Materials Included In Kit

Additional Materials Required

Prelab Preparation

It is important to review the potential dangers of mixing body fluids (e.g., disease, AIDS) prior to doing this lab. Student health records should be checked to make sure no students have AIDS, hepatitis, etc. Any student with a health problem should not be allowed to type their blood. Written parental permission should be secured for all participating students. This exercise cannot be mandatory and some teachers elect to do it as a demonstration using themselves as the blood source. It would be wise to demonstrate the entire procedure prior to doing the lab, emphasizing the sterile procedures as well as the disposal procedures.

Safety Precautions

It is extremely important that sterile procedures be followed during the entire blood typing procedure and that there is strict adherence to mature laboratory behavior. Blood typing materials must not be shared by two individuals! Each person needs to use his or her own sterile swabs, sterile lancets, blood typing card and mixing sticks. Provide clearly marked containers for student disposal of all used materials. A sharps container must be used for used lancets. Additional biohazard bags should be used for plastic toothpick stirrers and used cotton swabs. As indicated in the Prelab Preparation section, no one with any blood disease or other illness should be allowed to type his or her blood and parental permission and involvement should be encouraged.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All materials used in the lab should be sterilized before disposal. An autoclave or pressure cooker works best for sterilization. All used items should be steam sterilized (following autoclave or pressure cooker directions) in biohazard bags. If these items are not available, all items can be soaked for 24 hours in a 15% bleach solution and then disposed of in biohazard bags. If a local hospital or blood bank is available, they are likely to assist you in disposal of the materials.

Teacher Tips

• Success with this laboratory is dependent upon pre-lab organization. Clearly labeled items, disposal stations and pre-lab discussions about sterile procedures will increase confidence and maturity during this exercise.

• Discuss the entire procedure prior to the lab and demonstrate with a volunteer subject or yourself. Emphasize the need for individual use of materials and the need to be careful with blood samples. Some classes have been successful in typing one person’s blood at a time, cleaning up completely and then doing the next person’s sample. Some teachers have done the laboratory in the school clinic or nurse’s office by appointment. Student interest in blood typing is high; it just needs to be done very cautiously and deliberately.

Sample Data

Student results are usually very straightforward and clumping is obvious. Be sure students do not press on the paper card too hard when mixing. The fibers from the paper of their blood typing card could give a false looking clumping result. Even though the ABO blood typing procedure is reasonably accurate, emphasize that these test results are not absolute. Be especially sensitive if a blood type is secured that seems to be in conflict with genetic predictions based upon parental blood types. Be sensitive to possible social complications that can arise from blood typing discussions.

Teacher Handouts

10179_Teacher1.pdf

Introduction

A blood transfusion with blood of a mismatched blood type usually has serious consequences for the recipient of the blood. Today, complete blood analysis is done with sophisticated, costly equipment before transfusions are done. The basic principles of blood typing will be illustrated in this activity using ABO and Rh blood typing sera.

Concepts

• Antigens

• Multiple alleles
• Rh factor
• Antibodies
• Codominance

Background

General

Early attempts to transfer blood from one person to another produced varied results. Sometimes it seemed to help the recipient and other times it produced very serious consequences. Eventually, it was discovered that each individual has a unique combination of substances in his or her blood. Some of these substances may be compatible with another person’s blood and some may not be compatible. These findings led to the discovery and development of procedures to type an individuals’ blood. It is now known that safe transfusions of blood depend upon properly matching the blood types of the donors and the recipients.

Genetics of Blood Types

ABO blood type is determined by the presence or absence of specific proteins on an individual’s red blood cells. A basic genetic principle is that an individual’s inherited genes determines which proteins are produced in the individual’s body. In the ABO blood typing system (just one of many blood factors) the blood proteins (antigens) are called the A and B proteins. The presence or absence of the A and B proteins on the red blood cells determines the individual’s blood type in the ABO typing system. Individuals whose red blood cells contain protein A and lack protein B have type A blood. Those with protein B and lack protein A are called type B. Individuals with both protein A and protein B are called type AB and individuals with neither of the proteins is called type O.

