The Use of Electrophoresis and Western Blot in Testing the Patients with Pernicious Anemia - Case Study Example

PRACTICAL REPORT: THE USE OF ELECTROPHORESIS AND WESTERN BLOT IN TESTING THE PATIENTS WITH PERNICIOUS ANEMIA Name Institutional Affiliation Instructor’s Name Date Practical Report: The Use of Electrophoresis and Western Blot in Testing the Patients with Pernicious Anemia Introduction Pernicious anemia predominantly has hematological or neurological manifestations even though the disease is caused by an abnormality in the gastric mucosa. Initially the literature described pernicious anemia patients to be those with the inability to secrete gastric acid. However, further research later found that the pernicious anemia patients were unable to secrete the intrinsic factor that is essential for the absorption of vitamin B12 (cobalamin) at the lower part of the small intestine (Piper, Stiel, & Builder, 2016, 357). Schisk (2016a, 1) states that vitamin B12 deficiency is not the main cause of pernicious anemia except for the strict vegetarians who cannot consume any animal products including the products from dairy, eggs, or meat. The study of the etiology of pernicious anemia is of paramount importance to the medical practitioners due to its importance in differentiating the macrocytosis caused by pernicious anemia and that which develops from other causes. B12 deficiency anemia results in the presence of the immature and large red blood cells in the bloodstream which could as well result from other causes not associated with vitamin B12 deficiency. Macrocytic or megaloblastic anemia can result when the levels of either or the both of folic acid and vitamin B12 levels are sub-normal (Pacholok & Stuart, 2012, 89). In this practical report project, I will carry out experiments to demonstrate the need for understanding the morphology of the stomach wall along with its possible abnormalities that would result in pernicious anemia to avoid the misdiagnoses made in determining the causes for megaloblastic anemia for better patient outcomes. Purpose of the Practical I carried out the practical to elucidate the importance of the medical practitioners to understand the etiology and pathophysiology of the rare disease of pernicious anemia for us to avoid making the mistakes that could lead to unnecessary fatalities. Pernicious anemia occurs in the three forms of the congenital, juvenile, and adult-onset pernicious anemia. The main cause of pernicious anemia is through the stomach wall’s inability to produce the intrinsic factor necessary for the absorption of the vitamin B12 that is required for the normal hemoglobin formation (Escott-Stump, 2008, 640). This practical underscores the importance of understanding the constituents of a patients’ stomach wall lining before concluding that they are ailing or not ailing from pernicious anemia. Methodology Pernicious Anemia Electrophoresis To perform the tests, I added 50 µL of 5 * SDS-sample buffer to 200 µL patient protein and mixed it. I sealed the tube for some time and then loaded the MW marker into the first well. I then loaded 25 µL aliquots of the protein into the sample wells, connected it to a power supply and ran the gel for 1 hour. To transfer, I removed the gel from the apparatus after turning off the power supply and noted the position of the gel’s position on the protein marker and cooled the gel cassette under running water. I then opened the cassette and used the plastic wedge to cut the lanes from the gel. I then cu the corner from the gel’s top with the marker to recognize it. I then placed the gel plate in a tray with a transfer buffer to immerse the plate. I then detached the gel, removed the gel plate and set the gel aside. I set up an apparatus containing a nitrocellulose membrane to which the proteins were to be transferred. At the bottom of the stack I placed 1 piece of the ultra-thick filter paper, followed by the nitrocellulose membrane, then the gel and 1 piece of another ultra-thick filter paper at the top of the stack. I then placed a lid on the turbo-blot and locked it. I then inserted the cassette into the TurboBlot transfer apparatus and set it into a constant current of 25V for seven minutes before starting the transfer. I turned off the power supply once the transfer had finished, disassembled the apparatus and placed the nitrocellulose membrane in a container with mL 0.1% Poceau stain and placed it on a rocker for 1 minute. I rinsed the membrane with deionized water to enable the visualization of the lanes. I took note about the crispness and form of the bands. I then labeled the nitrocellulose membrane while still wet and marked out the lanes stained with ponceau S to enable the cutting of the strips of protein for accurate antibody testing. I then rinsed the membrane briefly in 50 mL NaOH on the rocker and when it disappeared I rinsed it briefly on the rocker in 50 mL TBS. I added 50 mL