LAB: Blood Exploration

Transcription

1 Name: Period: Date: LAB: Blood Exploration Introduction A liquid called plasma makes up about half (55%) of the content of blood. Plasma contains proteins that help blood to clot, transport substances through the blood, and perform other functions. Blood plasma also contains glucose and other dissolved nutrients. The other half (45%) of blood volume is composed of blood cells: Red blood cells, which carry oxygen to the tissues and remove carbon dioxide as waste, white blood cells, which fight infections, and platelets, smaller cells that help blood to clot. In this lab investigation, we will focus mainly on the structure and function of the RBCs, examining the various morphologies of blood cells, determining the ABO blood type of individuals, and exploring the ability of blood to buffer the circulatory system against drastic changes in ph. PART A Histology of Blood Smears Red blood cells (erythrocytes) have a unique structure and properties that allow them to fulfill crucial functions within the body. They are biconcave discs, having a depressed center on both sides. These depressed centers allow the cells to have more cell membrane surface which can be exposed to diffusing oxygen while transiting the lungs. This structure, along with a small size (about 7.8 micrometers) also allows them to be more flexible when negotiating tight passages. Sometimes structural changes occur in RBCs that indicate a possible pathological condition. For instance, one may observe enlarged RBCs with folate or vitamin B12 deficiency (macrocytic anemia). Small RBCs may be observed with iron deficiency or chronic blood loss (microcytic anemia). Sickle cell anemia is a condition where a genetic flaw leads to defective hemoglobin that changes shape under certain conditions. This shape change induces the cell to lose its original biconcave disc confirmation and take on a more sickled appearance. PART B Introduction to Blood Typing Red blood cells also play a role in an individual s immune system. The basic premise of your immune system relies on antigens and antibodies. Antigens are glycolipids that antigen your body uses to generate antibodies, which are proteins that attack foreign invaders that may enter your body, and prevent them from doing harm. Each antigen produces a specific antibody that will attack a very specific foreign invader. The surface of erythrocytes contains genetically determined glycolipids called agglutinogens. Antigens are categorized into blood groups, two of which are the ABO and Rh groups. There are at least 20 different blood groups in red blood cells; the major group is the ABO system. Type A has only the A-antigen. Type B has only the B-antigen. Type AB has both A- and B-antigens, and Type O has neither. Blood type denotes the class of antigens (glycolipids) found on the surface of the red blood cells. Each person inherits two genes that control the production of ABO antigens. A and B are codominant (I A and I B, respectively), and O (i) is recessive. A secondary group is the Rh factor. 85% of the population is known to have the Rh factor, and is said to be Rh+. The 15% that do not are said to be Rh-. A-antigens make antibodies (in the plasma) against B-antigens, etc. (This is believed to result from the presence in the plasma of preformed antibodies, made in response to some common bacteria in the digestive tract.) O produces both anti-a and anti-b antibodies. AB produces neither. The immune system does not attack its own red blood cell antigens. One person s antigens may be recognized as foreign if transferred into another person s body. This will trigger an immune response in which particular lymphocytes secrete proteins called antibodies (agglutinins), which bind to antigens.

