Laboratory Test for Sickle Cell Anaemia - Case Study Example

LABORATORY TEST FOR SICKLE CELL ANAEMIA s s email address) s s s ofLocation Date of Submission Abstract There are signs characterizing sickle cell anaemia. The symptoms may occur mildly or severely to a patient. The boy in this case, study had severe symptoms of the disease, which made him be hospitalized. Mostly, the patient suffers the disease from their birth. However, the disease takes time to manifest. It becomes visible at a minimum age of four months. The disease signs are linked to anaemia, pain, and disease`s complications. To diagnose sickle cell condition, it involved four screening test. That is, two haemolytic tests, one observation under a microscope and cellulose acetate electrophoresis. However, there are other sickle cell anaemia screening tests unavailable to test sickle cell anaemia in the small laboratory at the Mbetta Health Center. The tests require high cost, a lot of time, and they are by the high temperature and humidity. Physical Examination On the first day in Cameroon at Mbetta Village, the nursing staff referred to me a case of a boy aged 4 years. The boy was clearly unwell; he presented fever, headache, fatigue, pain in the arms and legs. On physical examination by a nurse, the boy`s blood pressure was in a normal range and he had no palpable lymph node. However, the higher left hand abdominal quadrant palpation, auscultation, and percussion signified an enlarged spleen. The nurse noted the boy`s mucus to be pale in colour on examination and the sclera portrayed some elements of Icterus. With no any other abnormal physical signs observed from the boy, the nurse took a venous blood sample and put it into anticoagulant to assist in carrying out a laboratory investigation of the disease. Interpreting Physical Examination Sickle cell anaemia affects bones, lungs, abdomen, joints, limbs, and lungs (Ohene-Frempong 2006). For instance, pain, swelling, and fever occur when sickle cells block limb veins (Center for children with Special Needs 2006). When spleen traps many red blood cells a mechanism meant to trap the abnormal red blood cells, it enlarges (Maakaron, E. J. n.d.). Lastly, life-threatening cases of sickle cell anaemia occur when red blood cells are trapped in the lung vein to cause Acute Chest Syndrome, which may damage vein in the lungs. It is detected through abnormal breathing and normal blood pressure (Ohene-Frempong 2006). Laboratory Sickle Cell Testing The laboratory in Mbetta village is small and equipped as a basic haematology laboratory. It contains microscope, slides, cover slips, and various stains like Romanowsky stain and immersion oil. It is also equipped with haematocrit centrifuge, glass capillaries, an electrophoresis tank, and a power pack. A small portable power generator supplies electric power. Finally, the laboratory has several reagents and chemicals. Hence, According to laboratory size, equipments and reagents, the laboratory tests for sickle cell on the sample of the boy`s blood must not be complex. i. Fluorescent Spot Test Screening for G6PD Deficiency There are various factors that made fluorescent spot test appropriate for testing sickle cell anaemia condition on the boy`s blood. Firstly, the fluorescent spot test is recommended for testing conditions showing a high level of G6PD deficiency considering it can test hemizygote, homozygote, and heterozygote (Wong, Boo, Ainoon, and Wang 2009). Secondly, the fluorescent spot test principle is simple and consumes little time to undertake. The principle of the test involves conversion of NADP+ to NADPH in the presence of G6PD. The speed of conversion depends on G6PD activity (Wong, Boo, Ainoon and Wang 2009). Thirdly, the fluorescent spot test uses a few reagents, which are inexpensive and easy to acquire. The reagents needed in the test included D-Glucose-6-Phosphate (G6P), NADP+, Saponin, Tri-Hcl buffer at pH of 7.8 and oxidized glutathione (GSSG) (Wong, Boo, Ainoon and Wang 2009). Lastly, the process of setting up the experiment is easy. It involved mixing two volumes of G6P with 1 volume of NADP+, 2 volumes of saponin, 