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Special stains are referred to all stains exclude H&E stain that is used in the histology laboratory in staining various tissues for microscopic views of various features of cells. Liver cirrhosis is regarded as a hyper-accumulation of fibrous tissue constituents and is frequently detected in later or fatal conditions of chronic hepatic diseases. Liver cirrhosis is well demonstrated with Masson's Trichrome stain. Collagen fibres will be stained blue, nuclei stained black and cytoplasm, muscle and erythrocytes stained red. Amyloidosis is extracellular accumulation of usually soluble autologous protein misfolded into an abnormal form of fibrils, resulting in major morbidity and is frequently fatal. Congo red is a common histology special stain for the detection of amyloid. Amyloid stains red with Congo red dye. Treatment for liver cirrhosis often dependent on the complications and cause of the disease with the aims of slowing down the development of scar tissue in the liver and treat or avoid the complications resulting from the disease. A liver transplantation is suggested when treatment is not capable to manage and control the complications. Although amyloidosis cannot be cured, drug therapy can be used to stabilise the native structure of the precursor protein to prevent its transition to further amyloid configurations and improve survival. Presently, therapies emphasizes on decreasing the supply of amyloid fibril precursor protein, while substituting and supporting the task of the involved organs. Liver cirrhosis and amyloidosis if not treated can lead to many complications.
Solid tissues require fixation and processing, to maintain their structures and later impregnated with a suitable hardening material to allow making thin slices appropriate for staining and evaluation under microscope (Morales et al., 2004). After processing, tissues do not retain enough of their natural colour to make their microscopic structures visible. It therefore becomes expedient to add colours to them by staining with proper dyes, which would stress the biological features with the impact of colours on tissues and thus increases contrast in the image examined under the microscope. In a routine histology laboratory, haematoxylin and eosin (H&E) stain is the standard stain for the basic diagnosis by staining merely nuclei and cytoplasm. Subsequently, various special stains have been developed to stain selective features of the tissue such as organelles, microorganisms and carbohydrates. Other histology stains exclude H&E stain, and antibody linked techniques are regarded as special stains. Giemsa, Periodic acid Schiff, VonKossa, Alcian Blue, Masson's Trichrome and Congo red stains are among the special stains used in histology laboratory.
The objective of this practical is to distinguish the histology of normal liver tissue and liver cirrhosis tissue stained with Masson's Trichrome, and also a normal liver tissue and amyloidosis liver tissue stained with Congo Red. The liver functions in the digestion of food, the formation of excretory products, the degradation of complex cellular materials (e.g., hemoglobin), the detoxification of unnatural compounds and the synthesis of plasma proteins. The liver is an important part of a human body, therefore it is important to diagnose and treat diseases involving the liver such as liver cirrhosis and amyloidosis of the liver.
Liver cirrhosis is a result of persistent liver disease represented by substitution of liver tissue by scar tissue, fibrosis and regenerative nodules (occurrence of lumps due to damage tissue undergone regeneration), thus heading to decrease function of the liver (Ueki et al., 1999). Common causes are hepatitis B, hepatitis C, alcoholism and fatty liver disease (Matsuda et al., 1997). Generally, cirrhosis is irreversible, and its treatment focuses on inhibiting complications and progression (de Jongh et al., 1992). The scar tissue that forms in cirrhosis damages the structure of the liver, progressively making the flow of blood through the liver more difficult and thus decreases the normal function of the liver.
Masson's Trichrome stain is a common histology special stain for liver and kidney biopsies. Liver cirrhosis is well demonstrated with this stain. In Masson's Trichrome staining method, three dyes are used selectively for staining collagen fibres, muscles, erythrocytes and fibrin. The principle of trichrome stain is that the smallest molecular size dye stains the less permeable tissues (Blanchard et al.,1987). However, if a large dye molecule is capable in penetrating the tissue, staining will take place in the rate of the smallest molecule. The tissue is initially stained with Biebrich Scarlet (acid dye), which binds to the acidophilic components of the tissue (Shoobridge, 1983). Then, when differentiated in phosphomolybdic acid, the less porous elements hold on to red colour, as the red colour is drawn out of the collagen and together providing a link for the collagen to be stained by aniline blue (Shoobridge, 1983). Collagen fibres will be stained blue, nuclei stained black and cytoplasm, muscle and erythrocytes stained red.
