Analytical and Molecular Techniques in Biomedical Sciences Assessment

laboratory-based practical work undertaken in this module was in relation to a case study of Systemic Lupus Erythematosus, SLE. SLE is a connective tissue disorder, which is autoimmune in nature. This disease affects multiple organs and its clinical manifestation is based on its severity and the organ involved. The pathogenesis of this disease is based on antigen-antibody complexes that circulate in the blood and deposit in the smaller blood vessels of organs. Through the deposition of these complexes and also through auto antibody mediated destruction, there is damage to the organ. (Boon et al., 2010)

The prevalence of SLE is influenced by certain factors, such as, gender, race and genetic predisposition. Like most autoimmune diseases, SLE is also a disease that primarily affects women. Sex hormones seem to play a positive role in this inclination, since most cases develop near menarche or before menopause. Patients, who develop this disease during childhood or after the age of 50, have an equal sex distribution. Racial differences have also been noted while studying the disease. White women appear to be affected much more than black women. Moreover, first degree relatives of patients affected with SLE, have a higher chance of developing the disease than the general population. This genetic predisposition has been attributed to the HLA DR2 and HLA DR3 genes. (Boon et al., 2010)

Before diagnosing SLE, it is important to exclude drugs that may cause a 'lupus-like' syndrome. Chlorpromazine, Hydralazine, Isoniazid, Methyl dopa, Procainamide and Quinidine have a definite association to this syndrome. There are a host of other pharmacological agents that could possibly cause a lupus like syndrome. (Boon et al., 2006)

SLE should be suspected in patients who have a multisystem disease with positive serology, that is, positive antinuclear antibodies and a false positive serologic test for syphilis. A set of criteria have been devised to make the diagnosis of SLE easier. Patients must fill at least 4 out of the 11 criteria to be a candidate for treatment of SLE. These criteria are; malar rash, discoid rash, photosensitivity, oral ulcers, arthritis, serositis, renal disease (if there is greater than 0.5 g/d of proteinuria or greater than 3+ dipstick proteinuria or cellular cast), neurologic disease (seizures or psychosis without any apparent cause), hematologic disorder (hemolytic anemia or leukopenia or lymphopenia or thrombocytopenia), immunologic abnormalities (positive LE cell preparation or antibody to native DNA or antibody to Sm or false positive serologic test for syphilis) and positive antinuclear antibody. (Boon et al., 2010)

The laboratory tests that can be performed to diagnose SLE apart from serologic tests are complete blood count, Coombs test, urine analysis and complement levels. Results may show, anemia, leukopenia, thrombocytopenia, positive direct coombs test, proteinuria, hematuria and hypocomplementemia. The laboratory tests done as part of this assignment were; protein and creatinine content assays, C reactive protein levels and PCR. (Boon et al., 2010)

PROTEIN AND CREATININE CONTENT ASSAYS:

Analysis of the protein and creatinine content in the urine is helpful in determining renal failure, a possible manifestation of SLE. The method used as part of this project was the colorimetric method. There are four colorimetric methods. The choice of method depends on the samples to assay. The main objective is to select a method that requires the least manipulation or pretreatment of the sample. For the purpose of this experiment, the Bradford protein assay method was used. This method is based on the amino acid composition of the protein. ("The colorimetric detection," 2001)

In this method, a red dye, called the Coomassie dye G-250, is used. Under acidic conditions, this dye changes its color to blue. The blue form of Coomassie dye binds to the proteins present in the sample. Several reactions take place during the formation of these complexes. First, the red Coomassie dye donates its free electron to the ionizable protein. This causes the protein to expose its hydrophobic center. The hydrophobic center binds to the non-polar region of the dye through van der Waals forces and ionic interactions. The bond between the protein and the dye stabilizes the blue form of the dye. The blue form of the dye is representative of the amount of protein present in the sample. This blue color has a specific absorption spectrum which is detected by a spectrometer. This method of protein analysis is more preferred for samples with a lower protein content, that is, between 1 and 2000 µg/ml.("The colorimetric detection," 2001)