3 volumes of buffer, 1 volume of GSSG, and 1 volume of water. Azide was added to prevent contamination (Wong, Boo, Ainoon and Wang 2009). Moreover, to read the results, control experiment and test experiment were compared. The process of preparing the experiment was as follows. Two tubes containing reagents were thawed and set ready. One tube was used as the control and the other as an examination tube (Jiang, Ma, Song, Lin, Cao, Wu, and Hsiao n.d.). They were left to attain room temperature before beginning the experiment. The blood from the boy`s body was added to the examination tube. Blood tested and proved negative on sickle cell anaemia was added in the control tube. Mixture from each tube was poured on a different Whatman No. 1 filter paper at the beginning and after 5-10 minutes of reaction (Oni, Johson, and Oguntibeju 2005). The filter paper was allowed to dry before examining under UV light (Wong, Boo, Ainoon and Wang 2009). Florescent was available for the filter paper that was soaked with the examination content. Hence, the boy had the sickle cell anaemia condition. ii. Mathaemoglobin Reduction Test Despite the long time taken to get results through this experiment, the experiment was chosen to determine availability of sickle cell condition, as its costs for buying required reagents and carrying the experiment are extremely low. The only requirement by the experiment is a water bath and reagents sodium nitrite, AR dextrose and NaNO2 dissolves in water and methylene blue solution. Moreover, the mathaemoglobin reduction test was chosen as its reagent may be set in various ways to match the convenience of the laboratory. For instance, batches of tubes are arranged and used to contain mixtures of similar volumes of sodium nitrite and methylene blue. From the solution, 0.2ml is poured into a tube by use of a pipette. At room temperature, the reagents are allowed to dry. Remember, glass tubes are used in place of plastic as plastics absorb some reagents. Another reason why the mathaemoglobin reduction test was employed is a possibility of using the reagents prepared in excess in the experiment within 6 months after preparation. The only requirement for storing the reagent is at room temperature and airtight container. These will prevent liquid from drying. Finally, fresh blood collected for sickle cell testing from the boy`s body encourage the use of the mathaemoglobin reduction test. This experiment was effective with fresh blood only; blood stored up to one week. After testing the boy`s blood under Mathaemoglobin Test, the colour of the tube containing his blood was similar to the colour of the normal reference tube. This is an indication that the boy was suffering from sickle cell anaemia condition. iii. Sickling Test On observing the boy`s blood placed on a slide through a microscope (X10), most of his red blood cells appeared to suffer from sickle condition. The test was considered appropriate as it reduced cost and it was more reliable (Okwi, Byarugaba, and Ocaido 2010). Moreover, it was easy to carry the experiment. The red blood cells on the slide containing blood were exposed to low oxygen while under microscope observation (Wajcman, and Moradkhani 2011). To expose red blood cells in blood to low oxygen, 20µl of blood sample was mixed with an equivalent amount of 2 percent Sodium Metabisulphite while with a cover slip. With the cover slip on, it was covered with wax and inverted (Wajcman, and Moradkhani 2011). The slide was left in a humidified chamber for 15 minutes. It was observed through a microscope (X10) and subsequent observations follow after every 15 minutes for 1 to 2 hours (Okwi, Byarugaba, and Ocaido 2010). The boy was considered suffering from sickle cell, as more than 25 percent of his blood cells observed turned out sickled. This is as shown in the below pictures (Okwi, Byarugaba, and Ocaido 2010). a. First observation b. Second observation c. Third Observation d. Fourth Observation e. Fifth observation iv. Hb Electrophoresis Screening Method The boy`s sickle cell condition was confirmed by the cellulose acetate membrane (CAM) Hb electrophoresis screening