Research Articles Base: Immunohistochemistry

The Accuracy of Immunohistochemistry in Duchenne Muscular Dystrophy

This article aims to discuss the accuracy of Immunohistochemistry in the diagnosis of Duchenne Muscular Dystrophy. This will also cover a brief discussion about this genetic disorder (Duchenne Muscular Dystrophy). In this regard, laboratory findings and results of some experiments will be presented to facilitate a better understanding of this topic. <p class="MsoNormal" style="BORDER-RIGHT: medium none; PADDING-RIGHT: 0in; BORDER-TOP: medium none; PADDING-LEFT: 0in; PADDING-BOTTOM: 0in; MARGIN: 6pt 0in 0pt; BORDER-LEFT: medium none; LINE-HEIGHT: 200%; PADDING-TOP: 0in; BORDER-BOTTOM: medium none; mso-border-alt: solid windowtext .5pt; mso-padding-alt: 12.0pt 6.0pt 12.0pt 6.0pt">Abbreviations <p class="MsoNormal" style="BORDER-RIGHT: medium none; PADDING-RIGHT: 0in; BORDER-TOP: medium none; PADDING-LEFT: 0in; PADDING-BOTTOM: 0in; MARGIN: 6pt 0in 0pt; BORDER-LEFT: medium none; LINE-HEIGHT: 200%; PADDING-TOP: 0in; BORDER-BOTTOM: medium none; mso-border-alt: solid windowtext .5pt; mso-padding-alt: 12.0pt 6.0pt 12.0pt 6.0pt">DMD           Duchenne Muscular Dystrophy IHC             Immunohistochemistry Introduction There are different human disorders caused by the mutation of certain genes. One of these is Duchenne Muscular Dystrophy. Duchenne Muscular Dystrophy (DMD) is the most common hereditary neuromuscular disorder in the first two decades of life (Araujo, et al., 2004). This disorder, which commonly affects male children since it is linked to the mutation of the X chromosome, is characterized by a number of signs and symptoms. Early manifestations include progressive muscle weakness (legs and pelvis), deteriorating muscle mass spreading to the other parts of the body, decreasing strength and stamina, enlargement of calf muscles and other problems associated with gait and posture. As the disease advances, muscle wasting occurs and is then replaced by fat leading the affected individual to even more difficulties. During the advanced stage of DMD, contractures occur and it may then lead to increased breathing difficulties, an inability to move, then death usually follows. Due to its intricacy, the possibility of the early diagnosis of this disorder can be very beneficial not only to the affected individual but to the family as well. Early detection of DMD can help prepare the family as to what plans will have to be taken into consideration. Aside from this, early diagnosis can influence the course of the disease process through early disease management principles that will be used. There are different diagnostic tests used to confirm the presence of DMD. These include: absent dystrophin on muscular biopsy or presence of a deletion in the dystrophin gene located on chromosome Xp21 (Araujo, et al., 2004). According to Araujo, et al. (2004, p. 180), One should be suspicious of DMD when creatine phosphokinase (CK) are elevated in boys with developmental delay or in neonatal screening procedures. Aside from diagnostic tests mentioned above, certain tests are also used in further studies of DMD. One of these tests is immunohistochemistry. Immunohistochemistry Immunohistochemistry (IHC) is widely used in surgical pathology and serves as a diagnostic, prognostic, and predictive tool (Goldstein, et al., 2007). Certain findings through IHC may support the presence of DMD in the affected individual. Linking IHC to the diagnosis of DMD can be explained mainly through the presence or absence of dystrophin in the muscle. However, there have been some recommendations to improve the standardization in the practice of IHC. Since different assays are used in the practice of IHC, it is only proper that these products be improved for more reliable results. But then, Goldstein, et al. (2007, p. 124) argued that despite the improvements of reagents and automation, authors over the years have consistently noted the inconsistent quality of IHC assays. In this regard, whether or not IHC is accurate in the diagnosis of DMD is now in question. To explore further the process by which IHC works in order to help diagnose DMD, here are some details on an experiment conducted to test the accuracy of IHC in the diagnosis of the aforementioned disorder. Freund, et al. (2007, p. 74 to 75) conducted an experiment with 106 unrelated patients referred for DNA analysis of the dystrophin gene between 1999 and 2005. Most had a clinical diagnosis of DMD or BMD based on clinical findings, electromyography (EMG) and elevated serum creatine kinase activity. The group then used methods such as DNA analysis, Muscle Biopsy, and IHC to determine any alteration or deletion in the patients’ muscle tissue/s. Here is a detailed look on the methods used in the aforementioned experimentation. DNA analysis – The DNA was isolated from peripheral blood leukocytes using the standard phenol/chloroform method16. PCR analysis was performed using primers previously described. An isolated reaction was carried out for each of the 20 exons. The multiplex reactions were not performed because the isolated one is more credible and compatible with the conditions of our laboratory The exons studied were numbers 3, 4, 6, 8, 12, 13, 17, 19, 42, 43, 44, 45, 47, 48, 50, 51, 52 ,53, 60 and Pm. The PCR products were analyzed on a 7% polyacrylamide gel and the bands visualized by silver staining. Muscle biopsy – The muscle biopsy was done in fifty one patients. Some had the DNA analysis before (normal) or during the work-up investigation (simultaneously muscle biopsy and blood draw for DNA analysis). These fifty one samples were freshly frozen and cut on cryostat into 8- micron sections and stained with hematoxylin-eosin, modified Gomori trichrome, oil-red O, PAS, cresyl violet and Sirius red. They were then processed with ATPase pH 9.4, 4.3 and 4.6, myophosphorylase, non-specific esterase, NADH-tetrazolium reductase, succinic dehydrogenase, cytochrome c-oxidase and acid and alkaline phosphatase. Afterwards, the biopsies were cut into 4-micron sections and submitted to immunohistochemistry for dystrophin proteins (rod and carboxyl- and amino–terminal domains), sarcoglycans (alpha, beta, gamma and delta) and dysferlin (Freund, et al., 2007) In this table of results from the experiment conducted by Freund, et al. (2007, p. 75), notice that there are seven who had alterations compatible with muscular dystrophy but had normal immunohistochemistry (unclassified limb-girdle muscular dystrophy). Discussion A normal IHC of an individual’s muscle tissue manifests the presence of dystrophin. On the other hand, its absence on the sarcolema is suggestive of DMD. Looking through the result of the experiment (as shown in Table 2), there were seven patients who had alterations suggestive of muscle dystrophy. But then, these patients had normal IHC results. What do these imply? There is an obvious inconsistency with regards to the result of the aforementioned detail of the experiment. As mentioned earlier, a person diagnosed with DMD will have to yield an absence of dystrophin. Yet, in the result, there were seven whose IHC results confirm that dystrophin is present. This alone is suggestive that the results of the IHC do not necessarily point out the presence of DMD. However, in a study conducted by Araujo, et al. (2004, p. 180), results of their experimentation (muscle biopsy immunostaining) confirm the presence of DMD. They were able to confirm it through the absence of dystrophin. In this regard, we can somehow conclude that the accuracy of diagnosing DMD through IHC is not yet perfect. But then again, there are several recommendations as to how the process of IHC can be improved. With the continuous effort of different scientists and agencies, IHC can soon be a perfect tool to diagnose diseases like DMD. After all, early diagnosis can contribute greatly to the outcome and treatment of any disease process. . . References Araujo, A., de Deco, M., de Sa Kloh, B., da Costa, M., de Gois, F., Guimaraes, A. (2004). Diagnosis Delay of Duchenne Muscular Dystrophy. Brazilian Journal of Mother and Child Health, vol.4 no.2, pp. 179-183, Instituto Materno Infantil de Pernambuco Freund, A., Scola, R., Arndt, R., Lorenzoni, P., Kay, C., Werneck, L. (2007). Duchenne and Becker Muscular Dystrophy: A molecular and Immunohistochemical Approach. Academia Brasileira de Neurologia, Sao Paulo , BRESIL (1943) Goldstein, S., Hewitt, S., Taylor, C., Yaziji, P., Hicks, D., and Members of Ad-Hoc Committee (2007). Recommendations for Improved Standardization of Immunohistochemistry. Applied Immunohistochemistry & Molecular Morphology, vol. 15 no.2, pp. 124-133, Lippincott Williams & Wilkins Credit:ivythesis.typepad.com

