by
Debra Stamper, Department of Biology
King's College
This case was designed to be implemented in the first semester of our introductory biology course. The majority of the students enrolled in this course are freshmen majoring in either Biology or our Pre-Physician Assistant Program. Each week, students attend three 50-minute lectures and one 50-minute problem hour. Most of the sections of the case were presented during the problem hour (although one section was done at the conclusion of a lecture period).
The different sections of this case all relate to various aspects of sickle cell anemia. In the first section, students are introduced to some of the key investigators responsible for determining the molecular basis of the disease. In the second section, students learn about the functioning of erythrocytes and are introduced to the notion that changes in the environment can influence the functioning of cells. The third section allows students to become familiar with the process of osmosis and how it can influence the sickling of the erythrocytes. In each section, students must address experimental design questions.
Fact or Fiction
This case is a work of fiction that refers to real events and people. All
of the discoveries mentioned in Section 1 were made by the individuals they are
attributed to, as were the observations made by Dr. Vernon Hahn described in Section
2. The time between discoveries has been dramatically condensed, however. It is
true that William Castle knew of Irving Sherman's spectrophotometric observations
of sickled cells, but the letter written in this case is fiction. Both Linus Pauling
and William Castle were members of the same medical advisory committee and it
has been reported that they discussed sickle cell anemia on a train; however,
their conversation as presented in this case is fictionalized. The initial separation
of HbA and HbS was done using a different technique than that presented in the
case. I elected to present it as gel electrophoresis for two reasons: 1) students
enrolled in the course will be doing gel electrophoresis later in the semester,
and 2) some of the students are already familiar with this technique. The electrophoresis
results are drawn in their correct relative position, but these specific samples
are a creation of the author. Irving Sherman's experiences at Boston are completely
fabricated. I have no knowledge that he ever lived in the Boston area. All graduate
students mentioned are also fictional, as is the intern and patient described
at the end of Section 3.
Unless otherwise noted below, students were divided into cooperative learning groups. Each group consisted of three to four students. The groups were pre-assigned by the instructor at the beginning of the semester and remained intact until the conclusion of the course.
The students were told that it would be highly beneficial to bring both their textbook (5th edition of Biology by Campbell et al.) and their lecture notes to the problem hours. Students were occasionally given supplemental material (such as overhead transparencies) with some of the sections of the case. Although the supplemental material is a nice addition, it is not required for successful completion of this case. Many introductory textbooks discuss some aspect of sickle cell anemia.
Section 1: The Investigation Begins
Learning Objectives for Section 1
After completing this section of the case, students will be able to:
Presentation of Section 1
Prior to this section's being presented, students have had lectures on the scientific method, inorganic chemistry, and organic chemistry. They have had no lectures on cellular components and have not been instructed about any aspect of sickle cell anemia. This section can be completed in a 50-minute period.
Students read the section individually. They are instructed to circle any words and/or phrases that they do not understand. After reading the section, they then assemble into their cooperative learning groups. As a group, they are asked to list all the experimental evidence that relates to sickle cell anemia, including why each piece of evidence is a significant finding. They then answer the questions posed at the end of the section. Their textbook contains a diagram of the molecular structure of the different amino acids (see Biology by Campbell, 5th ed., Figure 5.15, p. 69) which aids in answering the questions.
While the students were working, I moved around to the different groups. To initiate a conversation with each group I asked them if they would like me to clarify one of the terms they had circled. Since most of the students have not performed electrophoresis, this tends to be the most common explanation requested. For the groups that did not request electrophoresis to be explained, I asked them to explain the technique to me, filling in or modifying their explanation as needed.
I discovered that many groups had no idea where to start to answer the question relating the electrophoresis data with the data from Vernon Ingram. For these groups, I simply redirected their attention to the molecular structure of the different amino acids. This was sufficient for most of the groups that were stuck, although they still had a difficult time realizing that I wasn't going to point to a section in the textbook that simply spelled out the answer. After a few minutes of grumbling about the need to do their own thinking, they would finally figure it out.
At the end of the class, each group turned in a list of experimental evidence and answers to the questions.
Suggested Answers to Questions Posed in Section 1
In order to not bias the outcome. (This question gives me the opportunity to discuss blind versus double-blind experiments.)
Since there is a difference of one amino acid in the linear sequence of this protein, the primary level of structure will be altered. Altering the primary structure of any protein can then lead to possible (although not mandatory) changes in all other levels of protein structure.
Yes, the glutamic acid carries a negative charge and would therefore migrate to the positive pole quicker.
This sample is from an individual that possesses both forms of the hemoglobin protein. From this question you can then discuss whether this individual would have sickle cell anemia or not. This may lead to a discussion of what actually constitutes an "illness."