For the standard operating procedure, a urine sample and coomassie dye G. 250 was used. A dilution series was prepared from a known protein standard and the sample. 0.1 ml of each series was dispensed in test tubes and labeled. 5 ml of Coomassie dye agent was added to each test tube and was incubated for ten minutes at room temperature. Each tube was vortexed before measuring the absorbance at 595 nm. A standard curve was plotted based on the results. The sample protein concentration was determined by interpolation from the standard curve. ("The colorimetric detection," 2001)

As with all methods, this too has several advantages and disadvantages. Unlike other protein assays, this method is less susceptible to interference by other chemical agents that may be present in the sample. An exception to this advantage is the detergent, Sodium dodecyl sulfate, SDS. Concentrations of SDS that either too high or too low can interfere with the results of protein concentrations through this method. At low concentration, SDS tends to bind to proteins, decreasing its binding to the coomassie dye. This results in underestimation of protein levels. On the other hand, high concentrations of SDS can cause overestimation of protein concentrations due to the depletion of free protons from the solution by conjugate base from the buffer. This, however, is not a problem if the protein in the sample is at low concentrations. ("Thermo scientific pierce," 2009)

Moreover, this assay remains linear up till a concentration of 2000 micrograms/ml, making it necessary to use serial dilutions before analysis. Also, this protein dye complex has a tendency to stick to the glass surface, a property that can alter results. ("Thermo scientific pierce," 2009)

Despite the disadvantages, the Bradford method still remains the most popular method for protein analysis. It has simple protocols and results appear with in thirty minutes. The sample does not have to be incubated for long periods of time. There are also no temperature specificities and therefore, this test can be performed easily at room temperature. The reagent use does not have to be prepared and can remain stable for about twelve months. This technique is compatible with various agents, such as buffer salts, reducing agents, metal ions and chelating agents. ("Thermo scientific pierce," 2009)

Other methods used for protein analysis includes Pierce® 660 nm Protein Assay, BCA protein assay, micro BCA protein assay and the modified Lowry protein assay. ("Thermo scientific pierce," 2009)

For the next part of this assignment, creatinine levels were also measured. The Jaffe's method was used to measure creatinine levels. This method is based on the principle that creatinine reacts with picrate in an alkaline medium, producing a brick red color. This color is then assessed through a spectrometer. The intensity of this color is measured at 505 nm through a green filter. (Kanagasabapathy & Kumari, 2000)

For this method, three reagents were made.

Reagent A: 4.4 grams of NaOH was added to 400 ml of distilled water. 9.5 grams of trisodium phosphate and 9.5 grams of sodium tertraborate was added to this solution. The pH of the solution of checked and adjusted with NaOH. This solution was transferred to a 500 ml volume flask. Distilled water was added to the remaining volume, until it became 500 ml. (Kanagasabapathy & Kumari, 2000)

Reagent B: 20 grams of sodium lauryl sulfate was added to a final volume of 500 ml distilled water. (Kanagasabapathy & Kumari, 2000)

Reagent C: 7.0 grams of moist picric acid was added to 500 ml of distilled water. This was mixed and left overnight at 370 C. It was then filtered and stored in a brown glass bottle for use, at room temperature. (Kanagasabapathy & Kumari, 2000)

Equal volumes of all three reagents were mixed.

On the other hand, stock creatinine standard 100 mg/dl was prepared. For this, 100 mg of pure creatinine in 0.1 M. HCl was used and made up to 100 ml with distilled water. This stock creatinine standard was diluted to 2, 4, 6 and 8 mg/dl. This was done by taking 2, 4, 6 and 8 mL of stock creatinine standard and adding each to 100 ml with 0.1 M. HCl. (Kanagasabapathy & Kumari, 2000)

Each of the solutions were placed in separate test tubes and analyzed with a spectrometer. A standard curve was plotted based.....