Exercise 9: Blood. Readings: Silverthorn 5 th ed, , ; 6 th ed, ,

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1 Exercise 9: Blood Readings: Silverthorn 5 th ed, , ; 6 th ed, , Blood Typing The membranes of human red blood cells (RBCs) contain a variety of cell surface proteins called blood group antigens. The most important and best known of these are the A and B antigens, also called the ABO blood group, and the Rh antigen. When blood is transfused between a donor and a recipient, blood group antigens must be identified in order to ensure that their blood is compatible. Blood group antigens are found only on RBCs and their presence is determined by an individual's genetics. An individual who does NOT inherit a particular blood group antigen will produce an antibody that recognizes that antigen as foreign. Most commonly, an antibody is produced after an antigen is introduced into the body, as with the Rh system. The ABO blood group is unusual in that an individual lacking a blood group antigen will automatically produce an antibody against the lacking antigen, even if that antigen has never been introduced into the recipient. An antibody will specifically bind to the antigen it recognizes in an attempt to eliminate the antigen. This binding reaction is called agglutination (Fig. 1b). Agglutination of blood group antigens causes large clumps of RBCs and antibody to form, which can block and damage the small capillaries, especially in the kidneys. The resulting damage is called a transfusion reaction and can cause permanent kidney damage or even kill the recipient. So how are these blood group antigens identified before a transfusion is made? A sample of blood is removed from the recipient and mixed in a dish with purified antibodies that are known to recognize a specific blood group antigen. If the purified antibodies cause the blood sample to agglutinate, then the RBCs in the blood sample carry the protein recognized by the antibody. See Fig. 1a for examples of agglutination.

2 1a. 1b. Figure 1a. Agglutination reactions: Blood samples in the first column have been mixed with Anti-B antibody. Agglutination reactions with Anti-B antibody have occurred with Type B and Type AB blood, which both contain the B antigen. Blood samples in the second column have been mixed with Anti-A antibody. Agglutination reactions have occurred with Type A and Type AB blood, which both contain the A antigen. Figure 1b. Antibodies are Y-shaped. Only anti-a antibodies bind to the A antigen on the RBCs. A person with Type A blood has RBC's that carry the A antigen. Antibodies that recognize the A antigen are called anti-a antibodies. If Type A blood is mixed with anti- A antibodies, the RBCs will agglutinate. If Type A blood is mixed with anti-b antibodies, the RBCs will NOT agglutinate because the B antigen is not present. Conversely, if a blood sample agglutinates with anti-b antibodies but not with anti-a antibodies, then the blood sample contains Type B blood. 2

3 Where do these antibodies come from? They are naturally found in the blood of the opposite blood type. Remember that individuals with A antigen on their RBCs will carry anti-b antibodies and individuals with B antigen will carry anti-a antibodies. See Table 1 for clarification of which type of antigen and antibody is found in a given blood type. Type O blood contains neither A nor B antigen, and so can produce both Anti-A and Anti-B antibodies. Type AB blood contains both A and B antigen, and so will produce neither Anti-A nor Anti-B antibodies. At one time, Type O blood was considered a universal donor since it contained neither A nor B antigens and would therefore not react with any blood group antibodies produced by the recipient. Today, it is recognized that Anti-A and Anti-B antibodies found in the plasma of Type O blood have the potential to produce a transfusion reaction with a Type A or Type B recipient and so is no longer given to recipients with other blood types. Table 1. ABO Blood Groups Blood Type Antigen on RBC Antibodies in Plasma O Neither A nor B Both Anti-A and Anti-B A A only Anti-B only B B only Anti-A only AB Both A and B Neither antibody The Rh factor is separate from the ABO blood group. An individual carrying the Rh antigen is said to be Rh + while an individual without the Rh antigen is said to be Rh. The + or is added to the ABO blood type. For example, a person with Type A+ blood carries the A antigen, the Rh antigen, but not the B antigen. Individuals who are Rh will not produce Anti-Rh antibodies unless the Rh antigen has been introduced into their bodies. Rh antigen can be introduced into a woman's body if a fetus she is carrying inherits the Rh antigen from the fetus's father. Fetal RBCs carrying the Rh factor are too big to cross the placenta, so the mother is not exposed to the Rh factor until the baby is delivered. During delivery, or any break in the placenta, fetal blood can mix with the mother's blood and the mother can begin to produce Anti-Rh antibodies. When the mother becomes pregnant a second time, these antibodies proteins are small enough to cross the placenta and attack the Rh+ fetus, causing damage to fetus and possible miscarriage. This is referred to as erythroblastosis fetalis. Differential Blood Cell Counts In a sample of blood, various formed elements are present. The ratios between red and white blood cells allow us to evaluate the health of individuals. Disruption of these ratios is an indication of disease. Anomalies in the shape of the cells may also be 3

