Materials Included In Kit

Additional Materials Required

Prelab Preparation

It is important to make more of each simulated blood mixture than will be used by the lab groups. After thoroughly stirring the mixture, separation of the red sugar crystals and corn oil begins immediately. As a result, the final portion of the mixture in the bottom of the beaker may be significantly more concentrated than the rest, resulting in a falsely elevated PCV. The three simulated blood samples may be any concentration. The sample data given are from concentrations of 38%, 41% and 45%.

1. Obtain three 150-mL beakers and label them A, B and C, respectively.
2. Measure 30 mL of red sugar crystal into a 50-mL graduated cylinder. Pour the crystals into beaker A.
3. Add corn oil to beaker A, stirring constantly, to the 80-mL mark of beaker A.
4. Repeat steps 2 and 3 for beakers B and C, measuring 33 mL and 36 mL of red sugar crystals, respectively.
5. Place the three beakers at dispensing stations around the lab, along with the stirring rods and some paper towels.

Safety Precautions

The corn oil and red sugar crystals are considered nonhazardous. All food-grade items that have been brought into the lab are considered laboratory chemicals and may not be removed from the lab. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Remind students to wash their hands thoroughly with soap and water before leaving the laboratory. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. The simulated blood mixture may be disposed of according to Flinn Suggested Disposal Method #26a. Leftover corn oil and red sugar crystals may be stored for future use.

Lab Hints

• Enough materials are provided in this kit for 30 students working in pairs or for 15 groups of students. This laboratory activity can reasonably be completed in one 45- to 50-minute class period. The prelaboratory assignment should be completed before coming to lab. The calculations and data analysis may be completed the same day as the lab, and the class discussion may be conducted the day after the lab.
• Even though the corn oil and sugar crystals used to make the simulated blood samples are considered nonhazardous, it is recommended that students wear goggles, gloves, and aprons whenever chemicals are used (food-grade items brought into the lab are considered laboratory chemicals). In addition to reinforcing safe laboratory practices, wearing personal protection equipment enhances the students’ roles as lab technicians in the scenario.
• Determining outliers (any value significantly beyond the overall pattern of the data) may be a subjective exercise and difficult for students. For this activity, a simple method would be to calculate the difference between each PCV value and the average value for the tested sample. Any differences that vary significantly from the others may be considered outliers.

Teacher Tips

• This versatile lab may be used as part of a unit on the circulatory system, a study of different types of mixtures, human health or as a forensics activity.
• Cyclists often protested the “over 50% hematocrit rule” used to suspend contestants, claiming it was unfair and often inaccurate. A more accurate urine test has been in use since 2003. This test is able to distinguish between natural and synthetic EPO. However, a hematocrit is still often used as a preliminary screening device for the safety of the athletes and to determine if further testing may be necessary. The more expensive urine test is only conducted if preliminary blood tests indicate the possible use of performance-enhancing drugs.

Answers to Prelab Questions

1. After reading the Background section, what can be inferred about why increasing red blood cell volume may enhance performance in a competitive sport?

   Increasing red blood cell volume may increase the amount of oxygen available to the muscles, thus increasing the endurance levels of athletes.

2. A hematocrit measurement has a red blood cell level of 4.4 mL and a total blood level of 10.2 mL. What is the PCV of the blood sample? Does this fall within the average range? Show your work.

   (4.4 mL/10.2 mL) x 100 = 43% This percentage falls within the average range for both males and females.

3. Read through the Procedure. Why is it important for each group to thoroughly stir the simulated blood sample in the beaker just prior to dispensing it into the centrifuge tube?

   The simulated blood is a heterogeneous mixture, and the “red blood cells” separate from the “plasma,” settling to the bottom of the beaker. Stirring thoroughly helps disperse the solid “cells” more evenly throughout the mixture. A more uniform sample will result in a more accurate PCV value.

Sample Data

Blood Sample ___C___

Answers to Questions

1. Use Equation 1 from the Background section to calculate the percent PCV in each centrifuge tube from the blood sample your group was assigned to test. Record each PCV in the data table.