blocking solution to block non-specific binding of proteins to the membrane. I then heat sealed the membrane in a plastic pouch with a blocking buffer and labeled it while removing as many bubbles as possible. Finally, I compared my results with the other students and stored it in a buffer at 4°C. Demonstrating the Morphology of the Stomach To understand the morphology of the stomach, I stained a formalin-fixed, paraffin-embedded (FFPE) section of a mouse stomach with haematoxylin and eosin. I incubated a slide containing a slide with a mouse’s stomach in xylene and then ethanol for two minutes each. I rehydrated it in tap water and incubated in haematoxylin for minutes, rinsed it in tap water for 30 s and blotted the end of the slide. I dipped it in acid alcoholfro 3 s and rinsed immediately in water for 30 s. I incubated it in Scott’s tap water for 30 s and rinsed in water for 30 s. I then placed the slide in eosin Y for 4 min and blotted excess stain. I dipped the slide up and down in 90% ethanol for 30 s and drained excess liquid on a blotter. I incubated the slide in 100% ethanol for 2 min and let it dry. I then placed a drop of OrganoLimolene mounting medium on the section and covered it with a cover slip. Finally, I examined it under the microscope and recorded the morphology of the stomach with 2 drawings. Pernicious Anemia Western Blotting For Western Blotting, I removed the membrane that I had stored with electrophoresis from the pouch carefully. I then sliced the remaining membrane into strips to test the patients. I placed each strip onto a piece of parafilm and pipetted 100 µL of the sera to the strips. I then covered lightly with another piece of parafilm. I incubated the apparatus for 1 h at room temperature and then washed the strips 3 * 10 min in TBST with rocking. I then placed each strip to a fresh pience of parafilm and pipetted 100 µL of the sheep anti-human Ig-HRPO and incubated for 45 min. I washed the strips 3 * 5 min in TBS and shook. I left the strips with the TBS buffer for transport to the Chemidoc. The strips were then incubated in 4 ml of Lumi-Light substrate for 5 min and then aligned on a piece of parafilm. The Chemidoc is then used to place an image of the membrane on CloudDeakin. The result constituded the 2 images one of the marker and the other of the parafilm. I finally cropped the two images and created a figure for analysis. Results The results of my study showed that the patient that I was investigating has pernicious anemia as represented by patient number 3 in table 1. As illustrated by the diagram in figure 1, it can be concluded that the patient has pernicious anemia since the brown staining typical to parietal cells is not present. Figure three shows the diagram of my bench mates findings where patient 4 does not have pernicious anemia as proven by the presence of the parietal cells. Western blotting represented the findings accurately as shown in figure three except for the results for patient number 1. Table showing the 6 patients tested for pernicious anemia Patient No. + - 1 2 3 4 5 6 Presence of Pernicious Anemia  X    X X  Table 1: The table shows the results of the 6 patients tested for pernicious Anemia. I compared the results of my patient identified as number three with the results of my classmates. Figure 1: Patient 3 has pernicious anemia. Figure 2: Patient 4 does not have pernicious anemia. Figure 3: Findings for the 6 patients from western blotting. The findings from the experiment carried out to demonstrate the morphology of the stomach yielded positive results as demonstrated in the drawings in figures 4, 5, and 6. The parietal cells in a normal mucosa stain brown and the chief cells do not stain as shown in figure 4. Figure 5 shows the location of the parietal cells in the gastric or cortical glands layer of the stomach wall. Figure 6 represents the findings for the location of the parietal cells in the cortical glands layer of the stomach wall. Figure 4: Drawing demonstrating the positive staining of the parietal cells as compared to the chief cells. Figure 5: Diagram showing the cortical glands, mucosa, muscularis, and submucosal layers under low magnification. Figure 6: Diagram showing the parietal and chief cells in the stomach wall under high magnification. Discussion Pernicious anemia arises from vitamin B12 deficiency which most commonly arises from the lack of production of intrinsic factor that leads to the impaired or lack of absorption of vitamin B12 in the terminal ileum. Among other causes, the impaired absorption of vitamin B12 gives rise to megaloblastic anemia. The term pernicious comes from the high degree of fatality witnessed before treatments were developed and established (Schick, 2016b, 1). The practical that I have conducted underscores the importance of the Schilling’s test in determining the deficiency of the intrinsic factor. Pernicious anemia can be detected with 100% specificity