Anatomy

BIOLOGY

Blood Typing with Simulated Blood

Investigation Manual

BLOOD TYPING WITH SIMULATED BLOOD

Key Personal protective equipment (PPE)

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photograph results and

submit

stopwatch required

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Overview Blood groups are genetically determined, and not influenced by environmental factors. Blood typing is conducted to determine a person’s blood group. In this investigation, students are intro- duced to the ABO and Rh blood typing systems, the process of agglutination, and the use of agglutination in the typing of human blood. Students carry out a blood typing procedure using simu- lated (synthetic) blood and antisera.

Outcomes • Determine blood types using simulated blood and antisera. • Describe the factors determining ABO and Rh blood types and

their inheritance. • Identify compatible blood types for transfusion. • Explain how parental blood type may affect the health of a fetus. • Predict agglutination reactions when incompatible whole blood

encounters antibodies from blood serum.

Time Requirements Preparation .................................................................... 10 minutes Activity 1: Blood Typing with Simulated Blood ............. 30 minutes

Table of Contents

2 Overview 2 Outcomes 2 Time Requirements 3 Background 6 Materials 6 Safety 6 Preparation 7 Activity 1 7 Disposal and Cleanup

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Background When an individual must receive a blood trans- fusion, it is critically important to match his or her blood type to that of the appropriate donor. A mismatch could result in a serious and potentially fatal transfusion reaction. The reac- tion between mismatched blood types occurs because the recipient has antibodies, proteins generated by the immune system, in his or her blood serum, the liquid portion of the blood without clotting factors. These antibodies recog- nize and bind specifically to antigens, which are molecules on the surfaces of the donor’s red blood cells (RBCs). This causes clumping, or agglutination, of donor RBCs.

There are several different antigens on the surface of RBCs. The two most important types to consider for a blood transfusion are the A and/or B antigens and the Rh (D) antigen. The presence or absence of A and B antigens can be used to identify an individual’s blood type within the ABO group system. Someone with type A blood carries the A antigen on the surfaces of RBCs, and the immune system produces B antigen-specific antibodies. If he or she receives blood that was donated by someone with B or AB blood, the anti-B antibodies will bind to the B antigens and cause the RBCs to agglutinate. Likewise, a person with type B blood has the B antigen on the surfaces of RBCs and produces antibodies against the A antigen. Someone with type AB blood has both A and B antigens and does not produce antibodies against either antigen. An individual with type O blood does

not have A or B antigens and generates anti- bodies against both A and B.

The Rh antigen belongs to the Rh blood group system, which was named for the rhesus monkeys in which it was first studied. An Rh-positive individual has the Rh antigen on the surfaces of RBCs and does not produce anti-Rh antibodies. An Rh-negative person lacks the Rh antigen and will produce anti-Rh antibodies. The Rh antigen is not only important for matching the transfusion to the recipient’s blood, but it is also a consideration during pregnancy and birth. Hemolytic disease of the newborn (HDN) is an often fatal condition that may occur when an Rh-negative woman carries an Rh-positive fetus. This situation can only arise if the father is Rh positive. If both parents are Rh negative, there is no risk of the fetus being Rh positive. However, if the fetus is Rh positive, the moth- er’s anti-Rh antibodies may cross the placenta, enter the bloodstream of the fetus, and attack the RBCs. HDN does not usually occur with the first Rh-positive fetus, because the mother’s immune system is not exposed to the antigen until the time of birth. However, if the mother later becomes pregnant with another Rh- positive fetus, her immune system will produce anti-Rh antibodies. HDN once caused the deaths of many babies. However, an injection of anti-Rh antibodies during pregnancy can shield the antigen from the immune system, thus protecting the fetus.

BLOOD TYPING WITH SIMULATED BLOOD

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recipient’s anti-B antibodies would cause the donated cells to agglutinate. It is important to remember that ABO and Rh are only two of several different groups of blood types, and there are some rare blood types besides these two commonly used systems.

The antigens and antibodies that are associated with each of the different ABO and Rh blood types are summarized in Figure 1 below.

When the ABO and Rh blood groups are combined, the basic information needed to start cross-matching blood donors and recipients is obtained. For example, someone with type A+ blood could safely receive blood from another individual with type A+ blood. In an emergency situation with no A+ donor blood available, he or she could receive blood from certain other blood types: A–, O+, or O–. Types B+, B–, AB+, and AB– could not be used, because the A+

Blood A Anti-A B Anti-B Rh Anti-Rh Type Antigen Antigen Antigen Antigen Antigen Antigen

A+ ✓ ✗ ✗ ✓ ✓ ✗

A– ✓ ✗ ✗ ✓ ✗ ✓

B+ ✗ ✓ ✓ ✗ ✓ ✗

B– ✗ ✓ ✓ ✗ ✗ ✓

AB+ ✓ ✗ ✓ ✗ ✓ ✗

AB– ✓ ✗ ✓ ✗ ✗ ✓

O+ ✗ ✓ ✗ ✓ ✓ ✗

O– ✗ ✓ ✗ ✓ ✗ ✓

Figure 1.

