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ABO and Rh Blood Typing: An Important Clinical Application of
Biochemistry in the Modern Era with Ties to a Forensic Past
Written by Casey J. McCormick, candidate for the B.Sc. in Chemistry and Biology, as a joint
project between the Millard Oakley STEM Center and the ASBMB UAN of TTU for a
distance K-12 Outreach Model.
OPEN SOURCE – This document may be reproduced for educational purposes.
OBJECTIVES

The following will be the objectives of today’s activity.
 To recognize that antigen-antibody interactions are a type of biochemical reaction
that occurs as an immune response regularly and how this applies to erythrocytes.
 To relate the challenges of antigen-antibody interactions with respect to blood
cells in a clinical setting.
 To demonstrate the role of the central dogma of modern genetics as it applies to
these interactions.
 To learn some basic cells and vocabulary in anatomy, physiology, biochemistry,
and genetics.

BACKGROUND

Blood is the essence of life. The heart beats 75 times per minute in the average Homo
sapien. A normal heart rate is necessary in order to pump the special connective tissue
throughout the entire body, which supplies all of the body’s tissues with nutrients, removes their
waste products, and allows for the diffusion of salts and minerals throughout the system. One of
the most important homeostatic concerns is proper blood flow in the circulatory system. A
blocked blood vessel can result in tissue death due to lack of oxygen delivery and the build-up of
wastes in the tissues, as well as starvation from a lack of nutrients necessary for the cells in that
tissue to carry out their important functions.
Blood consists of plasma, erythrocytes, platelets, leukocytes, and blood proteins.
Proteins in the blood consist of antibodies, osmotic regulatory proteins (i.e. albumin), transport
proteins, and clotting factors. Erythrocytes are also known as red blood cells. These cells have
no nuclei or organelles and they consist mostly of hemoglobin (a special protein carrier for
oxygen and carbon dioxide transport). A leukocyte is a white blood cell. White blood cells are
involved in the immunological processes of the human body and also deal with maintenance
issues such as the degradation of old blood cells and the breakdown of dysfunctional tissues.
They play a major role in the lymphatic system, which filters interstitial fluid in the tissues and
keeps infections from spreading into the blood. Platelets are cell fragments, not cells, that are
involved in blood clotting and repair of vascular damage.
Although there are many aspects we could discuss in great detail about blood, our
discussion for this activity focuses on red blood cells or erythrocytes. The activity you will
perform today involves special structures on red blood cells called antigens. Antigens are
glycoproteins produced by the red blood cells, which can be recognized by the body’s immune
system. From the moment you are born, you have a certain genetic code that produces these
antigens in on the surface of your blood cells. The construction of these antigens consists of a
proteinaceous core with extensions of carbohydrates (sugar molecules—not necessarily sucrose,
the sugar you use in cooking). The antigens we will discuss in this activity are the famous A, B,
and O antigens, as well as the more recently discovered Rh antigen. Karl Landsteiner, M.D.,
received the Nobel Prize in Medicine and Physiology in 1930 for his discovery of the ABO
antigens on erythrocytes. In 1937, Dr. Landsteiner helped Dr. Alexander Wiener (also a
physician) discover the Rh antigens through work with Rhesus monkey test subjects. The Rh
factor is so named because it was discovered using Rhesus monkeys. Of the two discoveries, Dr.
Landsteiner’s discovery of the ABO antigens is considered more profound because it solved a
problem occurring during blood transfusions (the giving of donated blood to patients who have
had significant hemorrhage (blood loss) or other conditions that require replacement of blood).