method done at pH of 6.2. This method is accurate and reliable as when determining the presence of haemoglobin AA, AS and SS in the boy`s blood (Dogaru, Coriu, and Higgins 2007). Moreover, the experiment`s working principle is easy to apply. On application of electrical charge, the positively charged proteins were attracted on the negatively charged terminal. Similarly, the negatively charged proteins were attracted to the positively charged terminal. However, the size and shape of the protein differentiated the distance the protein bands are located between the positive and negative terminals as these electrically attracted proteins migrate at different rates (Dogaru, Coriu, and Higgins 2007). To determine the sickledness, the bands formed were stained by appropriate chromogen and they were compared with known controls (Dogaru, Coriu, and Higgins 2007). The requirements include power supplied by a generator, electrophoresis tank, plastic holders for CAM, sample plate and applicators, 3*0.5 inch tubes, plastic pot used to soak CAM and fix bands, bulbous plastic pipettes, Whatman No.1 filter paper, tube rack, scissors, small forceps, 5 percent Ponceau Red, barbital buffer at 9.2 percent pH, acetic acid at 5 percent, distilled water and saline (Dogaru, Coriu, and Higgins 2007). All of the above requirements require little capital to purchase and they are locally available. Moreover, the process of preparing reagents requires simple conditions. They include setting up buffer at 9.2 percent dissolve the appropriate amount of sodium barbital, and Tris in appropriate amounts of water, preparing 5 percent Ponceau red and preparing colour contrast, 0.05 percent acetic acid solution (Dogaru, Coriu, and Higgins 2007). Lastly, when undertaking this experiment, it is easy to read and follow the available guide. That is, first, there was soaking cellulose acetate strip in Tris buffer for a duration of 15 minutes as a way of making it soft (Hicks, Griep, and Nordschow 1973). Second, blood was spun for about 4000rpm in duration of 5 minutes (Hicks, Griep, and Nordschow 1973). The spun blood was separated into blood plasma and red blood cells. The red blood cells were washed by 1ml of saline and centrifuged as above. This washing process was repeated two times. Third, a few ml of water were added to the washed red blood cell. They were shaken to haemolyse the red blood cells. This released haemoglobin (Hicks, Griep, and Nordschow 1973). Fourth, 20µl of red blood cells were put in each well. Fifth, CAM was stripped from each buffer and blotted using Whatman No. 1 filter paper. The resultant film was secured firmly on stand set aside for the strips (Hicks, Griep, and Nordschow 1973). Sixth, sample applicators were used to put haemoglobin in the sample being tested. The control experiment was aligned into cellulose strip (Hicks, Griep, and Nordschow 1973). Seventh, CAM was put into electrophoretic tank, power supply set at 200v, switched on, and left running until the band clearly separated. Note that, in a case of oversupply of power, the strips would be destroyed (Hicks, Griep, and Nordschow 1973). Eighth, by use of ponceau red, the strips were stained. The excess ponceau red was washed off by 5 percent acetic acid until the bands were clear from the unstained parts of the strip (Hicks, Griep, and Nordschow 1973). This gave the below results. Finally, the test samples were compared against control bands. According to colours of test band and matching band, it was confirmed the boy was suffering from sickle cell anaemia (Dogaru, Coriu, and Higgins 2007). Other Test that would have been helpful if Available i. Cation Exchange High Definition Chromatography Cation Exchange High Definition Chromatography quantifies the normal and abnormal red blood cell fractions. Using the charges on proteins due to amino acids and different pH, the proteins in a blood sample will be grouped and quantified. In future, it is expected this method will incorporate densitometry, which will scan the electrophoretic patterns (Wajcman, and Moradkhani 2011). ii. DNA Analysis The analysis of DNA is done to establish the presence of β