1. A Simulated Experiment for ABO Blood Typing
Amanda J. Powell
Department of Chemistry, Bowling Green State University, Bowling Green, OH 43403; *powelaj@bgsu.edu
Dr. Stephania Jo Messersmith
Department of Chemistry, Bowling Green State University, Bowling Green, OH 43403; *messers@bgsu.edu
Abstract
One of the greatest challenges of
teaching the concept of blood typing is the
incorporation of common experimental
techniques into the teaching laboratory.
Instructors will often run into problems with
finding both legal and environmentally safe
chemicals to teach popular practices used by
forensic scientists, biology technicians and
others who use this type of testing. This
laboratory exercise will specifically focus on
using practical methods to teach students
about the concept of blood typing by
eliminating the need to use real blood and
utilizing reactions from common chemical
demonstrations. This lab uses chemical
reactions that have commonly been used
as demonstrations in order to simulate
blood typing—reducing the biological
hazards. It also uses chemicals that can
be disposed of down the drain after the
solids have been removed.
Background
The concepts behind blood typing are
essential to almost all science courses.
Blood typing is used in almost every
forensic and biology laboratory and it is
critical that students understand the theories
behind the procedures used in determining
blood types. This experiment will focus on
accomplishing this without using human
blood.
Blood typing relies on the interaction
between cell-surface proteins on
erythrocytes and antibodies. The antigens on
red blood cells correlate directly with the
blood type. Researching activities on blood
typing gave detailed examples of the process
behind agglutination and coagulation, these
articles can be found in the supplied
references1-5
. Antibodies, proteins specific
for an antigen, are produced when the
erythrocyte antigens are recognized as
“foreign.” A response will be elicited that
results in the clumping together of the red
blood cells by the antibodies, a process that
is referred to as agglutination. Specific
antigens have corresponding antibodies. For
example, antigen A will have proteins
present on its surface that will recognize and
adhere to proteins present in Antibody A.
No clumping takes place if the antibodies do
not recognize their corresponding antigen.
Blood type O will have no antigen
proteins present on its surface and therefore
both Anti-A and Anti-B will be produced in
response against it. Blood type AB is the
opposite in that it has both antigens A and B.
The immune system will not produce any
antibodies against type AB.
Table 1: Blood Types and Corresponding Antibodies and Antigens
1
BLOOD
TYPE
ANTIGEN ANTIBODY
A A Anti-B
B B Anti-A
AB A and B Neither
O None Anti-A and
Anti-B

2. Experimental
Overview
Table 2: Chemicals used in Simulated Blood Agglutination
The purpose of this exercise is to
demonstrate the concept of agglutination as
relevant to determining a person’s blood
type. We focus on using chemicals common
to most laboratories in order to simulate the
reaction between blood antigens and
antibodies. This experiment allows
instructors to teach the theory of blood
typing without the dangers of actually using
real blood.
Chemicals
In order to imitate the reaction between
blood cell antigens and their corresponding
antibodies, chemicals were chosen that gave
similar results. Great care was used to
ensure that the correct antibodies
precipitated with the corresponding antigens
and not with any others. The reaction for
blood type A will be a chemical reaction for
the production of Sterno, a gel, as found in
Chemical Demonstrations: A
Sourcebook for Teachers1
. Similarly,
the chemical reaction for blood type B will
be taken from the same source.
We first focused on simulating the
agglutination of blood type A with its
corresponding antibody, anti-A. In order to
imitate this reaction, a solution of calcium
acetate was used to replace blood type A and
100% ethyl alcohol was used as anti-A.
Similarly, we researched chemicals that
would react to imitate the agglutination of
blood type B with its corresponding
antibody, anti-B. A mixture of Borax®
detergent and aluminum sulfate was reacted
with sodium bicarbonate to give the
anticipated precipitate. High purity (98%)
egg albumin was also tried in place of the
Borax® and gave a similar result, a lower
purity sample (62-88%) was tried but it did
not work. The egg albumin is not
recommended due to the high price
compared to the Borax®.
Simulation of the AB blood type was
simply done by combining the mixtures used
for the separate blood types A and B at a
one-to-one ratio. The resulting mixture gave
precipitate with the above-mentioned anti-A
and anti-B chemicals. Water was used for
blood type O, giving no reaction with either
the anti-A or anti-B serums.
Procedure
The procedure for this experiment is
straightforward. Similarly, the chemical
reaction for blood type B will be taken from
the same source. The “clumping” together of
antigens due to antibodies will be simulated
by simple chemical reactions that produce a
solid-like precipitate. Food coloring is used
for convenience and the sake of
demonstration purposes. Red food coloring
can be added to the blood serums and yellow
to their corresponding antibodies. A problem
that arose was the coloration of ethanol. It
does not seem to pick up the food coloring
well.
The instructor will begin by first
preparing the solutions of fake blood and
serums before hand. First, start with blood
2
CHEMICAL VOLUME/MASS
BLOOD
TYPE A
Calcium
Acetate
17g
Tap Water 100mL
ANTI-B 100%
Ethanol
2mL
BLOOD
TYPE B
Borax® 1.0g
Aluminum
Sulfate
7.0g
Tap Water 50mL
ANTI-A Sodium
Bicarbonate
1.5g
Tap Water 150mL