2 Blood typing tests are really quite simple: expose samples of blood to known antibodies and record the reactions. If a blood sample agglutinates (clumps) in response to one (or more) particular antibody, then you know that specific antigen (agglutinogen) must be present in the blood. For example, if agglutination occurs when anti-b agglutinin serum is mixed with a sample of blood (+ reaction), there had to be B agglutinogens for which to bind, and therefore the person has type B proteins on their RBCs. Non-Agglutinated Blood Smear Agglutinated Blood Smear Blood transfusions can be fatal if the antigens and antibodies are incompatible because the clumping causes blockage of small blood vessels. The red blood cells begin to hemolyze (rupture), releasing their hemoglobin into the bloodstream and causing severe kidney damage. Before transfusions are performed, a cross match is made by mixing serum from the recipient with blood cells from the donor. Antibodies seeking specific antigens Antibodies agglutinating red blood cells PART C The Buffering Capacity of Blood The main function of the circulatory system is to transport oxygen to our cells (to make energy) and to remove carbon dioxide (a waste product). The blood must remain very close to its ideal ph, and if it deviates too much, a person could get very sick or even die. Yet, when we transport carbon dioxide from our cells to our lungs, it turns into carbonic acid in the deoxygenated blood. RBCs also contain an enzyme called carbonic anhydrase which takes carbon dioxide and water and catalyzes the creation of a stable molecule called bicarbonate (HCO 3- ). Bicarbonate dissolves much better than carbon dioxide in the fluid part of blood and great quantities can be transported this way without needing to be in the small red blood cells. Bicarbonate is an important buffer for blood, keeping it at a stable ph of about 7.4. Buffers can be thought of as chemical shock absorbers. They keep the fluid of the blood from being too acidic or basic, maintaining an environment where cellular machinery works best. When the bicarbonate dissolved in the blood arrives in the capillaries of the lungs it is converted back to gaseous carbon dioxide by RBCs and then diffuses out into the lungs where it is breathed out. Materials Station 1/3 - Part A Human blood smear, normal Human blood smear, sickle cell Frog blood smear Dolphin blood smear Station 2/5 - Part B Blood samples of Mr. Smith, Mr. Jones, Ms. Brown, and Mr. Green Anti-A, Anti-B, and Anti-Rh serums Blood typing well plates Toothpicks Station 3/6 Part C 20mL non-buffered blood solution 20mL buffered blood solution Dropper bottle of 0.1M HCl ph paper

3 Procedure PART A: Histology of Blood Smears 1. Plug in the compound light microscope and turn on the light source. Ensure you are on the lowest power (shortest objective lens) to begin this will be a total magnification of 40X. 2. Examine the normal human blood smear slide first. Start by centering the slide in your field of view and adjusting the coarse adjustment (larger knob of the microscope). This will move the objective lenses closer/farther away from the stage, bringing the object into rough focus. 3. Next, using the smaller fine adjustment knob, bring your sample into sharp focus. Ensure your specimen in the center of your field of view. 4. Switch to 100X total magnification WITHOUT touching the coarse adjustment knob. From this point forward, you can only adjust the fine adjustment. Bring your slide into sharp focus. 5. Carefully switch to 400X total magnification and observe the structure of the normal RBCs (they will be light pink/red under the microscope). Sketch an RBC in the data table. 6. Have other lab partners observe and sketch the RBCs on the sickle blood smear, frog blood smear, and dolphin blood smear. Sketch in the data table. 7. Next, identify/find and sketch each of the following types of white blood cells (use your HANDOUT: Blood to assist in morphology of WBCs. WBCs will be easily identifiable by their darkly purple-stained nuclei; you can distinguish between the five types based on the shape of the nucleus and whether or not it is granulated.) a. Eosinophil: Associated with allergic reactions. b. Basophil: Associated with allergic reactions. c. Neutrophil: Protect against pyogenic (pus-causing) microorganisms and participate in the inflammatory process. d. Lymphocyte: Includes T-cells and B-cells; generate specific responses that are tailored to maximally eliminate specific pathogens; remember antigens in memory cells. e. Monocyte: Largest WBCs; replenish macrophages. 8. Compare and contrast the shapes of the WBCs with that of RBCs. 9. Identify/find and sketch platelets. 10. Determine the % composition of your blood smear. Approximately how many RBCs are there for every WBC? For every platelet? Data Normal RBC Sickle RBC Frog RBC Dolphin RBC Eosinophil Basophil Neutrophil Lymphocyte Monocyte Platelet % Composition of Blood RBCs? WBCs? Platelets?

