Download BIOL242 Bloodtyping

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
From
A
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
Reaction
Well 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”).
Add 3-4 drops of the anti-A serum (A antibody) on the blood that’s in the “A” well of tray #1.
Add 3-4 drops of the anti-B serum (B antibody) on the blood that’s in the “B” well of tray #1.
Add 3-4 drops of the anti-Rh0 serum (Rh antibody) on the blood that’s in the “Rh0” well tray #1.
Use separate stirring picks to stir each sample of anti-serum and blood. Record your
observations and results in the Data Table.
3.
4.
5.
6.
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.
9. Place 3-4 drops of the Suspect’s #2 blood in each of the A, B, and Rh0 wells of Tray #4: Suspect
#2.
10. Place 3-4 drops of the Suspect’s #3 blood in each of the A, B, and Rh0 wells of Tray #5: Suspect
#3.
11. Place 3-4 drops of the Suspect’s #4 blood in each of the A, B, and Rh0 wells of Tray #6: Suspect
#4.
12. Add 3-4 drops of the simulated anti-A serum to each “A” well on the five trays.
13. Add 3-4 drops of the simulated anti-B serum to each “B” well on the five trays.
14. Add 3-4 drops of the simulated anti- Rh0 serum to each “A Rh0” well on the five trays.
15. Use separate stirring pick to stir each sample of serum and blood. Record your observations and
results in the Data Table.
Name(s)________________________________________
_________________________________________
Please turn in ONLY these pages. You may work hand in one answer sheet for your lab group.
Fill in the table below. I would suggest writing the words “clumping” or “no clumping” (or
agglutination or no agglutination) into each of the first three columns, but you can devise another
system. Clearly write in the Blood Type for each sample- be sure to include the ABO type AND if the
blood is Rh + or Rh-. All clumping reactions may not look identical! But the presence of ANY type of
clumping/turbidity/cloudiness is likely a positive result for agglutination.
Data Table
Blood Source
Anti-A
Serum
Anti-B
Serum
Anti- Rh0
Serum
Blood Type
Observations
Crime Scene
Victim
Suspect 1
Suspect 2
Suspect 3
Suspect 4
1. A. Based on the blood typing results, who would you say was the lead suspect?
2. Does the presence of someone’s blood at a crime scene directly prove that they are guilty of a
burglary? Defend your answer.
3. How would you respond- The burglar denies having the blood type you identified, since he has
proof that he had just received a large quantity blood transfusion with type O blood.
A. First, does a blood transfusion change your blood type, why or why not?
B. Specifically, how does the addition of Type O blood to the burglar’s bloodstream affect blood
typing results, or does it? Defend your answer for full credit.
4. Why is it necessary to type the victim’s blood?
5. What ABO antibodies are present in the plasma of the burglar? __________________________
6. A. What ABO antigens are present on the red blood cells of the burglar? ___________________
B. Is the Rh antigen present? ___________________
7. If the burglar needed a transfusion, what ABO type(s) of blood could he safely receive? (Include
Rh information!)