ABO blood type is a genetic example of multiple alleles. There are three alleles in the gene pool for ABO blood type (i.e., I^A, I^B, i). I^A codes for protein A, I^B codes for protein B and i codes for neither protein A nor protein B. Within this multiple allele pool the gene interactions illustrate both simple dominance as well as codominance. (Remember each individual has only two alleles for each trait even if there are multiple alleles in the gene pool.) When the I^Ai allele combination occurs, the individual is blood type A. When the I^AI^B combination occurs, the I^A and I^B alleles are codominant and the individual is blood type AB. The chart below illustrates the allele combinations, resulting blood type, proteins on the red blood cells, and antibodies in the blood for the four blood types in the ABO system.

Blood Transfusions

Blood groups are critically important with respect to transfusions. If someone with type A is given a transfusion of type B blood the two bloods will interact, clump and clog arteries which will have serious consequences to the individual. The clumping reaction is caused by the interaction of the proteins on the red blood cells and the antibodies present in the blood plasma. Antibodies are produced by the body in reaction to foreign proteins and are important in protecting the body against disease. Antibodies cannot distinguish a disease protein from protein on red blood cells. Individuals do not produce antibodies for pro-teins of their own red blood cells, but do produce antibodies for foreign proteins. Thus, a person with type A blood (A protein on surface of red blood cells) does not produce a antibodies. This person does produce b antibodies. If given the transfusion of type B blood, the antigens and antibodies of the mismatched blood will react and clump (a natural defense mechanism for foreign proteins). The illustrations below, in a very oversimplified way, illustrate the makeup of each of the four blood types.

Using the same illustration scheme, a transfusion of type B blood into an individual with type A blood might be illustrated as follows:

Similarly, a person with type B blood must not be given a transfusion with type A blood.

Because type AB blood lacks both a and b antibodies, it would appear that an AB person could receive a transfusion of blood from any other type. For this reason, type AB persons are sometimes called universal recipients. It should be noted, however, that type A (b), type B (a) and type O (a and b) blood still contain antibodies (either a or b) that could cause clumping of type AB cells. Consequently, even for AB individuals, it is always best to use donor blood of exactly the same type as the recipient blood. If the matching type is not available and type A, B or O is used, it should be transfused very slowly so that the donor blood is well diluted by the recipient’s larger blood volume.

Similarly, because type O blood lacks antigens A and B, it would seem that this blood type could be transfused into persons with blood of any other type. For this reason, persons with type O blood are often referred to as universal donors. Type O blood, however, does contain both anti-a and anti-b antibodies, and thus, if it is transfused into a person of a different blood type it should be done slowly to minimize large clumping reactions.

The bottom line for transfusion is that blood types should be matched for transfusions.

Blood Typing

ABO blood typing is based upon the clumping phenomena of bloods of mixed types. Blood sera antibodies can be isolated from other components of the blood and then used as blood typing sera. Antibodies-b (called Anti-a sera), for example, would clump red blood cells containing A-antigens (type A). Anti-b sera would clump type B blood. Clumping will occur in both sera with type AB blood and in neither sera with type O blood.

In the ABO blood typing procedure, drops of blood are first secured following sterile procedures. A drop of blood is placed in a drop of anti-a sera and another drop is placed in a drop of anti-b sera. The drops are then observed for clumping. The pattern of clumping or non-clumping is interpreted and the blood type determined. The following patterns occur for the various blood types:

There are many other blood typing systems in addition to the ABO classification system. One commonly used system is the Rh factor.

The Rh blood group has several antigen factors on the surface of the red blood cells. If any of the antigens are present on the RBC surface, clumping can occur and the individual is said to be Rh positive (Rh⁺). Conversely, if the red cells lack Rh antigens, the blood is said to be Rh negative (Rh^–).

