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 on the results. The sample creatinine concentration was determined by interpolation from the standard curve. This method provides analytically reliable results for creatinine level estimation. (Kanagasabapathy & Kumari, 2000)
The limitation of this test is due to the fact that chromogens, other than creatinine can react with picrate. This can overestimate original values. Other methods for creatinine estimation include the enzymatic method. This method requires a smaller sample size and is faster. (Kanagasabapathy & Kumari, 2000)
SERUM PROTEIN ANALYSIS:
C reactive protein, CRP, is an acute phase reactant. This protein is produced by liver in response to ongoing inflammation in the body. The measurement of CRP is not specific for SLE and is produced in a variety of other inflammatory disorders, such as infection, myocardial infarction, inflammatory bowel disease, malignancy or other connective tissue disorders. A positive result for CRP provides evidence for SLE if there are other clinical features present that are specific for the disease. The measurement of CRP is more valuable when trying to decide whether the disease is active or not.
There are different cut off values for CRP in relation to SLE. In a study conducted on 41 patients, active SLE, without the presence of co-existing infection, CRP values did not rise beyond 60 mg/l. This study was further supported by other studies, such as those conducted by Pepys et al. And Borg et al. It was concluded that exacerbations of SLE, without serositis, does not raise CRP beyond a given value, which is 60 mg/l. However, presence of serositis in an active disease can raise the CRP value up to 375 mg/l. Moreover, lupus serositis in a patient may not always be clinically apparent, in which case it is known as subclinical serositis. An elevated CRP beyond 60 mg/l, in the absence of a septic focus, may point to subclinical serositis. (Pyne, 2009)
CRP levels can be measured either qualitatively, semi-quantitatively or quantitatively. All these three methods are based on the ability of CRP to bind to different biologic ligands, forming CRP-ligand complexes. These complexes then precipitate and are detected through various methods. The Enzyme Linked Immunoabsorbent Assay, ELISA, is used to measure CRP quantitatively. This test uses enzyme marked anti-CRP antibodies. These antibodies bind to the CRP molecules in human serum. After all the complexes have been formed, the unbound antibodies are removed. A substrate is added to the enzyme marked CRP-ligand complexes. The enzyme breaks down the substrate causing the solution to change color. The specimen is then analyzed with a spectrophotometer, which calculates the net color absorbency. This test can be performed within fifteen minutes and can detect CRP levels up till 40 mg/l. (Hengst, 2003)
Immunofluorescence, laser and rate nephelometry quantitative tests are other techniques than can also be used for quantitative measurement of CRP levels. The immunofluorescence test is much like ELISA. This test uses fluorescent tracers for quantification. The laser and rate nephelometry quantitative immunoassays use infrared light emitting diodes and detectors passed through test tubes containing CRP-ligand complexes. The quantity of CRP-ligand complexes determine the degree of light scatter which is detected by detectors. (Hengst, 2003)
In a study conducted, the minimum level of CRP detected by ELISA was 0.005-0.022 ng/ml, with a mean minimum detectable dose of 0.010 ng/ml, which make it highly sensitive. However, since CRP levels are raised in a variety of pathological states, these values are not specific. ELISA can also be used to measure autoantibodies to double stranded DNA. This test is fairly cheap and is an indicator of disease prognosis. (“Quantikine,” 2010)
ELISA is a relatively quick test that can detect very low concentrations of antigens. The application of ELISA is also vast. It can be used to detect infection and other autoimmune conditions in the body. However, only monoclonal antibodies are used, which are more costly than polyclonal antibodies. Moreover, the enzyme-substrate reaction time is short and so results must be read as soon as possible. (Glen B, Susan, Abel & Andrew, 2007)
All of these tests are liable to biological and analytical variability. Biological variability is the variability in lab parameters based on differences in physiology amongst different individuals, in which case it is called inter-individual biological variability, or in the same person at different points in time, in which case it is known as intra-individual biological variability. Data on biological variation can be generated by applying specific experimental protocols. Once the data on it are achieved, it can be combined to the analytical variation for the calculation of critical difference. Analytical variation is the variability associated with the precision of the analysis technique. This technique may be different for different procedures. In this case, the analytical variation may be the variation associated with the spectrophotometer and the substrate-enzyme reaction. Pre-analytical variation can be due to phlebotomy techniques, sample transport, handling and storage techniques. (Fraser, 2012)
