Investigation of Amyloid Using Four Stains - Literature review Example

Investigation of Amyloid Using Four Stains (H&E, PAS, Cango Red and Sirius Red) Investigation of Amyloid Using Four Stains (H&E, PAS, Cango Red and Sirius Red) (a) Introduction Renal disease is a condition in which the kidneys fail to function properly or fail to function at all. The kidney is a very important organ and its removal or dysfunction can cause death (Michael, 2003), thus diagnosis through cellular pathology becomes essential. Renal diseases are as a result of amyloid fibrils in the liver. According to Ahmet (2012), Amyloid is a substance which is composed of different types of proteins. Ahmet (2012) notes that it is a proteinaceous substance in that it is composed largely of protein, in addition to other compounds like polysaccharides, lipids and even hormones. In amyloidosis, amyloid is deposited and builds up between the cells of tissues and organs. This is not a normal physiological process and is as a result of some pathology (disease, disorder and dysfunction). Amyloids are insoluble fibrous protein aggregates that are highly ordered and stable (Sigurdsson, 2005). Sigurdsson (2005) suggests that they arise from certain inappropriately folded proteins and polypeptides present naturally in the body. These proteins come together to form amyloids fibrils which cause certain renal diseases. The diseases include Alzheimer disease, spongiform encephalopathies and familial polyneuropathy (Sigurdsson, 2005). Traditionally, amyloid is detected in tissues by staining using the Cango red stain. It shows a demonstration of green birefringence under crossed polarizers (Elghetany and Saleem, 1988). Cango red has however gone through alterations and modifications to improve its sensitivity and thus enhance its reliability. Alkaline cango red for instance is a good example of some modification on cango red. Its specificity involved using a sodium chloride saturation solution. Amyloid can also be detected using fluorescent stains and metachromatic stains. Specifically, immunoperoxidase and immunofluorescence identify and classify amyloid’s presence in tissues. Types of amyloid: The type of amyloidosis depends largely on the amyloid fibril present or the protein affected and the resulting pathogen. The following table shows some of the classifications of amyloid. Primary amyloidosis: the amyloid affected protein causes the disease itself. The results are organ failure like kidney failure. Secondary Amyloidosis (AA): disease is created by deposition of AA fibril protein in tissues (Morie et al., 2010) and the disease is propagated as a result. Familial Amyloidosis: this is inherited Acquired amyloidosis: it is contracted through causing microorganisms, for example CJD, drugs etc. Localized Amyloidosis (Aloc): where the amyloid fibrils is situated and isolated in organs usually where the disease occurs. Staining Standard tissue staining is important in investigating amyloid where different stains are used to detect amyloid fibrils, including Cango red and surius, which show green birefringence upon detecting amyloid. Cango red gives a specific apple-green birefringence upon polarization, with amyloid fibrils which definitely distinguishes amyloid from collagen (Morie et al., 2010). Morie et al. (2010) notes that the Cango red stain reigns as the king-of-dyes in the diagnosis of amyloid. Haematoxin & Eosin: H&E is the product of reacting hematein with aluminum ions. Haematoxylin and Eosin staining procedure: Deparaffinize in hot air oven. Hydrate the section. i) 3 dips in xylene (2 Min. each) ii) ii) 3 dips in acetone / alcohol (2 Min. each) iii) iii) In running tap water for 5 Minutes. Mayers haemotoxylin for a duration of about 15 minutes Put in tap water which is running for about 20 minutes Counter stain with eosin for 2 minutes Dehydrate the section in alcohol and acetone (2minutes each). Clear in xylene (for about 120 seconds each) Mount or put inside DPX Results: Nucleus – blue Cytoplasm and background - pink Some detected poor quality arised probably from under ripen-haematoxilin or an over-used one. Periodic Acid – Schiff (PAS): Place section in water, rinse in distilled water, oxidize in 0.5% periodic acid for10min, rinse in distilled running water thoroughly, place the specimen in Schiff reagent for 20 min, and rinse again in water for 5 min. Stain it in Mayers haemalum for a minute and blue it in tap water. Place it in ethanol, clear and mount in DPX. Congo red (Highmans modification): place specimen in water, stain using Highmans Cango red stain for 5min, rinse and differentiate in Highmans differentiator, rinse in tap water, stain in Mayers haemalum for a minute, then blue in tap water. Dehydrate it and mount in DPX. Sirius red stain: place tissue in water, stain with Mayers haemalum for a minute, rinse and blue in tap water for 10min, rinse in 70% alcohol , stain in sirius red solution1 in a Coplin jar - 1 hour, wash well in tap water and then dehydrate, clear and mount in DPX (b) Results and discussion: H & E eliminates bluing of the specimen