and 73% sensitivity by using the useful surrogate markers for the parietal cell and intrinsic factor antibodies (Lahler & Annibale, 2009, 1). The antibodies required in the practical were the sheep anti-human antibodies that target the a-subunit and β-subunit that are normally found in the human gastric proton pump or the H+/K+-transporting adenosine triphosphatase (H+/K+ATPase) (Jones, Toh, Pettitt, Martinelli, Humphris et al, 2001, 51). The alternative for completing this practical could have been the Schilling test. In addition and as Andres et al (2015, 1) states, the “modified” Schilling test would use the radioactive cobalamin bound to the proteins of the animals that have been altered as opposed to the free radioactive vitamin B used in the normal Schilling test. The authors as well suggest that the Schilling and the modified Schilling tests have not been available to clinical practice and as such the cobalamin mal-absorption has been a diagnosis of exclusion. Still, some authors have used the Schilling test to confirm the presence of vitamin B12 mal-absorption to confirm the presence of the mal-absorption of B12 along with the diagnosis based on Kay’s augmented histamine test (Piper, Stiel, and Builder, 2016, 360). The serum vitamin B12 levels of less than 200 pg/mL are indicators of cobalamin deficiency and they constitute a high specificity of between 95 and 100%. However, the low serum cobalamin levels, Schilling test, and absence of cobalamin deficiency with the standardized level of 2.5 to 5 μg daily have not been validated in the approach of evidence based medicine. As such, it is safe to suggest that the standard and modified Schilling tests are the gold standard for testing the pernicious anemia that results from the steps of cobalamin absorption that lead to its serum deficiencies (Mazokopakis, 2012, 263). Moridani and Ben-Poorat (2006, 166) state that The well-known symptom of vitamin B12 deficiency include the hematological changes such as macrocytic anemia even though they become manifest in the later stages of the disease. More accurate diagnosis of the disease can be achieved with the newer methods of the metabolites methylmalonic acid, holo-transcobalamin II, antibody testing, homocysteine, and gastrin levels. As such, this project has outlaid the importance of the medical practitioners to understand the role of the macroscopic and microscopic features of the stomach wall in making the most appropriate decisions in managing the pernicious patients for the best patient outcomes. Finally, I suggest that further research should be conducted to standardize the use of electrophoresis and western blot for the patients’ protein to achieve clinical tests with a high level of sensitivity and specificity. Conclusion In this practical, I have demonstrated how the abnormalities in the stomach wall of the pernicious anemia patients lead to the inability of secreting the intrinsic factor essential for the absorption of vitamin B12 (cobalamin) at the lower part of the small intestine. The electrophoresis and western blot results show the lack of the parietal cells in the stomach wall that are responsible for secretion of the intrinsic factor. The microscopic study served as a modified control experiment to demonstrate the presence of the parietal cells and chief cells in the stomach wall. This practical has underscored the importance of understanding the constituents of a patients’ stomach wall lining before concluding that they are ailing or not ailing from pernicious anemia. References Escott-Stump, S. 2008. Nutrition and diagnosis-related care. Philadelphia: Lippincott Williams & Wilkins. Jones, C. M., Toh, B. H., Pettitt, J. M., Martinelli, T. M., Humphris, D. C. et al, 2001. Monoclonal antibodies specific for the core protein of the beta-subunit of the gastric proton pump (H+/K+ ATPase). An autoantigen targetted in pernicious anaemia. European Journal of Biochemistry, 197(1). Pp. 49-59. DOI 10.1111/j.1432-1033.1991.tb15881.x Mazokopakis, E. E. 2012. The old Schilling test as a necessary criterion at present for the diagnosis of food-cobalamin malabsorption (FCM) syndrome. Hellenic Journal of Nuclear Medicine, 15(3). Pp. 262-263. Accessed at http://nuclmed.web.auth.gr/magazine/eng/sept12/86.pdf Moridani, M. & Ben-Poorat, S. 2006. Laboratory investigation of vitamin B12 deficiency. LabMedicine, 37(3). Pp. 166-174. DOI 10.1309/CVHKLE2R4W68K2NQ Pacholok, S. M. & Stuart, J. J. 2012. Could it be B12?: An epidemic of misdiagnoses. Fresno: Linden Publishing. Piper, D. W., Stiel, M. C., & Builder, J. E. 2016. The electrophoresis of human gastric juice. BMJ. Pp. 357-360. Accessed at http://gut.bmj.com/content/3/4/357.full.pdf Schick, P. 2016b. Pernicious anemia: Background. Accessed at http://emedicine.medscape.com/article/204930-overview Schisk, P. 2016a. Megaloblastic Anemia: Etiology. Accesses at http://emedicine.medscape.com/article/204066-overview#a5 Read More