explain the biology of blood typing systems ABO and Rh
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BIOL 102: Lab 9
Simulated ABO and Rh Blood Typing
Objectives:
After completing this laboratory assignment, students will be able to:
• explain the biology of blood typing systems ABO and Rh
• explain the genetics of blood types
• determine the blood types of several patients
Introduction:
Before Karl Landsteiner discovered the ABO human blood groups in 1901, it was thought that all blood was the
same. This misunderstanding led to fatal blood transfusions. Later, in 1940, Landsteiner was part of a team
who discovered another blood group, the Rh blood group system. There are many blood group systems known
today, but the ABO and the Rh blood groups are the most important ones used for blood transfusions. The
designation Rh is derived from the Rhesus monkey in which the existence of the Rh blood group was
discovered.
Although all blood is made of the same basic elements, not all blood is alike. In fact, there are eight different
common blood types, which are determined by the presence or absence of certain antigens – substances that
can trigger an immune response if they are foreign to the body – on the surface of the red blood cells (RBCs
also known as erythrocytes).
ABO System:
The antigens on RBCs are agglutinating antigens or agglutinogens. They have been designated as A and B.
Antibodies against antigens A and B begin to build up in the blood plasma shortly after birth. A person
normally produces antibodies (agglutinins) against those antigens that are not present on his/her erythrocytes
but does not produce antibodies against those antigens that are present on his/her erythrocytes.
• A person who is blood type A will have A antigens on the surface of her/his RBCs and will have
antibodies against B antigens (anti-B antibodies). See picture below.
• A person with blood type B will have B antigens on the surface of her/his RBCs and will have antibodies
against antigen A (anti-A antibodies).
• A person with blood type O will have neither A nor B antigens on the surface of her/his RBCs and has
BOTH anti-A and anti-B antibodies.
• A person with blood type AB will have both A and B antigens on the surface of her/his RBCs and has
neither anti-A nor anti-B antibodies.
The individual’s blood type is based on the antigens (not the antibodies) he/she has. The four blood groups
are known as types A, B, AB, and O. Blood type O, characterized by an absence of A and B agglutinogens, is
the most common in the United States (45% of the population). Type A is the next in frequency, found in 39%
of the population. The incidences of types B and AB are 12% and 4%, respectively.
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Table 1: The ABO System
Blood Type
Antigens on RBCs
Antibodies in the Blood
Can GIVE Blood to Groups:
Can RECEIVE Blood from Groups:
A A Anti-B A, AB O, A
B B Anti-A B, AB O, B
AB A and B Neither anti-A
nor anti-B AB O, A, B, AB
O Neither A nor
B Both anti-A and anti-B
O, A, B, AB O
Blood Typing: Process of Agglutination
Blood typing is performed with antisera containing high levels of anti-A and anti-B antibodies/agglutinins. The
simple test is performed as follows:
Several drops of each kind of antiserum are added to separate samples of
blood. If agglutination (clumping of erythrocytes) occurs only in the
suspension to which only anti-A serum was added, the blood type is A. If
agglutination occurs only in the anti-B mixture, the blood type is B (see image).
Agglutination in both samples indicates that the blood type is AB. The absence
of agglutination indicates that the blood type is O.
Table 2: Agglutination Reaction of ABO Blood-Typing Sera
Reaction to Anti-A Serum Reaction to Anti-B Serum Blood Type
Agglutination (clumping)
No agglutination (no clumping)
Type A
No agglutination (no clumping)
Agglutination (clumping)
Type B
Agglutination (clumping)
Agglutination (clumping)
Type AB
No agglutination (clumping)
No agglutination (clumping)
Type O
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Rh System
In the period between 1900 and 1940, a great deal of research was done to discover the presence of other
antigens on human red blood cells. In 1940, an antigen designated as Rh factor, was discovered. Although it
exists as six antigens, the D factor is responsible for the Rh+ condition. The Rh factor is found in 85% of
Caucasians, 94% of African-Americans, and 99% of Asians. An individual who possesses these antigens is
designated as Rh+; an individual who lacks them is designated Rh-. The anti-Rh antibodies of the systems are
not normally present in the plasma, but anti-Rh antibodies can be produced upon exposure and sensitization to
Rh antigens.
The genetics of the Rh blood group system is complicated by the fact that more than one antigen can be
identified as the result of the presence of a given Rh gene. Initially, the Rh phenotype was thought to be
determined by a single pair of alleles. However, there are at least eight alleles for the Rh factor. For the
purpose of simplicity, consider one allele: Rh+ is dominant over Rh-. Thus a person with Rh+/Rh-
heterozygous genotype has Rh+ blood.
Importance of Blood Typing
Early attempts to transfer blood from one person to another produced varied results. If incompatible blood
types are mixed, erythrocyte destruction, agglutination and other problems can occur. For instance, if a person
with Type B blood is transfused with blood type A, the recipient’s anti-A antibodies will attack the incompatible
Type A erythrocytes. The Type A erythrocytes will be agglutinated, and hemoglobin will be released into the
plasma. In addition, incoming anti-B antibodies of the Type A blood may also attack the Type B erythrocytes of
the recipient with similar results. This problem may not be serious, unless a large amount of blood is
transfused.
The ABO blood groups and other inherited antigenic characteristics of red blood cells are often used in
medico-legal situations involving identification or disputed paternity. In paternity cases a comparison of the
blood groups of mother, child, and alleged father may exclude the man as a possible parent of the child. For
example, a child of blood type AB whose mother is Type A could not have as a father a man whose blood
group is Type O. Blood typing does not prove that an individual is the father of a child, it merely indicates
whether or not he is a possible parent.
