Pierce College Putman/Biol 242 Name: Lab 02: Blood Cytology (20 points) Reference: Marieb & Mitchell 9 th Ed: 29A (Activities 1, 2, 3, 4, 7); 10 th Ed: Exercise 29 (Activities 1, 2, 3, 4, 7). Pierce College Student Outcomes: Lab Outcome 1: Correlate qualitative and quantitative information about the constituents of blood via microscopic examination. Lab Outcome 2: Generate a hematology slip following the performance of a hematocrit and a differential white blood cell count from a blood smear stained with Wright s stain. Lab Outcome 3: Recognize the ABO blood type and Rh factor from an agglutination test and describe the frequencies of blood types among the various ethnic populations applying genetic concepts. Objectives for Lab Exam: 1. State the ph, color, degree of transparency and consistency of plasma. 2. Explain the protocol of safely obtaining blood for observation under a microscope. 3. Explain the necessary protocols when working with human blood. 4. Explain how to correctly make a blood smear. 5. Explain how to correctly make a Wright s stain of a blood smear. 6. Give the components of Wright s stain and state what they stain, what they do not stain, and how this allows us to identify the various leucocytes. 7. Identify erythrocytes, neutrophils, eosinophils, basophils, lymphocytes, monocytes, platelets. 8. List the leukocytes in order from most to least common, and state their functions. 9. Describe hematocrit protocol. 10. State what normal hematocrit levels are. 11. Explain the theory behind ABO and Rh blood typing. 12. State why it s important to know blood types before blood transfusions are carried out. *Make sure you obtain the microscope you initially calibrated so you don t have to recalibrate your scope! Activity 1: Determining the Physical Characteristics of Plasma 1. From your textbook, answer the following questions about plasma (blood): a. ph range of plasma: b. Color: Putman/Biol 242 Lab 2/20140524/Page 1
c. Degree of transparency: d. Viscosity compared to water: Activity 2: Performing a Blood Smear and Wright s Stain. Lab safety advisory: Biohazard! Human blood is a BIOHAZARD and must be worked w with using strict protocols. Anything that has come in contact with human blood is a BIOHAZARD. At Pierce College, we use animal (dog) blood from our or another veterinary clinic. You are still expected to follow human protocols for this blood. About the Wright s Stain Wright s stain is made of eosin and hematoxyln. The granules of granulocytes (eosinophins, lymphocytes and basophils) are stained by Wright s stain. Eosinophils pick up eosin strongly, so their large granules stain red. Neutrophils pick up eosin weakly, so their small granules stain faint pink. Basophils do not pick up eosin; instead, they pick up hematoxyln, which stains their granules blue. Hematoxyln also stains the nuclei of all of the leucocytes, allowing us to differentiate nuclear morphology. The agranulocytes, lymphocytes and monocytes, pick up neither eosin nor hematoxyln, so their granules don t stain in Wright s stain, but their nuclei are stained by hematoxyln. Making a Blood Smear Step 1: Move unneeded materials off your lab bench. Step 2: Disinfect your lab bench with the provided sodium hypochlorite (bleach) solution and a paper towel. Step 3: Wash your hands, put on gloves. Step 4: At your work station, lay down a paper towel. Obtain two microscope slides. Step 5: If needed, gently rock the blood sample provided; do not shake the blood as this will damage the blood cells. Step 6: Dip the end of a pipette into the blood sample to obtain a droplet of blood. Step 7: Apply the droplet to the end of a microscope slide; with the second microscope slide, immediately draw the blood over the slide so that it covers a maximum area. To do this, place the edge of the second slide so that it touches the drop of blood; then draw the second slide across the first slide steadily, with one motion, without starting/stopping, so that the blood is PULLED across the first slide. Do this once only; do NOT keep trying to spread the blood because this will cause the blood to clump; the blood smear MUST be even and thin! Putman/Biol 242 Lab 2/20140524/Page 2