The reason there was a noted problem with transfusions was due to agglutination of blood
cells when different blood types were mixed with one another in patients. Current research
indicates that upon the second month post-birth, bacteria endogenous to the body (bacteria that
are a part of the human normal flora) aid in the production of antibodies to the antigens that are
not presented on an individual’s specific blood cell type. An antibody is a protein normally
secreted by the B Lymphocytes (a type of leukocyte) in the immune system. They bind to
antigens on foreign bodies, so that the immune system will eliminate the threats. In many cases,
however, binding of antibodies to antigens causes a phenomenon known as agglutination (a
clumping-type of action). Thus, it is extremely dangerous to receive mismatched blood during a
transfusion because it causes the blood to agglutinate, which impedes blood flow through the
small capillaries of the body. Aside from agglutination, antibodies also cause complement
protein complexes to target the transfused red blood cells for destruction by cytotoxic T cells and
other degradative immune cells, which causes a release of cellular debris in the blood, as well as
release of hemoglobin which causes damage to the kidneys and can result in deadly renal failure.
In regard to agglutination reactions, antigens are often called agglutinogens and
antibodies are often called agglutinins. These terms represent the importance of the proteins and
the glycoproteins in the plasma and on the erythrocytes that cause the agglutination reaction that
is not desired. Table I shows which antigens are present on certain blood types and the
antibodies the individual produces against the antigens on the erythrocytes of individuals with
other blood types. Blood types are related to specific genes in the population, as such there are
statistical trends in blood types based on race. Table II shows the breakdown of percentages of
blood types for different races.
Table I – Blood Types, Antigens Produced, and Antibody Production
A
B
Antigens (Agglutinogens) on
Erythrocyte Surface
A Antigens
B Antigens
O
None
AB
A Antigens
B Antigens
ABO Blood Type
Antibodies (Agglutinins)
Produced by the Individual
Anti-B Antibodies
Anti-A Antibodies
Anti-A Antibodies
Anti-B Antibodies
None
Table II – Blood Type Percentages in the Population by Race
ABO Blood Type
AB
B
A
O
Caucasians
4%
11%
40%
45%
Blacks
4%
20%
27%
49%
Asians
5%
27%
28%
40%
Native Americans
Less than 1%
4%
16%
79%
The Rh antigens are either present or not present. However, the body does not naturally
produce antibodies against the Rh antigen. An individual would develop antibodies to Rh
positive blood cells if they did not have the Rh antigen after one exposure to the blood cells.
This is apparent in a special neonatal condition known as erythroblastosis fetalis or hemolytic
disease of the newborn. In this disease, a mother who does not produce Rh antigens has a child
that will be born with Rh antigens. Her first child is born normally, pending no other conditions
related to pregnancy or neonatal conditions, but her second child is attacked by antibodies from
her immune system in the womb that formed from exposure to blood transferred from the
placenta during childbirth of the first child. This condition is now treated with RhoGAM, an
injection of antibodies that do not allow the mother to form immune responses to Rh antigen
presenting blood cells.
When reporting blood cell results clinically, the individual’s blood is tested to determine
what type of ABO antigen is present on their blood cells using antisera (solutions of prepared
antibodies). The result obtained is reported simply as A, B, O, or AB depending on the typing
scenario displayed in Table I. A second symbol of either “+” or “-“ indicates the status of Rh
antigens in the individual. A “+” indicates that the individual has Rh antigens, whereas a “-“
indicates that the individual does not present with Rh antigens. Therefore, a person with A+
blood has A type antigens, produces anti-B antibodies, and has Rh antigens. An A- individual
would have everything except the Rh antigens. Table III summarizes allowable transfusion
matches.
Table III – Blood Types and Transfusion Rules
Blood Type
Patient Can Donate Blood to
Patients with these Antigens
A
B
O
AB
A & AB
B & AB
O, A, B, & AB
AB
Patient Can Receive Blood
from Donors with these
Antigens
O&A
O&B
O
O, A, B, & AB
NOTE: During a transfusion, the patient receives only packed red blood cells. The antibodies
the individual produces reside within the plasma, which is separated from the blood mixture via
centrifugation.
One important thing to remember is that our genes control our antigen presentation since they are
a type of protein material. The central dogma of modern genetics is summed up by the following
statement: “Genes are made up of DNA, which is transcribed into messenger RNA (mRNA),
which is then translated into a protein by the ribosomes of a cell.” Different cells in the body
produce different proteins and have different genes that they normally express. Yet, all cells
contain the same amount of DNA and one can sequence the entire genome from only a single
cell.