Figure 1: Normal histology of liver stained with Masson's Trichrome (Morales et al., 2004)
Figure 2: Liver cirrhosis stained with Masson's Trichrome observed under 200x magnification. (Morales et al., 2004)
Proteins are pivotal building blocks for all body part and are often harmless. However, in rare cases, abnormal proteins are produced by cells and tend to build up in the body tissue resulting in deposits called 'amyloid' that will eventually cause a disease termed 'amyloidosis' (Sanchorawala, 2006). Amyloidosis may result in major morbidity and are frequently fatal (Westermark et al., 2002). The rigid, linear extracellular fibrils that make up the amyloid is characterised from the misfolding of a protein from its normal alpha helix configuration into antiparallel beta-pleated sheets (Merlini and Bellotti, 2003 ; Glenner, 1980 ). The beta-pleated sheets (figure 3) when observed under the electron microscope, forms long, non - branching fibrils (figure 4) (Kisilevsky, 2000). The amyloid production results from the defective proteolysis of the precursor proteins (Merlini and Bellotti, 2003). Once the beta-pleated sheets have developed, the body's intra and extracellular proteolytic enzyme systems find it almost impossible to digest; therefore, it accumulates inevitably to basement membranes (Perfetti et al., 2001).
Figure 3: Beta pleated model of amyloid protein (Sipe, 2005).
Figure 4: Amyloid fibrils viewed under electron microscope (Hirschfield and Hawkins, 2003)
Congo red is a common histology special stain for the detection of amyloid. The affinity of Congo red binding to amyloid fibrils in Î²-sheet reflects the specificity and efficacy of this staining. Congo red, an equally linear molecule as shown in figure 5, has a molecular weight of 696.7g/mol and its formula is C32H22N6Na2O6S2, with a chemical name of benzidinedflandersiazo-bis-1-naphtylamine-4-sulfonic acid (Frid et al., 2007). Two phenyl rings in the hydrophobic centre linked to two charged end naphthalene moieties containing amine groups and sulfonic acid, via diazo bonds. Congo red has a chinone structure in an acid solution and sulphonazo structure in a basic solution as shown in Figure 4, thus changes the blue colour (<pH 3) to red (>pH5). Histologically, amyloid stains pink in Congo red dye (figure 6) (Warrell et al., 2006).
Figure 5: Chemical composition of Congo red. End groups are attached to the hydrophobic centre (linker centre) via diazo bonds (A). Based on the pH of the solution, end moieties can have a sulphonazo appearance (acid solutions) (B) or chinone appearance (basic solution) (C) (Frid et al., 2007).
Figure 6: Congo-red-stained section (medium power) from a slightly less involved liver parenchyma. Congophilic material (amyloid) in portal connective tissue is seen in a nodular configuration compressing the portal structures. In addition, congophilic material is seen surrounding the hepatic cords diffusely within the lobules (Shaz et al., 2001).
