsius for 60 seconds Celsius
for 60 seconds
2 drops of anti-human
globulin(AHG)

7. Question for Research

8. Differentiate naturally occurring
antibodies from immune antibodies.
Natural Antibodies Natural antibodies
• RBC antibodies found in the serum of • RBC antibodies are considered
individuals never been previously
exposed to RBC antigens by immune when found in the serum of
transfusion, injection, or pregnancy. individuals who have been
• Produced in response to substances transfused or pregnant.
in the environment that are highly • Not generally found in nature
similar to RBC antigen such as pollen
grains and parts of bacteria • Molecular makeup is unique to
membranes. human RBCs.
• Found in nature • IgG antibodies that react best at 37˚C
• Have a repetitive complex pattern.
• Require the use of antihuman
• IgM cold agglutinins, which reacts
best at room temperature or lower, globulin sera (Coombs sera) for
activate complement, and when detection.
active at 37˚C maybe hemolytic. • React with the Rh, Kell, Duffy, Kidd,
• Antigens of the ABH, Hh, Ii, Lewis, and Ss blood groups systems.
MN, and P blood group systems

9. What is zeta potential? Discuss it’s
effect in the blood banking test
results
ZETA POTENTIAL
• A potential is created because of the ionic cloud of cations (positively charged ions) that are
attracted to the zone of negative charges on the RBC membrane.
• The potential around the RBC is called the zeta potential.
• The difference in electrostatic potential between the net charge at the cell membrane and
the charge at the surface of the shear.
• Zeta potential is the difference in charge density between the inner and outer layers of the
ionic cloud that surrounds red blood cells in an electrolyte solution.
• Its the electron cloud that surrounds the RBC and with it there would be less sentization and
resulting to no subsequent agglutination.
• IgM and IgG antibodies have differences in how they react to the same zeta potential.
Reducing the zeta potential allows the more positively charged antibodies to get closer to the
negatively charged RBCs
• Increases RBC agglutination by IgG molecules.

10. http://www.horiba.com/scientific

11. http://onlinelibrary.wiley.com/doi/10.1
111/j.1751-2824.2008.00185.x/full

12. What is zone phenomenon? How does it
affect blood banking tests? How do we
remedy this?
Prozone phenomenon
• An excessive amount of antibody in
serum, relative to the constant antigen in the
testing well. In the presence of excessive
antibody, cross-linking of antigen is not
observed because there are so many
antibodies that each antigen molecule can
bind to a single antibody, such that cross-
linking and lattice formation do not occur.

13. What is zone phenomenon? How does it
affect blood banking tests? How do we
remedy this?
Postzone phenomenon,
• Excessive antigen in serum.
• Serum that are very dilute show little or no
agglutination because there are insufficient
antibodies for cross-linking to occur.

14. What is zone phenomenon? How does it
affect blood banking tests? How do we
remedy this?
Zone of equivalence
• Optimum precipitation occurs because the
number of multivalent site of antigens and
antibodies are approximately equal.
• For a precipitation reaction to be detectable, the
reaction must occur in the zone of equivalence.
• In this zone, each antibody or antigen binds too
more than one antigen or antibody, respectively,
forming a stable lattice or network.

15. What is zone phenomenon? How does it
affect blood banking tests? How do we
remedy this?
• This phenomenon can overcome by serial
diluting of antibody containing serum until
optimum amounts of antigen and antibody
are present in the test system.
• To correct the phenomenon , a repeat blood
specimen be collected a week or more.
• In either situation, the lattice formation and
subsequent agglutination may not occur,
which can give false-negative results.

16. http://www.microbiol.unimelb.edu.au/tea
ching/iLab/antigen_antibody/precipitation
_reactions.html

17. What are the stages of an agglutination
reaction? What steps/ techniques will help
enhance each stage?
Sensitization- the first phase
• Represents the physical attachment of antibody molecules to antigens
on the erythrocytic membrane.
• In this initial reversible interaction, antibodies combine rapidly with
antigenic particles.
• The amount of antibody that will react is affected by the equilibrium
constant, or affinity constant, of the antibody.
• In most cases, the higher the equilibrium constant, the higher the rate
of association and the slower the rate of dissociation of antibody
molecules.
• The degree of association of antigen with antibody is affected by a
variety of factors and can be altered in some cases in vitro by altering
some of the factors.

18. Lattice formation-the second phase
• Establishment of cross-links between sensitized
particles such as erythrocytes and antibodies
• Resulting in aggregation (clumping)
• Much slower process than the sensitization phase
• Depend on the ability of a cell with attached
antibody on its surface to come close enough to
another cell to permit the antibody molecules to
bridge the gap and combine with the antigen
receptor site on the second cell.
• Cross-linking is influenced by factors such as zeta
potential.

19. What is Rouleaux formation?
What is/ are its cause/s?
• Erythrocytes appear as rolls resembling stacks
of coins.
• Presence of rouleaux formation.
• Patients with high or abnormal types of
globulins in their blood, such as in multiple
myeloma, or after receiving dextran as a
plasma expander.
• Pseudoagglutination

20. Name the different potentiators used
in blood banking and give its
mechanism in enhancing
Reagent agglutination? of Antibody ID
Action Type
Saline enhances sensitization of an Primarily IgM; IgG if
antigen. incubated at 37˚C
AHG Cross- links sensitized cells, 1. Polyspecific; anti- IgG +
resulting in visible anticomplement
agglutination 2. IgG monospecific: Anti-
IgG only
22% Albumin* Causes agglutination by IgG
adjusting zeta potential
between RBCs
LISS* Low ionic strength IgG
environment causes RBCs
to take up antibody more
rapidly
PEG* Increases test sensitivity; IgG
Aggregates RBCs causing
closer proximity of RBCs to
one another assisting in
antibody cross -linking
Enzymes Reduces RBC surface Destroys Fya , Fyb , MNS;
charge; Destroys or enhances reactivity to Rh,

21. Reference/s:
Mary Louise Turgeon: Immunology and Serology in
Laboratory Medicine, 4th Edition 2009.
Denise M. Harmening: Modern Blood Banking and
Transfusion Practices, 4th Edition 1999.
Denise M. Harmening: Modern Blood Banking and
transfusion Practices, 5th Edition 2005.
Mary Louise Turgeon: Fundamentals of
Immunohematology, 2th Edition 1995.