The Accuracy of Immunohistochemistry in Duchenne Muscular Dystrophy

This article aims to discuss the accuracy of Immunohistochemistry in the diagnosis of Duchenne Muscular Dystrophy. This will also cover a...

DNA replication

Cell division exists to produce more cells for growth and repair processes. It is essential that all cells of the body have the same genetic material. For this reason, an important event always precedes cell division: the exact duplication of the genetic material in a process called DNA replication. Only after this has occurred can mitosis take place.             The primary function of DNA replication is understood to be the provision of progeny with the genetic information possessed by the parent. Thus, the replication of DNA must be complete and carried out with high fidelity to maintain genetic stability within the organism and the species. The process of DNA replication is complex and involves many cellular functions and several verification procedures to ensure fidelity in replication. About 30 proteins are involved in the replication of the E Coli chromosome, and this process is almost certainly more complex in eukaryotic organisms.             DNA in eukaryotic cells is associated with a variety of proteins, resulting in a structure called chromatin. The double-stranded DNA helix in the chromatin of each eukaryotic chromosome has a length that is thousands of times the diameter of the cell nucleus. The precise trigger for DNA synthesis is unknown, but once it starts, it continues until all the DNA has been replicated.             Eukaryotic DNA in nondividing cells is located in chromosomes, which typically are associated as identical pairs. DNA replication begins as the DNA helix uncoils and gradually separates into its two nucleotide chains.     The DNA in each chromosome is exactly replicated according to the rules of base pairing during the S phase of the cell cycle. Each strand of the double helix is replicated simultaneously but by somewhat different mechanisms. Nucleotides join in a complementary way: adenine always bonds to thymine, and guanine always bonds to cytosine. Hence, the order of the nucleotides on the template strand also determines the order on the new strand.             A complex of proteins replicates the leading strand continuously in the 5’ to 3’ direction. The lagging strand is replicated discontinuously, in short pieces of 150-250 nucleotides, in the 3’ to 5’ direction. The principal enzymes for replicating DNA are a complex of multiple enzymes called DNA polymerase which attaches and moves along the DNA template strand. These are joined by DNA ligase. DNA ligase causes bonding of successive DNA nucleotides to one another, using high-energy phosphate bonds to energize these attachments. DNA replication occurs at several sites, called replication bubbles, in each chromosome. The entire process takes about 9 hours in a typical cell. A variety of mechanisms, employing different enzymes, repair damaged DNA, as after exposure to chemical mutagens or ultraviolet radiation. The end result is that two DNA molecules are formed that are identical to the original DNA helix, and each consists of one old and one newly assembled nucleotide strand. Each newly formed strand of DNA remains attached by loose hydrogen bonding to the original DNA strand that has been used as its template. This new set of nucleotide strand then serves as a template, or set of instructions, for building a new nucleotide strand. Credit:ivythesis.typepad.com

DNA replication

Cell division exists to produce more cells for growth and repair processes. It is essential that all cells of the body have the same genetic...