4 pathological. We will use blood smears and a compound microscope to identify four of these anomalies. When looking at slide of healthy blood under the microscope one sees mostly red blood cells (erythrocytes), but also white blood cells (leukocytes) and platelets (thrombocytes). Less than 1% of the blood cells are white blood cells. The five types of white blood cells include neutrophils, eosinophils, basophils, monocytes and lymphocytes (Fig. 2). Each type has specific functions and a distinctive appearance. Doing a differential white blood cell count involves determining the relative abundance of the different types of white blood cells. Observing increases or decreases in the numbers of a certain type of white blood cells is helpful in diagnosing certain conditions. Figure 2. All blood cell types arise from a common stem cell. Blood Cell Abnormalities Sickle Cell Anemia Inherited abnormalities in hemoglobin may cause anemias. The red blood cells that contain abnormal hemoglobin may lose their shape or lose their ability to deliver an adequate supply of oxygen. In Sickle Cell, the red blood cells contain an abnormal form of hemoglobin, reducing the amount of oxygen in the cell, causing them to become crescent shaped (Fig. 3). These cells block and damage small blood vessels in the spleen, brain, kidneys and bones, reducing their oxygen supply. Sickle-cell cells are fragile and break up easily causing blocked blood flow to these organs. Eosinophilia Increased numbers of eosinophils (Fig. 4), or Figure 3. Sickle-cell red blood cells 4 Figure 4. Normal Eosinophil

5 eosinophilia, are most often associated with allergic diseases and the presence of parasites (such as worms). Cancers of the Blood Chronic Myelogenous Leukemia (=chronic myeloid leukemia) is a condition where cell division becomes excessive in myeloid stem cells. This results in an abnormal release of leukocytes from bone marrow, spleen and lymph nodes. The blood slides that you observe contain myeloid cells at all stages of maturation. Lymphomas are malignant neoplasms of lymphoid tissue (lymph nodes, spleen, and other organs of the immune system). Lymphocytes are found in great numbers on these blood smears. Keep in mind that a high lymphocyte count in the blood could also be an indicator of infection or even a leukemia. Today s Objectives 1. Blood typing with simulated blood. 2. Observe microscopic slides portraying various blood pathologies. 3. Perform a differential blood cell count Blood Typing Materials: 4 blood typing slides 12 toothpicks 4 unknown blood samples: Mr. Smith Mr. Jones Mr. Green Ms. Brown Simulated Anti-A Serum Simulated Anti-B Serum Simulated Anti-Rh Serum Getting Started 5

6 Procedure 1. Label each blood typing slide with the name of the sample: Slide #1 Mr. Smith Slide #2 Mr. Jones Slide #3 Mr. Green Slide #4 Ms. Brown 2. Place 3 drops of Mr. Smith's blood in each of the A, B, and Rh wells of Slide #1. 3. Repeat with Slides #2 #4 and their respective samples. 4. Place 3 drops of Anti-A serum in each A well on all four slides. 5. Place 3 drops of Anti-B serum in each B well on all four slides. 6. Place 3 drops of Anti-Rh serum in each Rh well on all four slides. 7. Use a separate clean toothpick to stir each well for 30 seconds. Do not press too hard on the typing tray to avoid splattering. 8. Record the occurrence of agglutination in Table 2. If the sample appears grainy or opaque, assume agglutination has occurred. Determine the blood type based on your agglutination results. 9. When finished, throw away all toothpicks and rinse off and stack the typing trays. Slide #1: Mr. Smith Slide #2: Mr. Jones Slide #3: Mr. Green Slide #4: Ms. Brown Table 2. Blood Typing Observations Anti-A Serum Anti-B Serum Anti-Rh Serum Mr. Smith Mr. Jones Mr. Green Ms. Brown Table 3. Blood Typing Results Antigens present Antibodies present Blood Type 6