   See sample data table.

2. What is the range of PCV values for the tested blood sample? Would any values be considered an outlier? What are possible sources of error in this lab activity?

   For simulated blood sample C, the range of PCV values is 42–55%. The lowest and highest values might be considered outliers. Possible sources of error include a less than uniform dispersal of the components of the simulated blood mixture, the precision of the centrifuge tube, some trapped “plasma” in the “red blood cell” volume, and the height of the “red blood cells” may not be perfectly level.

3. Together with the other lab groups who tested the same simulated blood sample, determine the average PCV of the cyclist. Hint: The groups may decide to eliminate one or more outliers, if any, or average the three closest values. Show your work below and include the reasoning for the average calculation.

   Since the highest and lowest PCV values of the simulated blood sample may be considered outliers, the other three values were averaged. (48% + 50% + 48)/3 = 49%

4. In your opinion, did the average PCV of your tested simulated blood sample indicate cheating by the cyclist?

   The PCV of Cyclist C is less than 50%, indicating the contestant was not cheating by blood doping.

5. Other than blood-doping, what factors might result in a higher-than-usual red blood cell volume in endurance athletes?

   An athlete may genetically have a high PCV, may be dehydrated, or may have trained at a high elevation prior to the contest.

6. Why is engaging in strenuous physical activity dangerous with a low or high red blood cell volume?

   Engaging in strenuous physical activity with a low red blood cell volume may direct needed oxygen to the muscles and away from vital organs. Strenuous activity with a high red blood cell volume is also dangerous, as higher viscosity blood may clog small capillaries, limiting the blood supply to the heart and brain, resulting in a possible heart attack or stroke.

7. Find out the average PCV of the other two simulated blood samples. As a class, determine if any cyclist was cheating. Discuss the pros and cons of using a hematocrit to determine whether or not a contestant should participate in a race.

   For simulated blood samples prepared according to instructions given, accurate PCV measurements will show no athlete was cheating. Varying results may lead students to conclude that using a hematocrit exclusively is not an accurate indicator of blood doping. However, the use of a hematocrit prior to a race may prevent athletes from competing under dangerous health conditions and may warrant further testing.

References

Special thanks to Diane Sweeney, Punahou School, Honolulu, HI, for providing the idea and the instructions for this activity to Flinn Scientific.

Blood Testing for Professional Cyclists: What’s a fair hematocrit limit? http://www.sportsci.org/news/news9703/AISblood.html (accessed June 2010).

Introduction

The bicycle race is over and the fourth place winner has accused the front-runners of cheating by blood-doping, a means of increasing the number of red blood cells in the bloodstream. Blood-doping to enhance athletic performance is banned by competitive sports. Your group is assigned to test the blood of one of the top three finishers to see if the contestant has an unusually high red blood cell volume, an indication of blood-doping.

Concepts

• Hematocrit
• Anemia versus polycythemia
• Red blood cell volume
• Blood-doping

Background

Red blood cells are biconcave, disc-shaped cells (see Figure 1). Red blood cells contain a protein called hemoglobin that binds oxygen for transport between the lungs and body tissues. Each red blood cell contains millions of hemoglobin molecules. Since hemoglobin in red blood cells provides the mechanism for oxygen transport in the blood, the amount of red blood cells present is one indicator of the oxygen-carrying capacity of the blood.

Blood is a heterogeneous mixture of red blood cells, white blood cells, platelets and liquid plasma. A hematocrit is a measurement of the red blood cell volume, which is also known as the packed cell volume (PCV). The PCV is the proportion of the total blood volume that is occupied by the red blood cells expressed as a percent. For example, if a 100-mL blood sample contains 30 mL of red blood cells, the PCV is 30%. To determine the PCV of a patient, a sample of the blood is placed in a small tube and centrifuged. The red blood cells are more dense than the plasma and other components of the blood mixture and settle to the bottom of the tube (see Figure 2). The PCV can be calculated by measuring the volume of the layer of red blood cells and dividing it by the total volume of the sample, then multiplying by 100 (Equation 1). A normal PCV is 41–50% for adult males and 38–46% for children ages 12–15 and adult females. A person with a low PCV is considered to have anemia, a condition that leads to fatigue and weakness, and in extreme cases may even cause a heart attack. A high PCV value, known as polycythemia, may be caused by an excess of red blood cells or a decrease in blood plasma as a result of dehydration. Endurance athletes may increase their PCV values temporarily by training at high altitudes before competition. Severe polycythemia causes the blood to be thicker and more viscous than normal, which can clog the small capillaries and lead to a heart attack or stroke.