Section 2: To Sickle or Not to Sickle: What's a Cell to Do?
Learning Objectives for Section 2
After completing this section of the case, students will be able to:
Presentation of Section 2
Prior to being presented with this section, students had been given lectures on the various organelles found within a cell and the structure of the plasma membrane. They had not received the lectures on membrane transport. They were directed to a photo in their textbook (see Biology by Campbell, 5th edition, Figure 5.19, p. 72) comparing the microscopic appearance of red blood cells from a normal individual and those from an individual with sickle cell anemia. They were also shown an overhead transparency of a photo of normal red blood cells passing through a capillary (from Understanding Sickle Cell Disease by M. Bloom, Figure 3.1, p. 40). This photo depicts the red blood cells passing through a capillary in single-file, bending into a bullet shape in order to "squeeze" through.
This section is broken down into three distinctive parts. The students receive Parts A and B in one class and Part C the next. Students must finish Part A prior to receiving Part B. Many of the groups required the entire 50 minutes to complete Part A. Because the first part took so long to complete, Part B was given to the students as homework to do either individually or as a group. A couple of the groups were able to complete both Parts A and B during the 50-minute period. The vast majority of the students who completed Part B outside the class period worked in their cooperative learning groups. Only four of the 31 students in the class elected to work independently; at least two of these students had outside commitments that hindered their ability to meet with other students outside of class.
Most of the students had difficulty initially addressing the questions in Part A. This was related to their expectation that the answers to the questions would simply be found in the textbook. Most groups were able to generate limitations for cells without a nucleus, but had a harder time coming up with any benefits. I talked to the groups who had difficulty with this a little more about the developmental process that erythrocytes undergo. Often I commented that since the cells lose the nucleus just prior to entering the plasma, that this may enhance their functioning in the plasma. If that didn't work, I redirected their attention to the overhead (which was displayed throughout the entire period).
Part C was done separately during the last 15 minutes of a lecture period. It was during this section of the case that many of the students realized that they could answer the question being posed (even if the answer wasn't given in the textbook).
Suggested Answers to Questions Posed in Section 2
Part A
The major limitation that most students are able to come up with is the inability for RBCs to continue synthesizing new proteins (over a prolonged period). I usually request that they describe what impact this would have on the functioning of the cell. The benefit to the RBC is that it is more flexible, which allows it to "squeeze" through the capillaries better.
A sickled RBC will not be as flexible as a normal RBC. This may lead to the sickled cells being unable to readily traverse the narrow capillaries.
The life span of a cell located in the nerve cell should be longer since these cells retain their nucleus.
As the spikes puncture the plasma membrane, the membrane will become more permeable. This may result in the cell not being able to regulate what molecules pass across the membrane, resulting in a disturbance in the intracellular concentration of many molecules. This could also result in reducing the life span of the RBCs.
Part B
Initially all the variables should be equal, but over time the RBCs will sink to the bottom of the tube since they are denser than plasma. Since the density of the cells will increase at the bottom, it would be expected that at the bottom of the tube the nutrients will be lower (RBCs utilizing them) and there would be an increase in waste products (generated by the RBCs). The pressure at the bottom of the tube will be greater (due to the height of the column of plasma). Temperature should be relatively the same due to the high thermal capacity of water (it should be able to readily absorb any metabolic heat generated by the cells).
Shaking the tube would help to re-equilibrate the contents of the bottom versus the top of the tube.
It has been shown that the major environmental factor is a decrease in the concentration of oxygen. Since the students do not have this information at hand, I am relatively lenient in allowing any sensible answer.
Cells that undergo repeated sickling have a reduced life span, most likely due to problems involving membrane selectivity.
Part C
As a control. He needed to establish that it was the hemoglobin molecule that was responsible for the sickling and not some other component of the cell (such as the membrane or cytoskeleton).
Section 3: Throwing Water at the Problem
Learning Objectives for Section 3
After completing this section of the case, students will be able to:
Presentation of Section 3
This section of the case is presented to the students after the lectures on membrane transport. It is given to the students to work on in their cooperative learning groups during a 50-minute problem hour. They are instructed that in order to complete the second half of the section (the clinical problem), they must first be able to answer questions 1 through 6. At the end of the period, each group turns in their answers to the questions.
Areas in which students needed some additional guidance included determining which molecules dissociate in water and how changes in the volume of water contained within the plasma membrane of a cell can alter the concentration of intracellular molecules (such as hemoglobin).
Suggested Answers to Questions Posed in Section 3
500 mM sucrose = 500 mosm sucrose; 300 mM NaCl = 600 mosm NaCl; 100 mM NaCl = 200 mosm NaCl; and saline is approximately 300 mosm.
