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1 Bio 151 Laboratory Manual Human Anatomy & Physiology II #3 - Objectives: Determine the hemoglobin content of sheep blood Know the normal values of hemoglobin for males and females Be able to calculate % hematocrit and know the normal values for males and females Perform a simulated differential white blood cell count and associate these counts with various pathologies Equipment: Remember to bring gloves, goggles, & proper shoes. I. Hemoglobin Content A. Introduction Hemoglobin is a molecule within the red blood cells that binds oxygen. Oxygen associates with the central iron of the heme portions of each hemoglobin molecule. The hemoglobinometer directly measures the amount of hemoglobin in a sample in grams per deciliter (g/dl). Normal hemoglobin Males Females values have a wide range (see table) 1,2, g/dl g/dl Figure 1. The hemoglobinometer. B. Measuring Hemoglobin with a Hemoglobinometer 1. Wear your gloves, goggles, and apron. 2. Turn ON the hemoglobinometer. 3. Wait until you see the number code in the display (for example 950) that corresponds to the test card number (found on the bottle). 4. Wait until the flashing test card symbol is visible to insert the white test card into the hemoglobinometer. You will feel it click into place. 5. Wait until you see the blood drop symbol to add a small drop sterilized sheep s blood in the center of the test card. Be careful not to overload the well. 6. Wait until the reading is stable. This may take a minute. Read and record the value in the machine s display in Table 1 of the laboratory report. 7. Remove the white test card by lifting up slightly and pulling it out 8. Discard the test card and gloves in the red BIOHAZARD box. Page1

2 Bio 151 Laboratory Manual II. Hematocrit Human Anatomy & Physiology II A. Introduction A hematocrit measures the percentage of packed red blood cells in a blood sample. A blood sample in a capillary tube is centrifuged to compact the RBCs. This provides an indirect measure of the amount of hemoglobin in a person s blood. The normal values for hematocrit have a wide range for males and females 2,3. Males % Females % B. Procedure: The samples in the following activity simulate female, male, normal, and anemic blood. 1. Chose one of the simulated blood samples. Write down which sample you chose in Table Gently invert the bottle to mix the simulated blood. 3. Remove the rubber stopper. Figure 2. Loading the capillary tube. 4. Place the capillary tube inside the blood in the bottle. 5. Tilt the bottle and the tube until you can see the blood climbing up the tube. Be careful not to tip it too far or the blood will drip out of the bottle and/or the tube (Figure 2). 6. Fill the tube until it reaches about 2/3 full. 7. When full, put your finger tip over the clean end to help keep the blood from dripping out of the tube and place the end in clay (critoseal) to block the end for centrifuging. 8. Record which centrifuge slot your tube went into (Table 2) C. Calculating Hematocrit 1. Measure the length of the packed RBCs from the end (do not include the clay plug) to the buffy coat / clear plasma in mm (Figure 3). Record in Table Measure the total length of the blood column in mm (the packed RBCs and clear plasma together). Record in Table Calculate the percentage of packed red blood cells, record in Table 2. Use Figure 3 to practice measuring the packed RBCs: cm = mm Figure 3. The capillary tube after centrifugation. Length of packed blood cells in tube (mm) Total length of blood column in tube (mm) x 100 = % hematocrit 4) Draw and label your capillary tube in the lab report show your measurements (mm) 5) Dispose of the capillary tube as your instructor directs. Page2