Laboratory tests are performed to either diagnose a patient’s illness, monitor one’s disease progression or for epidemiological research purposes. Analytical precision is most important for laboratory data generated for the purpose of monitoring disease progression or treatment response. The ELISA test for the quantitative measurement of CRP is usually for monitoring disease activity, serositis and to check the presence of concomitant infection. (Pyne, 2009)
If monitoring serial CRP readings in a single patient, changes in result could mean either that the patient is improving or getting worse or these changes could be due to pre-analytical variation, biological variation or analytical variation. If pre-analytical variation is minimized by good phlebotomy and standard sample transport, handling and storage techniques, then, in order to monitor patient progress, the change in CRP values must exceed the inherent variation due to biological and analytical variation, that is: 20.5 Z [CVA2 + CVI2]0.5 (Fraser, 2012)
(Z = number of standard deviates appropriate to the probability selected.
CVa = analytical variation.
CV1 = with in subject variation.) (Fraser, 2012)
The minimum performance is described as CVA as less than 75% of CV1. Desirable performance is CVA less than 50% of CV1 and optimum performance is CVA less than 25% of CV1. (Fraser, 2012)
In order to achieve certain analytical precision, it is important to create certain goals to attain the desired level of analytical quality. This should in include an interaction of three basic elements, that is, specification, creation and control of analytical quality. For quality specifications, three approaches can be used. The first is the analytical approach, in which the median quality achieved by a group of laboratories is taken as the level to be achieved. The clinical approach is the second approach, in which clinical uses of test results and their outcome, is used to set a target level. The third approach is the biological approach, in which quality specifications are based on inter-or intra-biological variations. After specifications are made, the quality nearest to the specified level is created by proper selection of measurement methods and an adequate control system is implemented. (Fraser, 2012)
MOLECULAR BIOLOGY:
The development of SLE is triggered by certain genetic and environmental factors. The HLA DR2 and HLA DR3 alleles are the genetic associations of SLE. Autoantibodies directed against native double stranded DNA, dsDNA, are present in 40-60% of patients with SLE. The presence of these autoantibodies has also been associated with decreased survival in patients suffering with the disease. The HLA DR3 allele has been associated with a greater risk of dsDNA positive SLE. Other genes such as the STAT, KIAA1542, BANK1, and UBE2L3 and ITGAM genes are also associated with positive dsDNA in SLE patients. These genes are more correctly considered to be “autoantibody propensity loci” rather than “SLE susceptibility loci.” Amongst the genes associated with SLE susceptibility involve the interferon and the T cell receptor pathways. (Chung SA, Taylor KE, Graham RR, et al., 2011)
In a research that was conducted to identify the genetic associations with SLE, 61 genes were identified to be expressed differently in patients with SLE. Out of these, 24 were up regulated and 37 were down regulated. These genes were related to a variety of cell processes and pathways. PCR can be used to identify disease susceptibility in relatives of patients with SLE. (GM, SL, N, et al., 2003)
Polymerase chain reaction, PCR, is a process through which analysis of short DNA or RNA sequences can be analyzed. A sample of chromosomal DNA is used as the starting material. Other ingredients include free nucleotides, DNA primers and an enzyme called Taq polymerase. Taq polymerase is used because it can tolerate high temperatures. The primers are about twenty bases long and are complementary to the sequence of the target DNA segment. The mixture is heated to 94 degrees Celsius. The heat denatures the double stranded DNA, breaking the hydrogen bonds holding them together. This leaves two separate single strands of DNA. The temperature is then reduced to about 54 degrees Celsius. This allows annealing of the primers with the single stranded template. The temperature is then again raised to 72 degrees Celsius. This allows optimum functioning of Taq polymerase. Polymerization begins as the Taq polymerase begins to add nucleotides to the 3′ end of each primer attached to a DNA strand. After each cycle, the number of double stranded DNA is doubled. (“Definition of pcr,” 2004)