Saline is isotonic; both the 300 mM NaCl and 500 mM sucrose solutions are hypertonic; and the 100 mM NaCl solution is hypotonic.
For both the sucrose and the 300 mM NaCl solutions, the extracellular concentration of solutes is greater than the intracellular concentration and the intracellular water concentration is greater than the extracellular concentration. The intracellular and extracellular concentrations of both total solutes and water should be relatively equal for the saline solution. With the 100 mM NaCl solution, the intracellular solute concentration should be greater than the extracellular concentration and the extracellular water concentration should be greater than the intracellular concentration.
Placing the RBCs into saline solution should result in no change in their shape/size since there should be an equal amount of water moving into the cell as out of the cell. With the hypertonic solutions, the cells should get smaller since water will be moving down its concentration gradient (via osmosis) and will leave the cell. The reverse will be true for the hypotonic solution (cells will get bigger and may possibly lyse). (I always emphasize that cells will not automatically lyse if placed in a hypotonic solution.)
The change in hemoglobin concentration is dependent upon the amount of water present in the cell. As water leaves the cell (as in the case with the hypertonic solution), Hb will become more concentrated within the cell. If there is a net inward flux of water (as with the hypotonic solution), then the concentration of Hb will decrease.
As the concentration of hemoglobin increases there is a decrease in the time required for the initiation of sickling.
(See answer under question 8).
Peter should try to use the 100 mM NaCl solution. With this solution there should be a net movement of water into the cells that would result in a decreased concentration of Hb. This may help to reduce the sickling. One possible drawback to this is that you may have some lysis of the cells, but hopefully this would be minimal. Saline would not help the situation, nor should it lead to any further complications. Distilled water would lead to more lysis of the cells than the 100 mM NaCl solution due to the greater inward flux of water (due to the higher concentration gradient). While there would be a reduction in Hb concentration within intact cells, there would be an increase in Hb concentration found in the plasma (which is detrimental to the functioning of the kidneys).
Genetics Problems
Since sickle cell anemia is a heritable trait, it is easy to continue this case into the genetics component of the class. Instead of writing another section to the case, I was interested in seeing if the students could continue on their own. Following the lectures on genetics, I included the following question on their lecture exam:
Sickle cell anemia occurs in individuals who are homozygous recessive. Individuals who are either homozygous dominant or heterozygous do not have the disease. Heterozygous individuals are said to have sickle cell trait since they are carriers. These carriers usually show no ill effects during their lifetime. However, if they find themselves in a situation where oxygen is limited (such as at high altitude), some of their red blood cells may show signs of sickling. Is this an example of complete dominance, codominance, or incomplete dominance? Provide a basis for your choice.
In their answer I expect to see something along the lines that since the phenotype of the heterozygous condition is not exactly like either of the homozygous conditions, then it should be an example of codominance.
I also had a section on their final exam (which is a comprehensive exam) that addressed different aspects of sickle cell anemia. For this exam, I give them a number of study questions which I draw upon to write the exam. Below are the study questions that I gave the class to coincide with this case. During the final exam, students are given a copy of the genetic code.
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For all of the genetics problems given to the students, the assessment of their performance was done on an individual basis.
Protein Synthesis Problem
After the lecture on translation, I gave the students the following problem set to complete during a 50-minute problem hour. Students were directed to the page in their textbook that contained the genetic code (Biology by Campbell, 5th edition, Figure 17.4, p. 299). They worked on this problem in their cooperative learning groups. At the end of the problem hour, each group turned in their answers to the questions posed.
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Letters from Section 1 in .pdf format (requires Adobe Acrobat Reader):
Letter 1 (from Irving Sherman): letter1.pdf
Letter 2 (from Linus Pauling): letter2.pdf
Web Sites
Joint Center for Sickle Cell and Thalassemic Disorders
http://sickle.bwh.harvard.edu
A site that provides information on many different aspects of hemoglobin, sickle
cell disease and current treatments.
"New Hope for People with Sickle Cell Anemia"
http://www.fda.gov/fdac/features/496_sick.html
Revised edition of article by same title by Eleanor Mayfield which originally
appeared in the May 1996 FDA Consumer.
Sickle Cell Information Center
http://www.emory.edu/PEDS/SICKLE
Another good overall source with information for both the layperson and the clinician.
Sickle Cell Syndromes
http://www.mc.Vanderbilt.Edu/peds/pidl/hemeonc/sickle1.htm
A site with information on a more clinical level.
Acknowledgements: This case was developed as part of a National Science Foundation-sponsored Case Studies in Science Workshop held at the State University of New York at Buffalo on June 7-11, 1999 (NSF Award #9752799).