Mendelian Genetics can be used to explain the passing of ABO blood traits. However, they do
not follow traditional “true breeding” traits. Rather, the ABO genes are an example of
codominance, especially in the AB phenotype. The following notations are used to describe the
phenotypes present in an individual with respect to the ABO antigens:
IA = A antigens
IB = B antigens
i = no antigens
Note: i is a recessive allele of the gene.
In codominance, both traits of a dominant allele are expressed simultaneously in the
individual. Thus, AB blood has a genotype of IAIB, and both glycoproteins are produced by the
two different chromosomes present in the individual’s genotype. One chromosome came from
the mother of the individual and the other came from the father.
Examples of genotypes and their produced phenotypes:
IAIA = A antigens
IAIB = A antigens and B antigens
IAi = A antigens (heterozygous) IBi = B antigens (heterozygous)
IBIB = B antigens
ii = none (O Blood)
NEAT FACT – ABO antigens used to be used predominately in crime scene investigation
methods, but have now been replaced by PCR (polymerase chain reaction). PCR is more
effective because it can match DNA restriction fragment length polymorphisms that are identical
to an individual's DNA. These vary from person to person, except in the case of identical twins.
Blood typing; however, is inaccurate because so many individuals can share blood types. Thus,
it is less specific than PCR.
EXPERIMENT I
Directions: Read the following information carefully. Students should wear gloves and goggles
or safety glasses due to potential splattering of the reagents. Reagents are not supposed to be
toxic, but they may stain clothing or behave as an irritant.
Today you will be using microreaction plates to perform your ABO/Rh Antigen Assays on
simulated (fake) blood. These kits use a series of water soluble inorganic salts combined with
some red dye. The salts precipitate during positive reactions due to insoluble combinations of
ions in the solutions.
Figure 1 – ABO/Rh Microreaction Plates
Rh
Rh
Rh
A
A
A
B
B
B
Figure 1 shows three examples of what the plates will look like for different steps and reactions. These are
explained as follows. From left to right, plate 1 is what your plate will resemble after adding one drop of the
blood solutions we are using for each test. Plate 2 is an example of the known O- reaction. Plate 3 represents
the reactions that should be observed for the AB+ sample. The small circles are precipitate, which indicates a
positive agglutination reaction.
Materials Checkpoint: You should have two microreaction plates and six plastic micro-stir bars.
Before beginning the experiment please put on a pair of gloves and goggles.
Experimental Procedure
1. Have one person from your group take one of the microreaction plates to the nearest
station with the known blood samples. They will place one drop of the known AB+
blood sample control in each section of the plate and bring it back to the group.
2. Add one drop of Anti-A antisera into the section labeled “A” on the microreaction plate.
Then use the micro-stir bar to gently stir the antisera and the blood sample in the “A”
section together. DO NOT stir so hard that chemicals splatter. It is ok to gently touch the
bottom of the plate in this process with the stir bar, but don’t scratch so hard that plastic
scrapes into the mixture. This could give a false result. Do not contaminate any of the
other sections/wells on the microreaction plate with chemicals from section A.
3. Observe the reaction that occurs in the well. Note you may have to stir for 30-45 seconds
to see some of the reactions.
4. Add one drop of Anti-B antisera into the section labeled “B” on the microreaction plate.
Then use the micro-stir bar to gently stir the antisera and the blood sample in the “B”
section together. DO NOT stir so hard that chemicals splatter. It is ok to gently touch the
bottom of the plate in this process with the stir bar, but don’t scratch so hard that plastic
scrapes into the mixture. This could give a false result. Do not contaminate any of the
other sections/wells on the microreaction plate with chemicals from section B.
5. Observe the reaction that occurs in the well. Note you may have to stir for 30-45 seconds
to see some of the reactions.