In 1997, the Union Cycliste Internationale (UCI) determined that any bicycle race contestant with a PCV over 50% was considered as being at risk to compete, temporarily suspended from the race, and subjected to further testing. In 1998, the cycling world was enveloped in scandal as an entire team competing in the Tour de France was expelled from the race for cheating by using performance-enhancing drugs, including erythropoietin (EPO). EPO is a natural hormone produced by the body to stimulate red blood cell production. The use of synthetic EPO for blood-doping is prohibited in competitive sports. Synthetic EPO is nearly identical to the body’s natural EPO and difficult to detect; therefore, a hematocrit is used to test the cyclists’ blood for elevated red blood cell volumes.

Experiment Overview

The purpose of this activity is to analyze red blood cell volumes in simulated blood samples obtained for the following scenario. Accusations of blood-doping with synthetic EPO have been brought against the top three finishers of a bicycle race. Blood samples were taken from each contestant prior to the race and will be used to conduct a hematocrit. Each lab group will test a portion of the sample from one of the three competitors. Groups testing from the same sample will determine the average PCV of the sample. All groups together will discuss their respective results and determine if any cheating took place.

Materials

Prelab Questions

1. After reading the Background section, what can be inferred about why increasing red blood cell volume may enhance performance in a competitive sport?
2. A hematocrit measurement has a red blood cell level of 4.4 mL and a total blood level of 10.2 mL. What is the PCV of the blood sample? Does this fall within the average range? Show your work.
3. Read through the Procedure. Why is it important for each group to thoroughly stir the simulated blood sample in the beaker just prior to dispensing it into the centrifuge tube?

Safety Precautions

The components of the simulated blood are considered nonhazardous. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. Wash hands thoroughly with soap and water before leaving the laboratory. Please follow all laboratory safety guidelines.

Procedure

1. Write the letter of the blood sample that your lab group has been assigned to test on the Who Cheated in the Race? Worksheet.
2. Obtain a marker and a centrifuge tube and cap.
3. Write the letter of the blood sample which your lab group has been assigned to test on the cap, along with your group number (see Figure 3).
   {11036_Procedure_Figure_3}
4. Take the centrifuge tube and cap to the group’s assigned dispensing station.
5. Use a stirring rod to thoroughly stir the simulated blood in the designated beaker so the components of the mixture are as uniformly dispersed as possible. Note: Do not skip this step even if another group has just recently stirred the mixture.
6. Lay the stirring rod on a paper towel and quickly, yet carefully, pour the simulated blood mixture into the centrifuge tube to the 10-mL mark (see Figure 4). Note: It is fine if the simulated blood level is a little above or below 10-mL mark. Do not pour any of the blood mixture from the centrifuge tube back into the beaker.
   {11036_Procedure_Figure_4}
7. Place the filled centrifuge tube in a test tube rack and let the mixture settle for 5 minutes. Questions 5 and 6 on the worksheet may be answered during the wait time.
8. After 5 minutes, remove the centrifuge tube from the test tube rack, being careful to keep it upright so as not to mix the contents.
9. Observe the top level of the simulated red blood cells. Record the volume to the nearest half-milliliter in the data table on the worksheet. Note: Holding the tube up to a light or window may make the gradations on the tube easier to see.
10. Observe the total volume of the mixture in the test tube. Record the volume in milliliters on the worksheet.
11. Confer with the other groups that were assigned to test the same simulated blood sample and record all of the groups’ data in the table.
12. Complete the calculations and answer the questions on the worksheet.
13. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

11036_Student1.pdf