Materials and Method
Solutions and Reagents:
Weigert's Iron Haematoxylin
Phosphomolybdic acid solution
1% Acetic Acid
Sections of two liver tissues were given- Masson's Trichrome stain was carried out by the laboratory assistant. First, both the slides were deparaffinized and rehydrated through a descending manner of alcohol, 100% alcohol, 95% alcohol and 70% alcohol. The slides were washed in distilled water. Then, the slides were stained in iron hematoxylin working solution for 10 minutes. The slides were then rinsed in running tap water for about 10 minutes followed by distilled water. Next, both the slides were stained in Biebrich scarlet-acid fuchsin solution for 15 minutes- this solution can be saved to be used in future. The slides were washed again in distilled water. Both the slides were further differentiated in phosphomolybdic-phosphotungstic acid solution for about 15 minutes or until collagen was not red. Both the sections then were transferred directly (without rinse) to aniline blue solution and stain for 5-10 minutes. Both the sections were rinsed briefly in distilled water and further differentiated in 1% acetic acid solution for 1 minute and then washed in distilled water. Following that, the slides were dehydrated very quickly (gently dipped in and out for few seconds) through 95% ethyl alcohol, absolute ethyl ethyl alcohol. Biebrich scarlet-acid fuchsin stain was wiped off in this dehydration process; the sections were then cleared in xylene. Subsequently, the slides were mounted with resinous mounting medium. Finally both the slides were examined under the microscope.
Solutions and Reagents:
Congo Red Stock Solution
1% Sodium Hydroxide
Congo red Working Solution
Alkaline Alcohol Solution
Sections of two liver tissues were given- Congo red stain was carried out by the laboratory assistant. First, the sections were deparaffinized and hydrated with distilled water, then the sections were placed in Congo red working solution for 10 minutes. Following that, the slides were rinsed in distilled water. The slides were further differentiated (5-10 dips) in alkaline alcohol solution, then again the slides were rinsed in tap water. Next, the sections were counterstained in Gill's hematoxylin for 30 seconds. Following that, the slides were rinsed in tap water for another 2 minutes. The slides were then dipped in ammonia water for about 30 seconds or until section turned blue. The slides were rinsed in tap water for 5 minutes. After that, the slides were dehydrated in ascending manner of alcohol, 95% alcohol and then 100% alcohol, they were then cleared in xylene and further mounted with mounting medium. Finally, the slides were examined under the light microscope.
Microscopy observation of two liver sections stained with Masson's Trichrome.
Observation date: 29/ 01/ 2010
Figure 7: Drawing of the microscopy observation of the normal histology of the liver tissue stained with Masson's trichrome, x400 magnification.
Figure 8: Drawing of the microscopy observation of liver cirrhosis tissue stained with Masson's Trichrome, x400 magnification.
Microscopy observation of two liver sections stained with Congo Red.
Observation date: 29/ 01/ 2010
Figure 9: Drawing of the microscopy observation of the normal histology of the liver tissue stained with Congo Red, x400 magnification.
Figure 10: Drawing of the microscopic observation of the amyloid stained with Congo red, x400 magnification.
The first slide, a normal liver tissue stained with Masson's trichrome showed single layered well defined hepatocytes with nucleus stained black, cytoplasm and muscle stained red. The second slide stained with Masson's indicates liver cirrhosis. Cytoplasm stained red, massive tissue fibrous tissue and area of macrovesicular fatty change were observed along with hepatocytes structure being compressed and atrophic. The third slide, a normal liver tissue stained with Congo red showed no nodules and congophilc material present, which indicates no amyloidosis. Whereas, the fourth slide stained with Congo red indicates amyloidosis. Amyloid stained red with Congo red stain. Compressed and irreguloar shaped hepatic cells were observed. Congophilic material seen surrounding hepatic cords diffuse within lobules indicate presence of amyloidosis. Generally, branches of the portal vein and hepatic artery are located in the connective tissue of the liver (Ross et al., 2003). These vessels combine to form the hepatic sinusoids (figure 11). Hepatocytes are multifunctional cells that make up about 80% of the cells in the liver and are typically large polyhedral cells, with large round centrally located nuclei (Hubscher, 2002).
Figure 11: Normal liver histology stained with H & E stain (Shaz et al., 2001)
The initial thing to observe when diagnosing liver cirrhosis is the loss of typical structure. Therefore, the specimen should be sufficient containing numerous intact areas. Bouins solution is fixative of choice for masson's trichrome (Jones, 2007). The reason for Bouins and acidified staining solutions (aniline blue) is because the acid pH is essential to increase the selectivity for the collagen fibers. The Weigerts iron hematoxylin is best when made up fresh each time, it continues to oxidize and if kept a long time as a working solution it loses strength, resulting in weak nuclei staining (Jones, 2007).