6. Add one drop of Anti-Rh antisera into the section labeled “Rh” on the microreaction
plate. Then use the micro-stir bar to gently stir the antisera and the blood sample in the
“Rh” section together. DO NOT stir so hard that chemicals splatter. It is ok to gently
touch the bottom of the plate in this process with the stir bar, but don’t scratch so hard
that plastic scrapes into the mixture. This could give a false result. Do not contaminate
any of the other sections/wells on the microreaction plate with chemicals from section
Rh.
7. Observe the reaction that occurs in the well. Note you may have to stir for 30-45 seconds
to see some of the reactions.
Describe the reactions you observed in each plate below (i.e. do you see agglutination, was there
a color change, etc).
A Well –
B Well -
Rh Well –
8. Have one person from your group take one of the microreaction plates to the nearest
station with the known blood samples. They will place one drop of the known O- blood
sample control in each section of the plate and bring it back to the group.
9. Add one drop of Anti-A antisera into the section labeled “A” on the microreaction plate.
Then use the micro-stir bar to gently stir the antisera and the blood sample in the “A”
section together. DO NOT stir so hard that chemicals splatter. It is ok to gently touch the
bottom of the plate in this process with the stir bar, but don’t scratch so hard that plastic
scrapes into the mixture. This could give a false result. Do not contaminate any of the
other sections/wells on the microreaction plate with chemicals from section A.
10. Observe the reaction that occurs in the well. Note you may have to stir for 30-45
seconds to see some of the reactions.
11. Add one drop of Anti-B antisera into the section labeled “B” on the microreaction plate.
Then use the micro-stir bar to gently stir the antisera and the blood sample in the “B”
section together. DO NOT stir so hard that chemicals splatter. It is ok to gently touch the
bottom of the plate in this process with the stir bar, but don’t scratch so hard that plastic
scrapes into the mixture. This could give a false result. Do not contaminate any of the
other sections/wells on the microreaction plate with chemicals from section B.
12. Observe the reaction that occurs in the well. Note you may have to stir for 30-45 seconds
to see some of the reactions.
13. Add one drop of Anti-Rh antisera into the section labeled “Rh” on the microreaction
plate. Then use the micro-stir bar to gently stir the antisera and the blood sample in the
“Rh” section together. DO NOT stir so hard that chemicals splatter. It is ok to gently
touch the bottom of the plate in this process with the stir bar, but don’t scratch so hard
that plastic scrapes into the mixture. This could give a false result. Do not contaminate
any of the other sections/wells on the microreaction plate with chemicals from section
Rh.
14. Observe the reaction that occurs in the well. Note you may have to stir for 30-45 seconds
to see some of the reactions.
Describe the reactions you observed in each plate below (i.e. do you see agglutination, was there
a color change, etc).
A Well –
B Well -
Rh Well –
Your teacher or laboratory proctor will now wait for the group to catch-up and we
will complete a CPS Clicker Activity to check for understanding.




EXPERIMENT II
Directions: Read the following information carefully. Students should wear gloves, goggles or
safety glasses, and aprons or lab coats while completing the activity due to potential splattering
of the reagents. Reagents are not supposed to be toxic, but they may stain clothing or behave as
an irritant.
Materials Checkpoint: You should have one microreaction plate and three plastic micro-stir
bars. Before beginning the experiment please put on a pair of gloves and goggles.
Experimental Procedure
1. Obtain an unknown blood sample from you lab proctor or teacher.
2. Add one drop of the unknown into each well of the microreaction plate.
3. Add one drop of Anti-A antisera into the section labeled “A” on the microreaction plate.
Then use the micro-stir bar to gently stir the antisera and the blood sample in the “A”
section together. DO NOT stir so hard that chemicals splatter. It is ok to gently touch the
bottom of the plate in this process with the stir bar, but don’t scratch so hard that plastic
scrapes into the mixture. This could give a false result. Do not contaminate any of the
other sections/wells on the microreaction plate with chemicals from section A.
4. Observe the reaction that occurs in the well. Note you may have to stir for 30-45 seconds
to see some of the reactions.