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The Genetics of Blood Types
Alleles are different versions of the same gene that can occupy the same locus (gene location on a
chromosome). There are usually two alleles of each gene. Humans have two copies of each gene because
they receive one copy from their mother and one copy from their father. If they receive two of the same alleles,
they are considered homozygous. If they have two different alleles, they are considered heterozygous. Alleles
can also be dominant and recessive. Alleles are dominant when the presence of one allele is sufficient to
express the trait and recessive when two copies of the allele must be present to express the trait.
The human blood types A, B, AB, and O are inherited by multiple alleles. Multiple alleles refer to three or more
genes that occupy a single locus. In the case of blood types, there are three versions of the gene which
encodes agglutinogens: A, B and O. The A and B alleles are both dominant and are considered co-dominant.
The O allele is recessive to both A and B alleles.
The alleles for blood types are often designated with the letter I with a subscript:
• The A allele is designated IA and codes for the synthesis of agglutinogen A
• The B allele is designated IB and codes for synthesis of agglutinogen B
• The O allele is designated i or IO and does not produce any antigens.
The phenotypes listed in the table below are produced by the combinations of the three different alleles IA, IB,
and IO.
Using Punnett Squares to Determine Future Genetic Combinations
A Punnett square is a chart which shows/predicts all possible gene combinations in a cross of parents (whose
genes are known). Punnett squares are named for an English geneticist, Reginald Punnett. He discovered
some basic principles of genetics, including sex linkage and sex determination. He worked with the feather
color traits of chickens in order to quickly separate male and female chickens.
Punnett squares can also be used to predict the blood type of future offspring between two people with a
known genotype. When creating the chart, the first step is to designate letters for dominant and recessive
alleles. It has been previously mentioned that A (IA) and B (IB) are both dominant alleles while O (i) is
recessive; therefore, this step is complete. The second step is to write the genotype (genetic combination) of
each parent and the third step is to list the alleles that each parent can contribute. If the parent is homozygous
(both alleles are either dominant or recessive), then she/he can only pass on the dominant allele that she/he
possesses. If the parent is heterozygous (one allele is dominant and the other allele is recessive or she/he has
both A and B dominant alleles), then he/she can pass on either allele. The fourth step is to draw the Punnett
square (one large square containing four smaller squares) and write the possible genes of one parent along
Table 3: Phenotypes and Possible Genotypes
Phenotype Possible Genotypes
A IA IA (homozygous dominant A) OR
IA i (heterozygous A)
B IB IB (homozygous dominant B) OR
IB i (heterozygous B)
AB IA IB (co-dominant AB)
O ii (homozygous recessive O)
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the top and the possible genes of the other parent along the left side. The fifth step is to fill the smaller square
by transferring in the parental letter above the square and the parental letter to the left of the square. The sixth
step is to list all of the possible genotypes (the combinations in each small square) and resultant phenotypes
(physical trait). Figure 1 below is of a cross (mating) between a person who is homozygous dominant A (type
A) and a person who is homozygous recessive (type O).
All of the children would have a heterozygous A genotype and blood type A phenotype.
IA IA
i IA i IA i
i IA i IA i
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LAB DATASHEET Purpose Each group will perform blood typing analyses to determine the unknown blood types of four patients using the
ABO and Rh factor systems.
Procedure
1. Obtain four (4) blood typing trays and use the wax pencil to label them as follows: P1, P2, P3, and P4.
2. Place five (5) drops of Patient 1 Simulated Blood Sample in each well (A, B, and Rh) of the P1 tray.
a. Place three (3) drops of Anti-A Simulated Serum in Well A and mix the blood and serum with a stirring
stick for ten (10) seconds.
b. Place three (3) drops of Anti-B Simulated Serum in Well B and mix the blood and serum with a stirring
stick for ten (10) seconds.
c. Place three (3) drops of Anti-Rh Simulated Serum in Well Rh and mix the blood and serum with a
stirring stick for ten (10) seconds.
d. Carefully examine each well to determine if the simulated blood in each well has clumped
(agglutinated). Record your results and observations in Table 4.
3. Place five (5) drops of Patient 2 Simulated Blood Sample in each well (A, B, and Rh) of the P2 tray.
Repeat directions “a-d” listed under Step 2.
4. Place five (5) drops of Patient 3 Simulated Blood Sample in each well (A, B, and Rh) of the P3 tray.
Repeat directions “a-d” listed under Step 2.
5. Place five (5) drops of Patient 4 Simulated Blood Sample in each well (A, B, and Rh) of the P4 tray.
Repeat directions “a-d” listed under Step 2.
6. Thoroughly rinse all trays and stirring sticks and return to their proper location.
Table 4: Agglutination Reaction Results
Anti-A
Serum
(+ or -)
Anti-B Serum
(+ or -)
Anti-Rh
Serum
(+ or -)
Observations
(Clumping?) Blood Type
Patient 1:
Mr. Smith
Patient 2:
Mr. Jones
Patient 3:
Mr. Green
Patient 4:
Ms. Brown
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Analysis of Results
1. What ABO agglutinogens are present on the red blood cells of Mr. Green’s blood?
2. What ABO agglutinins are present in the serum of Mr. Green’s blood?
3. If Mr. Jones needed a transfusion, what ABO type(s) of blood could he safely receive?
4. If Ms. Brown were serving as a donor, what ABO blood type(s) could receive her blood safely?
5. Why is it necessary to match the donor’s and the recipient’s blood before a transfusion is given?