STUDENT INVESTIGATION 3.2

How would you measure the volume of plasma in a person's body? If it were possible, the most accurate way to know how much plasma is in the body would be to take all of the blood out of a person's body, spin it in a large centrifuge (to separate the blood cells and the buffy coat) and then pour the plasma off the top of the cells into a large graduated cylinder to measure the total volume of plasma. Of course, this technique would be fatal to the poor individual who became involved in such a crazy effort! Therefore, a technique was developed to obtain a very accurate measurement of plasma volume in the body. The process for measuring plasma volume is called the "dilution method." Dr. Alfrey employed this method to determine the plasma volume for the astronauts who flew in space.

The dilution method involves injecting the astronaut intravenously (also known as IV, meaning in the veins) with a known quantity and concentration of a dye (also known as a marker or tracer because the dye is used to "mark" the plasma). This dye enters the blood stream and actually mixes with the blood (Figure 6a). After a short period of time, the dye has travelled and mixed with the blood throughout the entire circulation (Figure 6b). During this time, the blood dilutes the dye and then a sample of the blood is collected from the astronaut (Figure 6c). By comparing the concentration of the dye that was initially injected (X in units of mg) with the concentration of the dye in the blood sample (C in units of mg/liter of blood), the investigator can determine how much the dye has been diluted. Using this information in a mathematical equation will yield information about the volume of plasma in the body.

Figure 6: (a) To determine blood volume, a person is injected with a known quantity of dye or marker substance. (b) The dye circulates within the blood vessels and becomes evenly distributed throughout the blood. (c) A blood sample is taken to determine how much the dye has been diluted (yielding a new level of concentration of dye in the sample.) The blood volume can be determined based on how much blood must have been present to dilute the dye to its new concentration.

The dye that is used in this technique must be chosen very carefully so that none of the dye is lost in other parts of the body and so that an accurate plasma volume determination can be made. What would happen if the dye escaped from the bloodstream and seeped into the rest of the body? Remember that the blood serves as a vehicle to carry various substances to the cells and tissues of the body. Constant exchanges are taking place through the blood vessel membrane where oxygen, vitamins, minerals, and many other things pass in and out of the bloodstream. In order to make sure that the dye does not pass through the wall of the blood vessel and enter the rest of the body, a dye is chosen that binds directly to proteins in the blood that are too large to pass through the blood vessel wall. This insures that the concentration of the dye is not affected by the possibility of its disappearance into other parts of the body.

Figure 7: The three containers have different fluid volumes. A known amount of dye has been added to container A. It is your job to determine how much dye to add to containers B and C in order for the concentration of dye in each container to match the concentration in container A.

In this exercise, you will be asked to add a certain number of "drops" of dye to a certain volume of water. Then you will have to determine how many drops you must add to different volumes of water in order to obtain the same concentration of dye in the liquid (Figure 7). By referring to the equations above, Dr. Alfrey's investigation was designed to calculate V (the volume of blood) by measuring X (the amount of dye added to the blood) and measuring C (the concentration of dye per liter of blood). You will be calculating X (the amount or number of drops of dye) by measuring V (the volume of water) and measuring C (the concentration of dye per volume of water). It takes some very sophisticated analysis equipment to determine the concentration of dye in a blood sample. You probably do not have access to this equipment, so your determination of the concentration of dye in the water will have to be made by your eyes! This exercise is simply to point out how different volumes of liquid can affect the concentration of a dye. In the final part of this exercise, you will use what you have learned to calculate the actual plasma volume for some of the astronauts in Dr. Alfrey's study before, during, and after a space flight mission.

Materials

Three large glass beakers, each capable of holding at least one liter and all three the same size.

• A bottle of dye
• A stir stick
• Water

1. Students should work in groups. All students must read the entire lab together before beginning their activities.
   
2. Fill beaker #1 with one liter of water. Fill beaker #2 with a 1/2 liter of water. Fill beaker #3 with 1/4 liter of water. Be as accurate as possible with your volume measurements.
3. Put 24 drops of dye into beaker #1 and stir it until the dye is completely mixed. Count the drops carefully and then record the number on your data sheet.
   At this point, each group should select two individuals to step aside and calculate how many drops should be added to beakers #2 and #3 so that the concentration of dye in those beakers will be the same as beaker #1. They will use the equation shown in the background section. For this equation, V is known and C = 24 drops/liter. These two individuals should keep their results to themselves until the rest of their group can finish adding dye to each of the remaining two beakers. At the end, the calculated results will be compared with the actual number of measured drops of dye that were added to the beakers. The rest of the group will continue with the next step.
4. Set beaker #2 next to beaker #1 so that you can compare the two. Begin adding dye slowly, drop by drop, to beaker #2. Stir as each drop is added. Add enough drops until you have determined that the concentration of dye in beaker #2 matches the concentration of dye in beaker # 1. Don't forget to count the drops as you add them to the beaker. Record the number of drops you added to beaker #2.
5. Set beaker #3 next to both beaker #1 and #2. Again, begin adding dye slowly, drop by drop, to beaker #3. Follow the same procedure for beaker #3, counting and stirring after each drop, until you have determined that the concentration of dye in all three beakers is the same. Record the number of drops you added to beaker #3.

Now, compare the calculated results with the results obtained by adding drops of dye. Hopefully, the two results come very close to matching. Any difference in the two results can be attributed to the lack of an accurate way to "measure" the concentration of dye in the liquid.

Now, we will look at some actual raw data and results that Dr. Alfrey obtained during his space flight investigation. Table 2 includes various data including the amount of radioactive iodine (dye) that was originally injected into the bloodstream of each astronaut (x), and the concentration of that dye in the blood sample that was removed from the astronaut at different times during the mission (c). From these data, you are to calculate the plasma volume (v) using the equation v= x/c. You will be able to see how the plasma volume changed over time during the mission. Your teacher will review with you: the various parts of the table; how to carry out your calculations; and, later, what the correct answers are. You know, people are actually paid to do this sort of analysis!