Since PCR is a process that is widely applicable to different scenarios, no single set of conditions can be applied to all PCR amplifications. Therefore, appropriate and optimal primer sequences and concentrations are essential for maximal specificity and efficiency. The 3′- terminal sequence of the primer is responsible for PCR specificity and sensitivity. A sequence of three consecutive guanine and cytosine bases should be avoided since it could promote the non-specific annealing of the primer. Moreover, a thymidine base at the 3′ end is not recommended since it is more prone to miss-priming than other base pairs. Primer pairs should also be checked for complementarity. Complement primers may form bonds resulting in primer-dimer formation. This reduces primer availability to the template, causing failure of PCR. (“Troubleshooting guide for,” )
An optimum primer length consists of 18-30 base pairs. A shorter primer holds the risk of annealing at more than one complementary site, thus leading to the amplification of non-specific base pairs. The chances of a longer primer annealing at more than one site is lesser, allowing the generation of only the specific amplification. (“Troubleshooting guide for,” )
The annealing temperature also varies for different primers, even when using pairs of primers with a similar annealing temperature. The desired approach to this problem is to start at a temperature that is five degrees below the required temperature and to gradually adjust the value to improve specificity and yield through a series of experiments. Certain buffer systems, such as the QIAGEN buffer renders such optimization techniques as unnecessary. This buffer system provides a wider window of annealing temperatures. (“Troubleshooting guide for,” )
Primer concentration is another condition that determines optimization. The recommended concentration should be between 0.1-0.5 µM. Higher concentrations increases the risk of miss-priming, which eventually results in the production of non-specific PCR products. (“Troubleshooting guide for,” )
PCR is becoming increasingly popular as a diagnostic tool for clinicians and for researchers. However, the usefulness of PCR can be greatly handicapped due to the demanding assay protocols and the need to follow quality assurance protocols. If these protocols are not followed, a great risk of contamination exists by previously amplified products or environmental samples. This can lead to false negative results which may entirely change a doctor’s diagnosis and treatment. It may also have serious commercial consequences.
False negative results due to contamination can be reduced by the use of uracil N. glycosylase, UNG, anti-contamination technology. The basic concept of this technique is to label the amplicons that are to be produced during PCR. The amplicons can be labeled by replacing the base pair Thymine with Uracil. The UNG enzyme recognizes this label. Upon subsequent heating at an alkaline pH, this enzyme destroys the labeled apmplicon DNA but not the target DNA. This allows an originally true-negative specimen to turn negative, which would otherwise have been regarded as positive. For false negative reactions, UNG causes it to turn positive because during annealing, when the temperatures are elevated beyond UNG’s capacity, amplicons accumulate and a positive sign is obtained. (Burkardt, 2000)
Human error, insufficient PCR amplification and detection are other factors that may decrease the usefulness of this technique. Therefore, for the purpose of quality assurance, standard PCR protocols have been established to minimize error. These protocols provide five sections which assess laboratory quality, provide guidelines for products and methods used during PCR and also provide guidelines for data recording, record keeping and evaluation. (“Quality assurance/quality control,” 2004)
CONCLUSION:
SLE is an autoimmune, connective tissue disorder. The diagnosis of this condition is based on a combination of clinical findings and laboratory tests. Four tests undertaken in this course, in association to the disease, were creatinine and protein assays, ELISA for the quantitative detection of CRP levels and PCR for identifying gene susceptibility loci.
Creatinine and protein content assay in the urine is a useful diagnostic test for the development of renal failure, a possible manifestation of SLE. The Bradford method was used to measure the protein concentration, whereas the Jaffe method was used to measure creatnine.
