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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?