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Homework answers / question archive / Jerry Santiago MLT2194L May 1, 2010 Helicobacter Pylori Antibody Principle & Application Anti- Helicobacter pylori is an antibody That detects the presence of the Helicobacter pylori organim that can be found on the surface of the epithelium in the stomach and rarely, the duodenum

Jerry Santiago MLT2194L May 1, 2010 Helicobacter Pylori Antibody Principle & Application Anti- Helicobacter pylori is an antibody That detects the presence of the Helicobacter pylori organim that can be found on the surface of the epithelium in the stomach and rarely, the duodenum

Health Science

Jerry Santiago MLT2194L May 1, 2010 Helicobacter Pylori Antibody Principle & Application Anti- Helicobacter pylori is an antibody That detects the presence of the Helicobacter pylori organim that can be found on the surface of the epithelium in the stomach and rarely, the duodenum. H. pylori is a Gram-negative curved bacillus. It produces an enzyme that can create a low-acid microenvironment on the surface of the gastric epithelial cells (more prominently in the gastric antrum), thus being one of the few bacteria that can survive the harsh acidic environment of the stomach. It is estimated that two-thirds of the world’s population carries the bacteria and that in the United States approximately 20% of the people under the age of 40 or over the age of 60 are infected with the bacteria. However, in the U.S. it has been observed that the infection rates are declining. H. pylori can cause chronic gastrtistis, lead to gastric lymphoid proliferations and evne localized gastric lymphomas. A diagnosis of the presence of H. pylori can be detected by invasive (biopsy) and noninvasive methodologies including breath analysis, serum serology, urease testing and cultures. None of these tests have 100% sensitivity. Histologically, the identification of the H.pylori can sometimes be made using a routine Hematoxylin & Eosin (H&E) stained sections of the stomach. However, the average detection rate is only 66%. The utilization of histochemical stains for the demonstration of the H. pylori organism increases the sensitivity compared to the H&E stain. However, such stains can be difficult to read and time consuming to review. Immunohistochemistry has become a valuable tool for improving the rate of identification of H. pylori by the use of a highly specific and sensitive antibody. This stain is especially useful in specimens with only a few number of organisms. Selection & Interpretation of Controls The selection of controls for Helicobacter pylori should include two types of controls. Generally, one positive control that contains the H. pylori organism and a negative control where the primary antibody has been omitted and no organisms are detected. Positive Control Tissue ? ? The use of a positive control will test the validity of the antibody as well as the protocol used. The staining observed should include the detection of the cel wall of the organism when the chromogen is applied. For the demonstration of H. pylori, many pathologists prefer a red chromogenic reaction over the brown detection as it seems to provide a relative better contrast with the selected counterstain. ? Select a positive control that contains numerous H. pylori organisms that can be easily visualized under light microscopy. Negative Control Tissue ? The use of a negative control will test for the specificity of the antibody and should not detect any H. pylori organisms. There are several ways to perform the negative testing: - Omitting the rpimary antibody on the patient tissue by using a wash buffer or normal serum from the same species used for the primary antibody. - The use of an antibody that will not stain the cell wall of the H. pylori organisms. Example: Using and antibody such as Prolactin that is specific for brain tissue and not developed to detect the H. pylori organism. Visualization of a stained slide The staining of H. pylori is concentrated on the surface of the mucinous epithelial cells, either on the luminal urface or in the crypts. Ocassionally, a slight blush may be seen on the cytoplasmic membrane of the mucosa. The staining should be as strong as possible, without significant reaction with other structures. Troubleshooting Problem: False positive staining of non target cells. Cause: Cross reactivity of the antibody with cellular components. Solution: ? Use a purified primary antibodythat has had non-specific immunoglobin removed. ? Use a monoclonal antibody if available. ? Compare H. pylori stained slide to H&E slide and identify the areas containing the organisms. Problem: Non specific staining of patient tissue with over stained positive control and over stained specific targets in patient tissue. Cause: a) Concentration of the antibody is too strong. Solution: ? Check diltuion of the antibody and repeat using freshly prepared antibody. ? Re-titer the primary antibody; testing at least three consecutive dilutions of the antibody on control and patient tissue. ? Use a purified primary antibodythat has had non-specific immunoglobin removed. Cause: b) Section too thick. Solution: ? Cut all sections at the same thickness as section used to validate antibody dilution. Safety & Precaution 1. Personal Protective equipment should be worn when handling all chemicals. 2. Follow manufacturer’s instructions for the use and operation of all equipment. 3. Disposal of all waste solutions should follow MSDS and local or state disposal requirements. References: 1. Baron, Samuel. “Helicobacter Pylori and other Gastric Helicobacter-Like Organisms.” 1996. Medical Microbiology, 25 January 2010 http://www.ncbi.nlm.nih.gov. 2. Boenisch, T et al, Educational Guide: Immunohistochemical Staining Methods. 4thed. Carpinteria, CA: Dako Corporation, pp. 41-45. 3. Curran, R.C and J Croker. Curran’s Atlas of Histopatology. 4th ed. London: Oxford University Press, 2000. 4. Dabbs, David J. Diagnostic Immunohistochemistry. Philadelphia, PA: Churchill Livingston, 2002. 5. Wheater, Paul, et. al. Basic Histopathology. 2nd ed. New York: Churchill Livingston, 1991.

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