Step 8: Allow the blood on the slide to air dry. This should take only a few minutes Wright s Stain Protocol Step 1: Once the blood is air dried, put the slide on a staining tray over a sink and saturated with Wright s stain. Stain for 1 minute. Step 2: After 1 minute, dip the slide, down and up, into distilled water rinse 1, then down and up into distilled water rinse 2. Step 3: Wipe the back of the wet slide with a paper towel and keep the slide in a vertical position on a paper towel until it is dry. Step 4: Don t put a cover slip on this slide as this is a temporary slide. Step 5: After the slide is dry, examine the cells under the microscope. Make sure you begin with lowest power, 40x or 32x, and work your way up to 400x. After you have focused on your cells at 400x, apply a drop or two of immersion oil, then slide the 100x objective (1000x total magnification) into the oil and observe. Step 6: Locate a white blood cell and show your instructor. Have your instructor sign below so you receive credit for your properly-made Wright s stain: Instructor s signature Step 7: After your instructor has checked your work and signed above, place the slide in the indicated biohazard container (red box). Step 8: If you are finished, triple-clean the 100x lens. If you have time to continue on to the next section, you may wait to clean the 100x lens. Activity 3: Identifying Leucocytes 1. Using a commercial slide of human blood, identify, draw and label erythrocytes, thrombocytes (platelets), neutrophils, lymphocytes, monocytes, eosinophils and basophils. Be sure to include a size rule and have your instructor sign your drawings for credit. If you make all of your drawings to the same scale, one size rule will suffice. You may include your drawings in the space provided on page 4. Use 1000x total magnification with immersion oil; be sure to triple clean both the microscope slide and 100x objective when finished! Putman/Biol 242 Lab 2/20140524/Page 3
Do drawings of blood cells on this page! Putman/Biol 242 Lab 2/20140524/Page 4
2. Answer the following questions: 1. List the leukocytes in order from most to least common. 2. Which two leucocytes does the eosin in Wrights stain stain? 3. Regarding granule size the how strongly eosin is taken up by those granules, how can you use eosin to differentiate between the two leucocytes it stains? 4. How can eosin be used to identify monocytes and lymphocytes? 5. How can hematoxyln be used to identify basophils? 6. How can hematoxyln be used to differentiate between neutrophils, lymphocytes, monocytes, eosinophils and basophils? 7. What is the function of neutrophils? 8. What is the function of eosinophils? 9. What is the function of basophils? 10. What is the function of lymphocytes? 11. What is the function of monocytes? Putman/Biol 242 Lab 2/20140524/Page 5
Activity 4: Conducting a Differential WBC Count Differential counts are used in clinics to determine if certain blood cell types are higher or lower than normal, thereby providing valuable clues as to the health of a patient. Step 1: Using a commercially-prepared blood slide, perform a differential WBC count, identifying at least 50 leucocytes. Work with your lab partner. Start with a corner of the slide and work your way up, over, down, over, up, etc. until you have identified at least 25 leucocytes; have your lab partner keep a tally for you in the table below. After you have identified at least 25 cells, switch roles and record data for your lab partner as she or he identifies at least 25 leucocytes. WBC Tally Number % Normal % Neutrophil Explanation Lymphocyte Monocyte Eosinophil Basophil Total: Step 2: Combine data with your lab partner. Add up the number of each leucocyte counted, then add up the total of all leucocytes counted. Calculate the percent of each leucocyte. Finally, look up the normal % for each leucocyte in reference materials given to you and add it to the table. Step 3: Compare the percentages of each type of WBC. Are all of the counts within normal range? If any are high or low, explain what might be going. If values are within a normal range, simply write within normal range. Activity 5: Determining the Hematocrit (PCT) Hematocrit (packed-cell count or PCT) allows us to determine what percentage of a blood sample is composed of blood cells, thus giving us information on the possible diagnosis of various blood pathologies, including polycythemia. Step 1: Put gloves on. Obtain a heparinized capillary tube. Heparin is an agent that inhibits blood from coagulating within the capillary tube. Putman/Biol 242 Lab 2/20140524/Page 6