thalassemia, which mutates to cause sickle cell anaemia. DNA methodologies commonly employed are PCR Restriction analysis and ARMs. However, people have reduced DNA tests through these tests. Another test used for analyzing DNA are α and β globin gene analysis. The methods use dye terminator sequencing methodology. The methods test for α thalassemia gap where PCR reactions determine the deletion of α thalassemia. In rare cases, Multiplex Ligation dependent Probe Amplification (MLPA) detects copy number. iii. Mass Spectrometry In sickle cell, testing, mass spectrometer measures the mass to charge ratio of the charged particles (Banta-Wright 2004). Compounds are ionized to produce molecules carrying charge. This identifies the composition to chemical ratio of the compounds (Banta-Wright 2004). There are different techniques available. Firstly, this process is used to screen a panel for variant for the first time. For instance, it is a test for the newborn screening and potential for antenatal screening (Banta-Wright 2004). Secondly, it is used for second time screening as a confirmation of Hb variants. Finally, it determines protein sequencing as a way of definitively identifying Hb variant (Banta-Wright 2004). iv. Capillary Electrophoresis This method is associated with all advantages of a high-resolution sickle cell testing process (Schwartz and Guttman n.d). It is semi automated. It uses online detection of the condition and directly quantifies the defect and normal red blood cells in terms of fraction (Schwartz and Guttman n.d). It has a capillary system with eight tubes running parallel, which gives a room for multiple and concurrent sample analysis (Wajcman, and Moradkhani 2011). References Banta-Wright, A. S. 2004, ‘Tandem mass spectrometry in newborn screening: A primer for neonatal and prenatal nurses’, Journal Pertinent Neonatal Nurse, vol.18 no.1, pp.41-48. Center for children with Special Needs (2006). Sickle cell Disease: Critical care of Elements. 1st March 2004. < http://www.nwsicklecell.org/uploads/file/Sickle%20Elements%20of%20Care.pdf >. Dogaru, M., Coriu, D., and Higgins, Trefor, 2007, ‘Comparison of two analytical methods (electrophoresis and HPLC) to detect thalassemias and haemoglobinopathies’, Revista Romana de Medicina de Laborator’, vol.9, pp.39-48. Hicks, Edwards, Griep, John, and Nordschow, S. J. 1973, ‘Comparison of results from three methods of haemoglobin identification’, Clin Chem, vol.19, no5, pp.533-535. Jiang, Jianhui, Ma, Xieqin, Song, Chengyan, Lin, Benheng, Cao, Weifeng, Wu, SHuzhen, and Hsiao, Kwang-Jen, n.d, Using the fluorescence spot test for neonatal screening for G6PD deficiency, viewed 1st March, 2014, < http://www.tm.mahidol.ac.th/seameo/2003-34-suppl-3/southeast-2003-vol-34-supp-3-p-140.pdf >. Maakaron, E. J, n.d, Sickle cell anaemia clinical presentation, viewed 1st March, 2004, < http://emedicine.medscape.com/article/205926-clinical >. Ohene-Frempong, Kwaku, 2006, Sickle Cell Center: Overview of sickle cell disease, viewed 1 March, 2013. < http://www.chop.edu/service/hematology/our-programs/sickle-cell-center/diagnosing-and-treating-sickle-cell-disease.html>. Oni, Ga, Johson, Rae, and Oguntibeju, Oo, 2005, ‘Detecting patients with glucose-6-phosphate dehydrogenase deficiency’, JIACM, vol.6 no.1, pp.42-4. Okwi, A. L.,Byarugaba, Parkes, A., and Ocaido, M., 2010, ‘The reliability of sickling and solubility test and peripheral blood film method for sickle cell disease screening at District Health Centers in Uganda’, Clinic in Mother and Child Health, vol.7, pp.1-5. Schwartz, Herb, and Guttman, Andras, n.d. Separation of DNA by capillary electrophoresis. Redwood City, USA: Hoffmann-La Roche, Inc. Wajcman, Henri, and Moradkhani, Kamran, 2011, ‘Abnormal haemoglobin: Detection and characterization’, Indian Journal of Medical Science, vol.134, no.4, pp.538-546. Wong, F. L., Boo, N. Y., Ainoon, O., and Wang, M. K., 2009, ‘Comparison of glucose-phosphate dehydrogenase deficiency using fluorescent spot test, enzyme assays, and molecular method for prediction of severe neonatal hyperbilirubinemia’, Singapore Medicine Journal, vol.50 no.1, pp.62-67. Read More