Ward’s Whodunit? Simulated ABO Blood Typing
Note: WARD'S has developed an alternative blood typing activity that does not use real blood. Students will follow a similar procedure used to type actual human blood and obtain results that closely approximate those obtained by real blood typing.
Introduction
To better understand the ABO and Rh blood groups, and also the procedure of “blood typing”, two important terms must first be defined. An antibody is a protein that is generated by lymphocytes and is a critical component in an immune response. An antibody has very high affinity for a single antigen. An antigen is any molecule that the body perceives as foreign and that generates an immune response (meaning its presence in the body can lead to rapid and prolific antibody production; so an antigen =“antibody-generator”). During an immune response within the body, antigen is bound by antibody and this leads to the swift destruction of the antigen by the immune system.
If very high numbers of antibody molecules bind to high concentration of antigen, “clumping” (also called agglutination) may occur as the mixture becomes insoluble and large. “Clumping” in the blood plasma is not a beneficial response as it can lead to kidney damage, kidney failure, and even death. However, the procedure of ABO blood typing utilizes this clumping response as a “read out” to help identify the blood type of a blood sample. Blood typing is only performed on a blood sample, OUTSIDE of the body, where a clumping response can be safely assessed. Again, clumping or agglutination is not a normal part of a typical immune response but it can unfortunately occur following an incompatible blood transfusion, tissue graft or organ transplant.
Around 1900, Karl Landsteiner discovered that there are at least four different kinds of human blood, determined by the presence or absence of specific glycoproteins on the surface of red blood cells (RBCs). These glycoproteins can function as antigens (as they can trigger an antibody-based immune response if transfused into someone with a different blood type). These RBC surface antigens have been designated as Type A antigen and Type B antigen.
Type A blood has A antigens on the cell surface.
Type B blood has B antigens on the cell surface.
Type AB blood has both A and B antigens on the cell surface.
Type O blood has neither A nor B antigens on the cell surface.
Shortly after birth, antibodies against Type A antigen or Type B antigen begin to build up in the blood plasma- the antibody (or antibodies) produced are OPPOSITE to the antigen(s) present on one’s own RBCs. The antibody levels peak at bout eight to ten years of age, and the antibodies remain, in declining amounts, throughout the rest of a person's life. The stimulus for antibody production is not clear; however, it had been proposed that antibody production is initiated by minute amounts of A and B antigens that may enter the body through food, bacteria, or other means. Humans normally produce antibodies against those antigens that are not on their own RBCs, and a person’s ABO blood type is based on the antigens, not the antibodies, a person possesses. It is important to know the antibodies present in the plasma for a specific ABO blood type:
--A person with Type A blood (type A antigens on the surface of their red blood cells) has anti-B antibodies circulating in their plasma.
--A person with Type B blood has anti-A antibodies circulating in their plasma
--A person with Type AB blood has neither anti-A nor anti-B antibodies in the plasma
--A person with Type O blood has both anti-A and anti-B antibodies in the plasma
IMPORTANT NOTE: During a blood transfusion, billions of RBC’s are transferred from donor to recipient (hence, billions of cell surface antigens are transferred). Only a minute, negligible quantity of donor antibody is transferred, and upon transfer this very small quantity of donor antibody is immediately diluted and thus we do not consider the transfer of antibodies, only antigen. Thus, for a successful transfusion the KEY concept is to only ever inject blood of a type that will NOT trigger an immune response in the recipient. In a transfusion, you need to consider the antigen(s) being administered and the antibodies present in the donor’s plasma. They must NOT bind one another!
An incompatible transfusion will result in antibody-mediated agglutination followed by hemolysis (the rupturing of RBCs). As mentioned above, this undesirable clumping reaction can lead to kidney failure and death.
Of the four ABO blood types (A, B, AB, and O), blood type O, characterized by the absence of A or B antigens, is the most common in the United States, in ~45% of the population. Type A is next in frequency, found in ~40% of the population. The incidences of types B and AB are ~10% and ~4% respectively.