3. type A. A solution of saturated calcium
acetate is prepared by dissolving calcium
acetate in distilled water with stirring. This
could take a few minutes. Anti-A is simply
100% ethyl alcohol. The exact masses and
volumes of the mixtures can be found in the
provided table.
Blood type B is a solution of Borax®
detergent, aluminum sulfate and water. After
measuring out the given quantities of
laundry detergent and aluminum sulfate,
grind them both together in a mortar until
the mixture appears as a fine powder with no
obvious clumps. Dissolve this powder in
warm water and then filter through
quantitative grade filter paper. Dissolving
sodium carbonate in water as well can make
antibody B.
To simulate blood type AB, a mixture of
the solution used for type A and for type B
was produced. Simply use a one-to-one ratio
of the two solutions and use the antibodies
as previously explained. Blood type O will
simply be distilled water, eliciting no
reaction from either antibody A or antibody
B.
After all of the solutions have been
prepared, it is a good idea to label the
beakers. For demonstration purposes, a drop
of red food coloring was added to the blood
serums and a drop of yellow food coloring
was added to the antibody solutions.
Students can begin the experiment either
with blood type A or B. They will mix
together the calcium acetate solution with
100% ethyl alcohol on a small watch glass
or well plate. A clear gel like substance will
precipitate out, imitating the agglutination of
blood type A with anti-A. A similar reaction
will occur with type B and anti-B but instead
of a clear gel, white foam will be produced.
Using a macro scale approach to this
experiment will have the reactions looking
like the clumping of erythrocytes.
Blood type AB should react with both
anti-A and anti-B, while type O will produce
no reaction. Students should take care to
record all results.
Clean-Up
As stated before, this experiment is
aimed to be an environmentally friendly
procedure, in which most of the materials
can be disposed of by simply disposing
down the drain, after the liquid is decanted
from the solids. The solids should be
collected for disposal and include calcium
acetate and aluminum hydroxide.”
Hazards
Most of the chemicals used are common
to most households, therefore there are a few
risks associated with the materials.
Ethanol is flammable and if inhaled,
respiratory tract irritation can occur and it is
known to cause skin irritation.
For more information on any of the
compounds used in this exercise, please
refer to the appropriate MSDS information.
Conclusion
After vigorous research, it was found
that very few, if any, experiments have been
published that can successfully demonstrate
the reaction between erythrocytes and their
corresponding antibodies. The method
outlined by Sharp and Smailes uses an
experiment with a lead compound to imitate
the agglutination of blood cells2
. Our
experiment seeks to use chemicals with
fewer health hazards and easier disposal
methods. This experiment seeks to provide
chemicals of low toxicity, and ones that are
inexpensive as well.
The reactions that occur do not fully
resemble the actual reactions agglutination
reactions of blood. However, this
experiment is useful in simply
demonstrating what takes place to entry-
level students. It is a good hands-on
experiment that better illustrates the
3

4. concepts associated with blood typing. This
experiment can be used at the high school or
college level.
Note
If watch glasses are not available,
instructors can use small test tubes as well.
This experiment is designed for a micro-
level approach and thus, the quantities are
small.
To the authors’ knowledge, all current
simulated bloody typing experiments use
chemicals that are either toxic to humans
and/or the environment3
.
Literature Cited
1. Summerlin, L.R. (1985) Production
of sterno: a gel. Chemical
Demonstrations: A Sourcebook for
Teachers, 134.
2. Sharp, J.D., & Smailes, D.L.
(1989). How-to-do-it: a simulation
of the blood type test. The
American Biology Teacher, 51(4),
232-33.
3. Inheritance and blood typing.
(2006). Vanderbilt Student
Volunteers For Science, Retrieved
from vaderbilt.edu/vsvs.
4. Summerlin, L.R. (1985) Production
of a foam. Chemical
Demonstrations: A Sourcebook for
Teachers, 135.
5. Wake, C. (2005). Abo/rh blood-
typing model: a problem-solving
activity. The American Biology
Teacher, 67(3), 158-62.
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