and clearly defines the nuclei leaving the background colorless. The advantage with H&E staining is that it gives consistent results even in the same tissue. It is simple and can easily be learned or taught. It takes a less time and less stain. Cango red on the hand has been said to be the king-of-dyes in terms of amyloid investigation and thus can be good in detecting renal diseases. Some other histological stains that can aid in diagnosis of other renal diseases include digested stain PAS-D, Gram stain, Coloidal iron stain and so on. They are important in their accuracy in detecting amyloid fibrils and any pathogens that may cause renal disease. Amyloid is relevant in the study of renal diseases as it is quickly detected in histological stains and so any renal disease caused by amyloid fibrils can easily be detected. Cells are the building blocks in all living organisms and they unite to perform specific functions in the body. When they unite, they are called tissues (Bancroft & Stevens, 1990). The study of tissues is called histology. According Bancroft and Stevens (1990), Histology is a branch of anatomy that deals with minute structure, composition and function of tissues, and there are three types of histological preparation that may be used to detect other renal diseases affecting tissues and cells. According to Bancroft and Stevens (1990), the following are histological preparations for a specimen: Whole mount; Sections; Smears. 1. Whole mounts- This is preparation for the entire specimen like animal eg. Fungus, parasite. The preparations should be no more than 0.2-0.5 mm in thickness. 2. Sections- The majority of the preparations in histology are sections. The tissue is cut in about 3-5 mm thick pieces processed, and 5 microns thick sections are cut on a microtome. These are then stained and permanently mounted. Microtomes are special instruments which have automatic mechanism for cutting very thin sections (Bancroft and Stevens, 1990). To cut the sections on the microtome, the tissue must be made hard enough not to get crushed. There are 2 methods of hardening the tissues. One is by freezing them and the other is by embedding them in a hard material such at paraffin wax or gelatin (Bancroft and Stevens, 1990). 3. Smears- Smears are made from blood, bone marrow or any fluid such as pleural or ascitic fluid. These are immediately fixed in alcohol to presence the cellular structures and are then stained. Smears are also made by crushing soft tissues between two slides, and an impression smear is made by pressing a clean slide in contact with the moist surface of a tissue. By doing this, the cells are imprinted on the slide and these may be stained for cytological examination (Conns, 1969). According to Lillie (1965), histopathology means study of diseased tissues and a pathologist responsible for the diagnosis of diseased tissues which may include some of these renal diseases. Specimen slides should be well sectioned and properly stained to provide detailed information of the tissues under examination (Pearse, 1960). Histopathological studies have proved to be one of the most effective in diagnosing tissue abnormalities, benign and malignant conditions (Bancroft and Stevens, 1990). Thus, they can be used to investigate most renal diseases. Dyes are very important in histopathological studies and investigation, but there are some diseases that do not require histological dyes. Dyes used in staining can be classified according to affinity to tissues, source and chemical composition. According to Bancroft and Stevens (1990), they include Acidophilic, Thiazinesb, Basophilic, Azo-dyesc and RosailinsSynthetic. Dyes are good as they have a good staining capacity and a greater spectrum of colors (Conns, 1969). According to Bancroft and Stevens (1990), Haematoxylin is the most popular dye used as a nuclear stain (Lillie, 1965). It is derived from the log tree, mainly found in Mexico. It develops staining property after oxidation, and it’s a weak dye and to make it, a sharp stain mordant is needed. References Ahmet, D., 2012. Amyloid and Related Disorders: Surgical Pathology and Clinical Correlations. London: Springer. Bancroft, J. D., & Stevens, A., 1990. Theory and practice of histological techniques. London: Churchill Livingstone Inc. Conns, H. J., 1969. Biological Stains. Baltimore: Williams and Wilkins. Elghetany, M. T., & Saleem, A., 1988. Methods for staining amyloid in tissues: a review” Stain Technology. Houston, Texas: Department of Pathology, Baylor College of Medicine. John, D. B., & Marilyn, G., 2008. Theory and Practice of Histological Techniques. New York: Elsevier health sciences. Lillie, R. D., 1965. Histopathologic technique and practice: histochemistry. New York: McGraw Hill. Michael, S. G., 2003. Renal Disease: Techniques and Protocols. London: Springer. Morie, A., Gertz, S., & Vincent, R., 2010. Amyloidosis: Diagnosis and Treatment. New York: Springer. Pearse, A.G.E., 1960. Histochemistry. Boston: Little Brown. Sigurdsson, E., 2005. Amyloid Proteins: Methods and Protocols. New York: Springer. Read More