7 Questions: 1. Explain how you were able to fill out Table For each patient, determine what blood type(s) could be used if an emergency transfusion was needed. It is understood that he/she should receive only his own blood type under optimal conditions, but assume that he/she was in an emergency situation, out of reach of a hospital, but close to a partially equipped clinic with some blood in its coolers. Blood Slides Obtain one of each of these microscope slides: 1. Sickle Cell Anemia 2. Eosinophilia 3. Chronic Myelogenous Leukemia 4. Lymphoma Observe the slides under the compound microscope. Use the descriptions below to help you to determine what to look for. You should use immersion oil at 1000X magnification. Make a drawing of each of these slides to hand in. Sickle Cell Anemia Notice the large numbers of misshapen RBCs. The affected cells have the characteristic sickle shape or will look like grains of rice. Eosinophilia Observe the large numbers of eosinophils on the blood smear. The lobed nuclei are stained purple and the cytoplasm has dark pink granulations. Neutrophils are also present. Disregard the clumped worm-like aspect of the RBCs. This clumping was the result of the preparation technique. Also disregard the many small purple-stained cell fragments throughout the slide. Chronic Myelogenous Leukemia- Notice the high counts of white blood cells throughout the blood smear slide. Here too, the red blood cells on these slides appear clumped and out of shape. Disregard this condition. Instead, concentrate on finding granulocytes (basophils, eosinophils and neutrophils) and agranulocytes (monocytes) in 7

8 all stages of development. The granulocytes have visible stained granules within the cytoplasm of the cells. Lymphoma Observe the blood smear and notice the large numbers of lymphocytes in all different stages of maturity. Neutrophils are also present and some of the red blood cells appear crenated. 8

9 Differential Blood Cell Counts For this activity you will first perform a differential white blood cell count to determine the relative abundance of the different types of white blood cells in normal human blood. You will then perform a similar analysis on a slide of blood from a person with a white blood cell abnormality. Using your text, and figures available in the lab you will identify the types of white cells. Some general characteristics to help in differentiating the types are described below. Neutrophil: Multi-lobed nucleus, clear cytoplasm (looks like a "Mickey Mouse Balloon"). Makes up 65% of WBC. Lymphocyte: Large round nucleus that fills most of the cell, a small amount of clear cytoplasm. Makes up 25% of WBC. Monocyte: Large cell with heart-shaped/ horseshoe shaped nucleus and clear cytoplasm. Makes up 2-4% of WBC. Eosinophil: Bi-lobed blue/purple staining nucleus with red staining granules in cytoplasm. Makes up 4-5% of WBC. Basophil: Bi-lobed nucleus with blue-purple staining granules in cytoplasm. Makes up <1% of WBC. Normal Blood Focus the normal blood slide at 400X magnification. Starting at one end of the slide, scan back and forth as shown in Fig. 5, systematically locating white blood cells, identifying them and tallying the different types you encounter in Table 4. Each group should count a total of 25 white blood cells. If a particular white blood cell is squashed or ambiguous looking, skip it and go on to the next one. We will compile the class data to get larger numbers to use for calculating the relative percentage of each type of cell. 9

10 Figure 5. Scanning the blood slide To determine relative percentage of neutrophils divide the number of those cells by the total number of white cells counted and multiply by 100. For example: Relative % = Number of neutrophils counted X 100 Total number of white cells counted 10

11 Table 4. Normal Blood Group data Class data Cell type # identified Relative % # identified Relative % Neutrophil Eosinophil Basophil Monocyte Lymphocytes Total = (25) Eosinophilia Blood Repeat the steps above now using the Eosinophilia blood slides. Record your data in Table 5. Analyze the abnormal blood smear as you did above after counting 25 white blood cells. Table 5. Eosinophilia Blood Group data Class data Cell type # identified Relative % # identified Relative % Neutrophil Eosinophil Basophil Monocyte Lymphocytes Total = (25) Based on your observations, describe what is abnormal about the blood: 11

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