In 1940, Landsteiner and Weiner discovered another class of glycoproteins on the surface of red blood cells called Rh factors. They are called Rh factors because they were first found in rhesus monkeys. About 85% of Caucasians, 94% of Blacks, and 99% of Asians and Native Americans have Rh factors on their red blood cells. An individual who possesses these Rh glycoproteins (which are also deemed antigens) is designated Rh+ (Rh positive); an individual who lacks them is designated Rh- (Rh negative). A person that is Rh+ does not produce Rh antibodies in the plasma (as this would lead to an instantaneous, life threatening condition). Unlike the ABO system, a person that is Rh- does NOT spontaneously produce antibodies to the Rh factors; but they will produce them after exposure to Rh factor. Exposure to Rh factors can occur during a mismatched blood transfusion if Rh+ blood is transfused into an Rh- recipient, or when an Rh- mother carries a fetus who is Rh+. For transfusion purposes (assuming a proper ABO match), Rh- blood can be safely transfused into someone with Rh+ blood. Rh+ blood cannot be given to someone with Rh- blood, as it will result in antibody generation and a subsequent agglutination and hemolytic response.
Table 1- ABO System
Blood Type / Antigens on Erythrocytes / Antibodies in Plasma / Can Give Blood To / Can Receive Blood FromA / A / Anti-B antibodies / A, AB / 0, A
B / B / Anti-A antibodies / B, AB / 0, B
AB / A and B / Neither Anti-A nor Anti-B
antibodies / AB / 0, A, B, AB
0 / Neither A nor B / Both Anti-A and Anti-B antibodies / 0, A, B, AB / 0
Process of Agglutination
There is a simple test to determine blood type, performed with antisera containing high levels of anti-A antibodies, anti-B antibodies, or anti-Rh antibodies. Several drops of each kind of antiserum are added to separate samples of blood (understand that when the term antiserum is used, it simply means a liquid that is almost entirely antibody molecules!). When analyzing the blood typing results in class, you will look at separate reactions performed in 3 separate wells (one well receives anti-A antibodies, one receives anti-B antibodies and the third well receives anti-Rh antibodies). If agglutination (clumping) occurs only in the well to which the anti-A serum was added, the blood type (of the blood in the dish) is A. If agglutination/clumping occurs only in the anti-B mixture, the blood type is B. Agglutination in both the A and B wells indicates that the blood type is AB. The absence of agglutination in the anti-A and anti-B serum wells indicates that the blood type is O.
The blood sample will also be tested for the presence or absence of the Rh factor. The blood sample will be mixed with Anti-Rh serum (remember that this is simply saying that the blood sample will be mixed with anti-Rh antibodies). If this mixture shows agglutination, this is evidence for the presence of the Rh glycoprotein (antigen) on the cells of the blood sample, and this blood would be termed Rh+. Conversely, a lack of agglutination is evidence for the absence of the Rh glycoprotein in this sample, and thus the blood is Rh-.
Table 2- Agglutination Reaction of ABO Blood-Typing Sera
ReactionWell containing Anti-A Serum / Well containing Anti-B Serum / Blood Type
Agglutination / No Agglutination / Type A
No Agglutination / Agglutination / Type B
Agglutination / Agglutination / Type AB
No Agglutination / No Agglutination / Type O
Importance of Blood Typing
As evidenced in the table above, people can receive transfusions of only certain blood types, depending on the type of blood they have. If incompatible blood types are mixed, erythrocyte destruction, agglutination and other problems can occur. For instance, if a person with Type B blood is given a transfusion of Type A blood, the recipient's anti-A antibodies (present in their plasma) will attack the incoming Type A erythrocytes. The Type A erythrocytes will be agglutinated, and hemoglobin will be released into the plasma.