4 PART B: Introduction to ABO Blood Typing 1. Place a piece of white paper (preferably with print) behind the blood typing plates. 2. On the paper behind the plates, label each plate as follows: a. #1 = Mr. Jones b. #2 = Mr. Smith c. #3 = Mr. Green d. #4 = Ms. Brown 3. Place 2-3 drops of simulated Mr. Jones blood into each well ( A, B, and Rh ) on plate #1. 4. Next, place 2 drops of simulated anti-a agglutinin serum in well A, 2 drops of simulated anti-b agglutinin serum in well B, and 2 drops of simulated anti-rh agglutinin serum in well Rh. 5. Using the clean end of a toothpick thoroughly mix each well for approximately 30 seconds for all the well plates. It is important to use a clean end of a toothpick for each well as you do not want to cross-contaminate your samples! 6. Observe each well and record results in the data table. If you are uncertain as to whether or not agglutination occurred, try reading a sample of text through the blood. If you cannot read the text, agglutination did occur. a. If agglutination occurred in a well, mark a + to indicate a positive reaction. b. If no agglutination occurred, mark a to indicate a negative reaction. 7. Use the results of your agglutination tests to determine the ABO blood type of the individual (A, B, AB, or O and Rh+ or Rh-). 8. Dispose of all blood samples in the sink. Thoroughly wash and dry the well plates and toothpicks for reuse before returning your materials to the baggie. Data Table 1: Agglutination Results Blood Sample Anti-A Serum (+ or ) Anti-B Serum (+ or ) Anti-Rh serum (+ or ) ABO Blood Type #1-Mr. Jones #2-Mr. Smith #3-Mr. Green #4-Ms. Brown

5 PART C: Investigating the Buffering Capacity of Blood 1. Obtain 20mL of non-buffered blood solution in a 100mL beaker. 2. Using tweezers, pick up a strip of ph paper. Dip the ph paper in the blood, remove the strip promptly, and match to the ph value on the tube. Record the value in the data table. 3. Your blood sample already has a buffer in it. Add 5 drops of HCl (acid solution) to your blood solution, and re-test the ph using a new strip ph paper. Record the value in the data table. 4. Add 5 more drops of HCl to your blood solution, re-test the ph, and record the value in the data table. 5. Add another 5 drops of HCl to your blood solution, re-test the ph and record the value in the data table. 6. Add 10mL of HCl to your blood solution, re-test the ph and record the value in the data table. 7. Rinse your beaker thoroughly, dry, and repeat steps 1-6 with the buffered blood solution. Initial After 5 drops After 10 drops After 15 drops After 10mL + 15 drops ph of Non-Buffered Blood Solution ph of Buffered Blood Solution Analysis Questions PART A 1. Draw the normal shape of an RBC. Why is this form important for its function? Be specific. 2. Compare and contrast the shape of the red blood cells observed in the normal human blood smear, the sickle cells smear, the frog blood smear, and the dolphin blood smear. Note any structural differences observed. 3. Sickle cell is actually an autosomal recessive trait. This means one must inherit two defective recessive alleles (one from each parent) to display the disease. However, being heterozygous for the sickle cell trait has been shown to increase an individual s resistance to the disease malaria (carried by mosquitos, common in Africa). If two parents, each heterozygous for the sickle cell trait, have an offspring, what is the probability the child will be born with sickle cell disease? Show a Punnett square with your answer. Use H as the dominant allele and h as the recessive, sickle allele.

6 PART B 4. Of the patients tested (Mr. Jones, Mr. Smith, Mr. Green and Ms. Brown) complete the following chart based on your knowledge of blood typing and blood transfusions. Use the names of each person (don t just write blood types!). Patient Name Can Donate To Patient(s) Can Receive From Patient(s) Mr. Jones Mr. Smith Mr. Green Ms. Brown 5. What would happen if Ms. Brown received blood from Mr. Smith? Explain the physiology. PART C 6. What is a buffer? Why are they biologically important, specifically in the cardiovascular system? 7. Did the ph value change significantly when you added small amounts (15 drops) of HCl to the buffered blood, when compared with the non-buffered blood? Why or why not? 8. Did the ph value change significantly when you added large amounts (10mL) of HCl to the buffered blood, when compared with the non-buffered blood? Why or why not? 9. Explain the role of carbonic anhydrase (an enzyme) in maintaining homeostasis within the cardiovascular system. 10. Ketoacidosis is a metabolic state associated with high concentrations of keto acids in the blood, formed when fatty acids and proteins are broken down excessively. It is most common in untreated type 1 diabetes mellitus. Discuss why ketoacidosis can be fatal if left untreated, using your new knowledge of the blood. Use the terms carbon dioxide, oxygen, carbonic acid, carbonic anhydrase, bicarbonate, enzyme, and denatured in your response.