Step 2: Pack the capillary tube with blood until it is at least ½ full by dipping it up and down in the blood sample. When finished, press tube down into Cryoseal clay and turn to seal. Step 3: With a small piece of wet paper towel, wipe the excess blood from the outside of the capillary tube. Step 4: Place capillary tube in centrifuge with clay plug facing outwards so blood remains in tube when centrifuge is turned on. Note the number of the slot in which you placed your tube. Also make sure it s balanced with a tube on the other side; your instructor will help you with this. Step 5: When everyone has loaded their tube into the centrifuge, your instructor will turn the centrifuge on for 5 minutes. This will spin the formed elements down, erythrocytes first, a very thin leucocyte line on top of the erythrocytes, the remainder being plasma. Step 6: When ready, pick up your centrifuge tube. Obtain a millimeter ruler. Measure the total height of the column of blood to the nearest 10 th of a millimeter. Also measure the height of the erythrocytes to the nearest 10 th of a millimeter. Be careful to begin your measurement at the bottom of the column of blood, not including the clay plug. Record your data below and determine the hematocrit, which is the %RBC in your sample. Place capillary tube in red biohazard box when finished. Total Height of Blood in Capillary Tube, mm Total Height of RBCs in Capillary Tube, mm Hematocrit, %RBCs Answer the Following Questions: 1. What is a normal hematocrit value? 2. If the hematocrit you determined is different than a clinically normal hematocrit value, propose an explanation! Otherwise, write hematocrit normal. Putman/Biol 242 Lab 2/20140524/Page 7
Activity 6: Typing for ABO and Rh Blood Groups. All cells contain antigens in their glycocalyx. The immune system reacts to non-self antigens by producing antibodies which attach to the non-self antigens. This causes cells involved in the immunological response to attack the non-self antigens and to cause agglutination or clumping together of the non-self antigens. If the non-self antigens are located on erythrocytes introduced into the body during a blood transfusion, serious clinical consequences, including death, may result as the blood agglutinates and blocks arterioles and venules. Individuals with type A, B or AB have A, B or AB on their erythrocytes; people who are type O have none of these antigens. During a transfusion, if someone who is type A receives any blood containing B antigen, as from type A or type AB, this will cause an antigen-antibody reaction and agglutination. Similarly, if an individual who is type B receives any blood containing A antigen, as from type B or AB, agglutination will result. Type AB can receive A, B, AB or O without danger of agglutination. Type O can only receive type O as type A or B antigens will elicit agglutination. The Rh blood group, or rhesus factor, works in a manner similar to the ABO blood group. An individual who is + for the rhesus factor carries the antigen on his/her erythrocytes; - individuals do not carry the rhesus factor. An Rh + individual can receive Rh + blood without fear of agglutination. If an Rh individual receives Rh + blood, then antibodies are produced and agglutination will occur. Typing for ABO and Rh Step 1: Obtain an ABO/Rh test kit. The kit will consist of blood serum from a patient, three antibody solutions (anti-a, anti-b and anti-rh), a plastic typing tray and plastic toothpicks. Write the patient s name down below (only type one of the patients). Patient s Name: Blood Type: Step 2: Place two drops of serum into each of the three wells. Place two drops of each of the antisera into each of the corresponding wells and stir each time with a clean plastic toothpick. Rinse the toothpicks when you re done and put them back into their holders (don t throw away). Step 3: After five minutes, place the plastic tray on some print such as the print of this page. If you can read through the serum/antibody combination, the test is negative for that antigen. If you can t read through the serum/antibody combination, that s a positive reaction. If you didn t get a positive reaction, examine positive reactions obtained by other students so that you learn what a positive reaction looks like. Putman/Biol 242 Lab 2/20140524/Page 8
Answer the following question: 1. Why is blood typing so important before surgical procedures are attempted! ~When you re finished, help clean up! 1. Is your lab bench clean and wiped down with antiseptic solution? 2. Are all materials returned to their proper place? 3. Is there any trace of oil on your microscope lenses OR microscope slides? TRIPLE CLEAN!!! 4. Is all refuse disposed of properly? 5. Is the lab generally in order? Putman/Biol 242 Lab 2/20140524/Page 9