ELISA is a sensitive test that can accurately measure CRP levels up to 40 mg/l. (Hengst, 2003) CRP levels in patients with SLE are mainly used for the purpose of monitoring disease progression and response to treatment. This makes analytical variation the most crucial factor in determining the degree of error. Other factors that may cause variation are pre-analytical variation and biological variation.
PCR is another test that was used to identify susceptibility loci. The role of diagnosing SLE with PCR is nil. However, future genetics might use PCR to screen patients who are prone to develop SLE. PCR is a sensitive and a specific test and is the preferred modality in diseases where diagnosis cannot be confirmed through other methods. The usefulness of PCR in SLE and in other diseases can be limited if proper protocols are not followed. This can be due to contamination or improper selection of primers or other PCR conditions. Optimization of PCR technique through the establishment of guidelines has greatly reduced human and machine error.
REFERENCES:
Burkardt, H. (2000). standardization and quality control of pcr analyse. Clin Chem Lab Me, 38(2), 87-91. Retrieved from http://www.gene-quantification.de/burkardt-2000.pdf
Chung SA, Taylor KE, Graham RR, Nititham J, Lee AT, et al. 2011 Differential Genetic Associations for Systemic Lupus Erythematosus Based on Anti-dsDNA Autoantibody Production. PLoS Genet 7(3): e1001323. doi:10.1371/journal.pgen.1001323 (genes of HLA DR2,3)
Definition of pcr. (2004, Feburary, 2). Retrieved from http://www.medterms.com/script/main/art.asp?articlekey=4807
Glen B, B., Susan, D., Abel, L., & Andrew, H. (2007). Handbook of neurochemistry and molecular biology. (3rd ed., Vol. 6, pp. 215-216). New York: Springer
GM, H., SL, C., N, S., S, Y., & CD, B, et al. (2003). Analysis of gene expression profiles in human systemic lupus erythematosus using oligonucleotide microarray.Genes and Immunity, 4(3), 177-186.
Franzini, C. (1995). Relevance of analytical and biological to quality and interpretation of test results: examples of application to haematology. Ann 1st Super,31(19-13), Retrieved from http://www.iss.it/binary/publ/cont/Pag9_13Vol31N11995.pdf
Fraser, C.G. (2012, January 24). biological variation data for quality specifications. Retrieved from http://www.westgard.com/index.php?option=com_content&task=view&id=201&Itemid=83
Kanagasabapathy, A.S., & Kumari, S. (2000, September). Guidelines for standard operating procedures in clinical chemistry. Retrieved from http://whqlibdoc.who.int/searo/2000/SEA_HLM_328.pdf
Nicholas A. Boon, Nicki R. Colledge, Brian R. Walker, John A.A. Hunter.(2010). Davidson’s Principles and Practice of Medicine . India, Elsevier.
Pyne, D. (2009). Interpretation of c-reactive protein values in systemic lupus erythematosus. Grand Rounds,9(4), 18-19. Retrieved from http://www.grandrounds-e-med.com/articles/gr090005/gr090005.pdf
Quantikine. (2010) Retrieved from http://www.rndsystems.com/pdf/dcrp00.pdf
Quality assurance/quality control guidance for laboratories performing pcr analyses on environmental samples. (2004, October). Retrieved from http://www.epa.gov/microbes/qa_qc_pcr10_04.pdf
Standard operating procedure for laboratory service. (1995, March 1). Retrieved from http://www.msh.org/Documents/upload/SOPs_for_Laboratory-1.pdf
The colorimetric detection and quantitation of total protein. (2001). Retrieved from http://www.nshtvn.org/ebook/molbio/Current Protocols/CPFAC/fab0101.pdf
Thermo scientific pierce protein assay technical handbook. (2009). Retrieved from http://www.piercenet.com/files/1601669_passayfinal_intl.pdf
Troubleshooting guide for pcr. (n.d.). Retrieved from http://www.fermentas.com/templates/files/tiny_mce/media_pdf/3_PCR_Troubleshooting.pdf
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