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The production of the H antigen is controlled by a gene locus with two alleles (H and h) on chromosome 19. These are independent of the ABO locus, which is on chromosome 9. Because the h allele is uncommon, the genotype hh is extremely rare. The hh individuals produce RBCs without the H antigen, and this is a condi- tion called the Bombay phenotype, as it was first discovered in Bombay, India. In the absence of the H antigen, A and B antigens have nothing with which to bind. As a result, when individuals possessing the Bombay phenotype are tested with anti-A and anti-B antibodies, they appear to belong to group O. However, these individuals cannot receive blood from type O donors who have H antigens, because their hh RBCs are agglutinated by anti-H antibodies.

Testing of Simulated Blood The test procedures used in this investigation are the same as those used to test real blood. However, only simulated blood and antisera are included. This eliminates any risk that may be associated with the exposure to real blood or blood products. The materials in this kit may be discarded after use. There are no biological materials in the simulated blood or antisera that would cause health hazards when discarded.

The inheritance of blood types is well under- stood. The inheritance of ABO blood groups is of particular interest, because it provides an example of codominance, which is a situation in which two different versions, or alleles, of the same gene are both expressed. The IA or IB allel codes for enzymes that catalyze the binding of the A or B antigen, respectively, to the surface o RBCs. The i allele eliminates the ability to bind or B antigens. Thus, an individual with the type A blood phenotype (observable trait) could hav the genotype (genetic makeup) IAIA or the geno- type IAi. Likewise, someone with type B blood could have genotypes IBIB or IBi. Type AB blood requires the IAIB genotype, and type O blood requires the ii genotype.

People with type O blood were once thought to lack antigens on the surface of their RBCs, and those with type O– blood were referred to as “universal donors.” We now know that all ABO blood groups have a weak H antigen. The presence of the IA or IB allele actually codes for enzymes that catalyze the binding of the A or B antigen to the H antigen. The i allele eliminates the ability to bind the A or B antigen to the H antigen. Therefore, type O RBCs have more unbound H antigens.

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BLOOD TYPING WITH SIMULATED BLOOD

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Materials Included in the materials kit:

Blood typing 4 Simulated Simulated slide blood dropper anti-A serum

vials dropper vial

Simulated Simulated 12 Mixing anti-B serum anti-Rh (D) sticks (4 blue, dropper vial serum dropper 4 yellow, and

vial 4 white)

Needed but not supplied: • Stopwatch or timer with seconds • Pen or pencil • White paper • Paper towels • Access to sink with running water • Digital camera (or mobile device with

camera)

Reorder Information: Replacement supplies for the Blood Typing with Simulated Blood investigation can be ordered from Carolina Biological Supply Company, kit 580172.

Call 800-334-5551 to order.

Safety Wear your goggles, gloves, and lab apron at all times while conducting this investigation.

Read all the instructions for this laboratory activity before beginning. Follow the instruc- tions closely and observe established laboratory safety practices, including the use of appro- priate personal protective equipment (PPE) as described in the Safety and Procedure sections.

Do not eat, drink, or chew gum while performing this activity. Wash your hands with soap and water before and after performing the activity. Clean up the work area with soap and water after completing the investigation. Keep pets and children away from lab materials and equip- ment. Do not attempt to use real blood in this investigation. The simulated antisera will not work with real blood.

Preparation 1. Review the background information and

activity instructions. 2. Obtain all items from Materials, and PPE.

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ACTIVITY

ACTIVITY 1 A Blood Typing with Simulated Blood

1. Put on PPE: gloves, lab apron, and goggles. 2. Using the dropper vial, place a drop of the

first simulated blood sample in each well of the blood typing slide. Replace the cap on the dropper vial.

Caution: Always replace the cap on one vial before opening the next vial to prevent cross-contamination.

3. Add a drop of simulated anti-A serum (blue) to the well labeled A. Replace the cap.

4. Add a drop of simulated anti-B serum (yellow) to the well labeled B. Replace the cap.

5. Add a drop of simulated anti-Rh serum (clear) to the well labeled Rh. Replace the cap.

6. Using a different color-mixing stick for each well (blue for anti-A, yellow for anti-B, white for anti-Rh), gently stir the simulated blood and antiserum drops for 30 seconds.

Caution: Remember to discard each mixing stick after a single use to avoid sample contamination.

7. Carefully examine the thin films of liquid mixture that are left behind. If a film remains uniform in appearance, there is no agglutination. If the sample appears granular, agglutination has occurred. Determine the blood type of the sample using the table below as a guide. Establish whether or not agglutination has occurred in each sample. A positive agglutination reaction indicates the blood type. Record your results in Data Table 1 for this activity.

8. Document your results by taking a picture of the slide. Document which simulated blood sample was used.

9. Thoroughly rinse the blood typing slide, and dry it with a paper towel.

10. Repeat steps 2 through 9 for the other simulated blood samples (2, 3, and 4).

Disposal and Cleanup 1. Dispose of solutions down the drain with the

water running. Allow the faucet to run a few minutes to dilute the solutions.

2. Rinse and dry the lab equipment and return the materials to your equipment kit.

3. All other investigation materials may be disposed of as household trash.

4. Sanitize the workspace.

ABO/Rh Sample Anti-A Anti-B Anti-Rh Blood Type

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2

3

4

Data Table 1.

BIOLOGY Blood Typing with Simulated Blood

Investigation Manual

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