Assessment: Laboratory-Based Practical Work Undertaken

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SAMPLE EXCERPT:

[. . .] 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… [END OF PREVIEW]

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