Just like the ABO system, the Rh factors are inherited. The genetics follow a simple dominant/recessive inheritance with Rh⁺ being dominant. Unlike a and b, antibodies for Rh do not occur continuously in the blood. Instead, they form only in Rh^– persons in response to special stimulation.

If an Rh^– person receives a transfusion of Rh⁺ blood, the recipient’s antibody-producing cells are stimulated by the presence of the foreign Rh antigen and will begin producing anti-Rh antibodies. Generally, no serious consequences result from this initial transfusion. But if the Rh^– person (who is now sensitized to Rh⁺ blood—has antibodies) received another transfusion of Rh⁺ blood at a later time, the donor’s red cells are likely to clump.

A related condition may occur when an Rh^– woman is pregnant with an Rh⁺ fetus for the first time. Such a pregnancy may not be harmful if the fetus’ blood and mother’s blood do not mix during birth. If, however, during birth or miscarriage, some of the infant’s Rh⁺ blood cells get into the mother’s Rh^– blood, it might sensitize her blood and begin the production of Rh antibodies.

If a mother who has already developed Rh antibodies becomes pregnant with a second Rh⁺ fetus, the Rh antibodies can pass through the placental membranes and react with the Rh⁺ fetal red cells, causing them to clump. The fetus often develops a condition knows as erythroblastosis fetalis, which is likely to be very serious for the fetus.

Materials

Safety Precautions

It is extremely important that sterile procedures be followed during the entire blood typing procedure and that there is strict adherence to mature laboratory behavior. Blood typing materials must not be shared by two individuals! Each person needs to use his or her own sterile swabs, sterile lancets, blood typing card and mixing sticks.

Procedure

1. Obtain your personal blood typing materials.

Caution: It is extremely important that materials are not shared or interchanged during any of the steps in the blood typing procedures.

2. Use the non-writing hand as the hand for the source of blood. Swing the hand vigorously several times trying to “shake” blood into the fingertips or massage blood to the fingertip with the thumb.
3. Wipe the middle finger of the non-writing hand with a new, sterile, individualized, saturated, alcohol pad. Allow the finger to air dry and do not contaminate this finger.
4. Using a new, individualized sterile lancet, puncture the tip of the finger once with a firm, quick, downward stroke.

Caution: Discard the lancet after the finger has been lanced in a biohazard container. A LANCET SHOULD NEVER BE REUSED. NEVER USE ANOTHER PERSON’S LANCET!

5. Force a drop of blood from the finger with a little pressure. Wipe away the first drop of blood with the alcohol pad.
6. Force out additional blood and place one large drop in each of the three indicated areas on the blood typing card.
7. Wipe excess blood from the finger with the alcohol pad. Apply light pressure to the lanced finger by placing an adhesive bandage on the finger.
8. Immediately discard the used alcohol pad as directed by your instructor.
9. Add one drop of anti-a, anti-b and anti-Rh sera to the appropriate place on the blood typing card.
10. Mix the anti-Rh sera and its drop of blood with the red colored toothpick. Mix and spread the blood and sera over the entire area allotted for the Rh test on the card. When completely mixed and spread, safely discard the red toothpick in the container designated by your instructor.
11. Mix the anti-a sera and its drop of blood with the blue toothpick in the designated area on the blood typing card. Safely discard the blue toothpick as directed by your instructor.
12. Mix the anti-b sera and its drop of blood with the yellow toothpick in the designated area on the blood typing card. Safely discard the yellow toothpick as directed by your instructor.
13. Allow the blood/sera mixtures to set for about one minute. After a minute, observe the mixtures and look for clumping in all three areas on the card. Results are best seen in the thin layer areas that have been created by mixing. Record your clumping observations on the blood typing card. Circle the ABO blood type and Rh status as indicated by the test results.