The ABO blood groups and other inherited antigen characteristics of red blood cells are often used in medical-legal situations involving identification of disputed paternity. A comparison of the blood groups of mother, child, and alleged father may exclude the man as a possible parent. 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. For example, a child with a blood type of AB, whose mother is type A, could not have as a father a man whose blood type is O. (NOTE: We will not be covering the genetics of the ABO blood groups in this lab).
Whenever blood has been shed, the identification and typing of the blood stains are of primary importance to the crime investigator. The ABO blood groups are used to screen out possible suspects involved in a crime. The first step in the investigation is to distinguish the bloodstains from other similar looking compounds such as fruit juices, jam, chemicals, paint, etc. Secondly, along with a number of tests to determine the sex of the individual from which the blood came from, and the age of the blood stain, a simple blood typing test is also performed. Although a positive match of the suspect’s blood type is not sufficient to convict someone of a crime, it is one type of evidence that is often obtained during a crime investigation.
CRIME SCENARIO
Crime investigators were called to the scene of a burglary. Mr. Smith had come home, only to find someone robbing his apartment. As the criminal rushed to leave the apartment, he ran into a glass door, cutting his arm and tearing his shirt. The crime investigators were able to remove small pieces of clothing that appeared to be blood stained from the broken glass door. The blood samples from the crime scene, along with the victim’s blood, were sent to the forensic lab to be analyzed. After the crime investigators carefully reviewed all of the available evidence, they apprehended four suspects. The last remaining piece of evidence needed to solve the crime is to match the blood type found at the scene of the crime with one of the suspects’. You, along with your classmates, have been chosen to provide this last piece of evidence and determine which of the suspects is most likely the burglar.
Objective In this investigation, you will assume the role of a forensic scientist as you attempt to solve a crime, using as evidence blood samples found at the scene of the crime. You will determine its blood type and match it to one of the four suspect’s and victim’s blood type.
Materials in minitrays for Who Done It lab
· Wax pencil (for labeling trays)
· 6 blood trays
· stirring picks
· 3 pcs. Blood stained cloth (NOTE: we will NOT use this!!)
· Anti-A Serum (a bottle of antibody, Anti-A antibody)
· Anti-B Serum (a bottle of antibody, Anti-B antibody)
· Anti-Rh0 Serum (a bottle of antibody, Anti-Rh antibody)
· Suspects 1,2,3, & 4 blood
· Victim’s blood
· Crime scene blood (we will use this in place of the piece of cloth)
Each team will determine the blood type of: the victim, the four suspects, and the blood found at the scene of the crime (aka crime scene blood).
1. Use a wax pencil to label each of your blood typing trays as follows:
Tray #1: Crime scene
Tray #2: Victim
Tray #3: Suspect #1
Tray #4: Suspect #2
Tray #5: Suspect #3
Tray #6: Suspect #4
* I recommend placing plain white paper on your lab bench, underneath the trays!
2. FIRST, to determine the type of blood found at the crime scene, place 3-4 drops of “crime scene” blood into each of the A, B, and Rh0 wells of your blood typing tray #1 (“crime scene”).
3. Add 3-4 drops of the anti-A serum (A antibody) on the blood that’s in the “A” well of tray #1.
4. Add 3-4 drops of the anti-B serum (B antibody) on the blood that’s in the “B” well of tray #1.
5. Add 3-4 drops of the anti-Rh0 serum (Rh antibody) on the blood that’s in the “Rh0” well tray #1.
6. Use separate stirring picks to stir each sample of anti-serum and blood. Record your observations and results in the Data Table.
Once you have determined the type of blood found at the scene of the crime, you will then “type” the blood of the victim and four suspects. (Refer to Table 2 if you need help!)
For the directions that follow, you are dispensing all of the blood samples first (to their respective trays) and are then adding the appropriate antibody (antiserum).
7. Place 3-4 drops of the victim’s blood in each of the A, B, and Rh0 wells of Tray #2: Victim.
8. Place 3-4 drops of the Suspect’s #1 blood in each of the A, B, and Rh0 wells of Tray #3: Suspect #1.