5. Add one drop of Anti-B antisera into the section labeled “B” on the microreaction plate.
Then use the micro-stir bar to gently stir the antisera and the blood sample in the “B”
section together. DO NOT stir so hard that chemicals splatter. It is ok to gently touch the
bottom of the plate in this process with the stir bar, but don’t scratch so hard that plastic
scrapes into the mixture. This could give a false result. Do not contaminate any of the
other sections/wells on the microreaction plate with chemicals from section B.
6. Observe the reaction that occurs in the well. Note you may have to stir for 30-45
seconds to see some of the reactions.
7. Add one drop of Anti-Rh antisera into the section labeled “Rh” on the microreaction
plate. Then use the micro-stir bar to gently stir the antisera and the blood sample in the
“Rh” section together. DO NOT stir so hard that chemicals splatter. It is ok to gently
touch the bottom of the plate in this process with the stir bar, but don’t scratch so hard
that plastic scrapes into the mixture. This could give a false result. Do not contaminate
any of the other sections/wells on the microreaction plate with chemicals from section
Rh.
8. Observe the reaction that occurs in the well. Note you may have to stir for 30-45 seconds
to see some of the reactions.
Describe the reactions you observed in each plate below (i.e. do you see agglutination, was there
a color change, etc).
A Well –
B Well -
Rh Well –
What is unknown did you receive from your teacher or lab proctor? ______________
What is the blood type of your unknown based on your conclusions about the agglutination
reactions?
Your teacher or laboratory proctor will now wait for the group to catch-up and we
will complete a CPS Clicker Activity to check for understanding.
ADVANCED CHALLENGES
Although not necessarily conventional, certain assumed circumstances could allow a
geneticist to prove parenthood by inheritance schemes using Mendelian genetics. You are Dr.
Aaron Greenburg, a biological anthropologist working with the Smithsonian Institution in
Washington, D.C., who has been investigating the Mayan civilization at a southern Mexican site
in Calakmul off of the Yucatan Peninsula. You have been called in by the archaeological team
to explain whether or not remains that they have identified are related to previous remains
identified by you that now rest in the Smithsonian’s collection. Despite your insistence that the
remains be transported to Washington for a more accurate method using PCR, the Mexican
government has refused your request to expedite the remains. You only have enough equipment
to base the preliminary identification off of blood typing to the other two remains. According to
your records, the set of female remains you had previously identified was blood type O. The
male set of remains was blood type A.
If you can make a substantial case for the percentages of similarity between known
genotype scenarios and the blood type of the remains you are identifying, then the U.S. State
Department will step in and fight the Mexican government on your behalf.
Guidelines:
A. In order for the match to be considered sufficient for the U.S. State Department to
intervene, you must prove that the percentage of possible genotypes is greater than
50% with respect to what you know about the possible parental remains.
B. The percentage of genotypes will be determined by finding the fraction of
phenotypically possible genotypes out of all possible genotypes added together from
the multiple Punnett squares. Thus, if there are two squares, then there are eight
possible genotypes. If there are three, then there are 12 possible genotypes total, etc.
1. Drawing several Punnett Squares, predict what the genotypes of the two individuals’
offspring could possibly be.
2. The remains you are identifying have been found to have type A blood. What is the
percentage of that representative phenotype from the combination of punnett squares?
During centrifugation of a blood sample, the antibodies an individual’s body makes against other
blood types stays within the blood plasma. You need to identify a patient’s blood type, but you
only have the plasma from their blood sample because their hematocrit was contaminated and
lost when the test tube containing it was dropped on the floor of the lab. There is no time to
draw another sample from the patient. You run back to the donor rack and grab a bag of A blood
and B blood. You make sure that the Rh factors are negative. You set up a reaction similar to
the ones performed previously in this lab.
Adding type A blood to the sample did not cause agglutination in the A well. Adding type B
blood to the sample caused agglutination. Based on these results, what is the ABO blood type of
the individual?
Since it is not possible to test Rh antigens using plasma, you would inform the blood bank to
deliver what type of blood to the emergency room before informing the phlebotomist to draw a
fresh sample for a better match?
Your teacher or laboratory proctor will now wait for the group to catch-up and we
will complete a CPS Clicker Activity to check for understanding.