3 Bio 151 Laboratory Manual Human Anatomy & Physiology II III. Differential White Blood Cell Count Note: This activity was originally designed and kindly donated for use by Mark Eberle (Central Oregon Community College). It was reported in Human Anatomy and Physiology Society (HAPS- EDucator Newsletter in 2009) 4. Some of the following text, diagrams, and report questions have been modified from their original version and added to for our specific needs. A. Introduction In today s medical and research labs, obtaining differential white blood cell counts (and all types of blood cell counts) is an automated process. However it is useful to perform these counts manually to become familiar with the various white blood cells. In this exercise, micrograph sets that represent a blood smear will be viewed. Each lab bench will be responsible for analyzing the leukocyte counts of all five patients. Work in pairs, doublechecking any questionable cells. Match the unknown micrographs to the WBC micrograph key until you are able to identify the cells without referring to the key. Identify and count only the WBC s. Use the following pages to record the counts for each WBC type Most of you will never do differential white blood cell counts on the job, but you should know how counts can be used to diagnose infections. You should suspect bacteria if the neutrophils are high, viruses if lymphocytes are high, parasitic worms if eosinophils are high (though allergy is a possibility too), and AIDS (HIV infection) if lymphocytes are low. The HIV virus infects T- lymphocytes (T-cells) and therefore can reduce their numbers and inhibit immune system function. You should know that high banded neutrophil counts suggest acute bacterial infections. Bands are replacement neutrophils just entering circulation. Their nuclei are band-shaped unlike the segmented nuclei of mature neutrophils, or segs. You should know that atypical lymphocytes are an important indicator of infectious mononucleosis. These cells are transformed T lymphocytes responding to B lymphocytes which are infected with Epstein-Bar virus. Atypical lymphocytes are irregularly shaped because the cytoplasm is frequently indented by the surrounding red blood cells. Mononucleosis can cause spleen enlargement and can be spread through saliva. Keep in mind that other WBCs will seem high when lymphocyte percentage is low. You can see pictures of all these WBCs in the following figure. Practice what you just read by filling in these blanks. High neutrophils suggest High lymphyocytes suggest infections. infections. High eosinophils suggest or. Low lymphocytes suggest. High bands suggest acute infections. Atypical lymphocytes suggest. Page3

4 Bio 151 Laboratory Manual Human Anatomy & Physiology II Figure 4. The white blood cell picture key. Segmented neutrophil Band neutrophil Lymphocyte Atypical lymphocyte Monocyte Eosinophil Basophil B. Simulated White Blood Cell Count Activity: 1. Each person will pick a Differential White Blood Cell Count card (1-4). Record your number in Table Identify all 100 white blood cells in your patient s two-page blood smear. (Note: more than one WBC may be visible in each photo, more cells are on the back of the page.) 3. Keep track of your count by putting marks next to the WBC names in Table 3 as you count. 4. Total up the marks and write the totals in the percentage column. (Counts are percentages because you counted a total of 100 cells.) 5. Indicate whether or not percentages are normal by writing yes or no. Normal percentages are given in parentheses. PLEASE DO NOT WRITE ON THE PAGES. WE NEED THEM FOR OTHER CLASSES. References 1. Beutler, E & Waalen, J The definition of anemia: what is the lower limit of normal of the blood hemoglobin concentration? Blood:107(5) Walker, HK; Hall, WD; Hurst, JW. Editors Clinical Methods: The history, physical, and laboratory examinations. 3 rd Ed. Boston: Butterworths Complete Blood Count. Mayo Foundation for Medical Education and Research Eberle, M Free simulated blood smears for differential white blood cell counts. HAPS EDucator. 13(2): 16. Newsletter of the Human Anatomy & Physiology Society. Page4

5 Bio 151 Laboratory Manual Human Anatomy & Physiology II Laboratory #3 Report Name: I. Hemoglobin Content Table 1 Sample hemoglobin value: Is this value normal (N), low (L), or high (H) for a male or a female? Male? Female? II. Hematocrit: Table 2 Draw and label your capillary tube here (include the measurements): Sample Centrifuge Slot Number: Letter: Length of packed red mm blood cells in tube Total length of blood mm column in tube hematocrit % Is this value normal (N), low (L), or high (H)? Male? Female? III. Table 3. Differential White Blood Cell Count Patient Number White Blood Cell Count Percentage Normal percentage Normal? (Y/N) Segmented neutrophils (50-65%) Band neutrophils (0-5%) Normal (15-40%) Lymphocytes Atypical (0-1%) Lymphocytes Monocytes (0-10%) Eosinophils (0-5%) Basophils (0-2%) Diagnosis: Decide whether your patient is normal, has parasites, has a bacterial infection, has mononucleosis, or has AIDS (Circle one). Page5

6 Bio 151 Laboratory Manual Human Anatomy & Physiology II Now share your diagnosis with the other members of your table team. Put your heads together and answer all of the questions on the next page. Patient 1 s diagnosis: Patient 2 s diagnosis: Patient 3 s diagnosis: Patient 4 s diagnosis: Patient 5 s diagnosis: (Your table team may not have diagnosed Patient 5 if there were only four of you it should be normal.) Questions: 1. More developed countries have a lower incidence of parasites than less developed countries. If your patient has a high percentage of eosinophils, what would be a good follow-up question to ask? 2. The immune system usually increases lymphocytes when fighting viral infections. Why then can lymphocytes be low in HIV patients? 3. Which one of patients 1-4 was most likely infected during surgery? Explain your answer. 4. What concern should you have if the patient with mononucleosis is a high school football player? Page6