Following staining with Congo red, amyloid emerge as numerous shades of red when observed under unpolarised light. The reason is that the maximum absorption of wavelengths of the dye falls on the blue/green area of the visible spectrum (Howie and Brewer, 2009). Red shades are the results of the white light being altered by the utmost absorption of these wavelengths. For red colour to appear, Congo red (disodium salt) should be in a weak acid or an alkaline solution. In a strong acid solution, the maximum absorption falls on the longer wavelengths in the orange and yellow area, thus emerging a blue or violet colour. This justifies the reason why Congo red is also used as pH indicator to observe changes of colour seen between pH 3 to 5 (Horobin and Kiernan, 2002). Bathochromic shift, the highest absorption moving to longer wavelengths following escalate binding to a substrate, may occur (Amelin and Tret'yakov, 2003). This red colour appears apple green under polarised - light, which is a unique property to amyloid (figure 12). The stain is unstable and must be freshly prepared every 2 months or less (Warrell et al., 2006).
Figure 12: Liver tissue stained with Congo Red tissue observed under polarised light, arrow indicates apple green colour of amyloid (Shaz et al., 2001)
Microscopy view of every type of amyloid is alike when stained with Congo Red, however, the structure of the fibrils are different for every type of amyloidosis which determines the different classes of amyloidosis (Warrell et al., 2006). Other special stains can be utilised to detect amyloid deposits. In some cases, a cardiac pathologist normally favours Sulphated Alcian Blue stain to detect amyloid accumulation in myocardium, whereas, a peripheral nerve pathologist would prefer using crystal violet stain in screening sural nerve biopsies and verify positive results with further staining with Congo Red (Mayo References Services, 2002). The original Congo Red stain in later stages have been modified as discovered in the Highman's and Alkaline techniques, resulting in a much more specific detection of amyloid (Cheung et al., 2006). In particular, false positive results may occur due to the fact that the birefringence is merely based on a consistently systematic Congo Red binding to the amyloid fibers, though being nonspecific (Pettersson and Konttinen, 2008). False negative results may also occur due to the factors related to staining and sample collection.
Liver cirrhosis and amyloidosis if not treated can lead to many complications. In liver cirrhosis, portal hypertension causes ascites which may further cause umbilical hernia (a protruding belly button) and also varices which may burst and cause blood loss (Vizzutti et al., 2009). Cirrhosis may also lead to jaundice and hepatic encephalopathy (de Jongh et al., 1992). Liver amyloidosis is a progressive disease with poor prognosis and may cause death in patients due to liver failure (Silverstein, 2005).
Treatment for liver cirrhosis often dependent on the complications and cause of the disease with the aims of the treatment to slow down the development of scar tissue in the liver and to treat or avoid the complications results from the disease. Combination of diuretics furosemide and spironolactone aids in treating patients with oedema and ascites, by operating in the kidneys which helps to eliminate water and salt into the urine. Beta-blocker medications aids in treating portal hypertension by reducing the pressure in esophageal varices, thus lower the chances of bleeding. Lactulose, a laxative to clean the bowel is often prescribed to hepatic encephalopathy patients. Cirrhosis patients with hepatorenal failure are required to undergo frequent haemodialysis treatment. Antiviral drugs like interferon are prescribed for treating viral hepatitis patients. A liver transplantation is suggested when treatment is not capable to manage and control the complications (Francoz et al., 2007). Stravitz and colleagues suggested that liver transplantation in patients diagnosed with hepatocellular carcinoma does prolong survival (Stravitz et al., 2008).
Systemic amyloidosis, according to Hirschfield and Hawkins, is the reason of mortality in 1/1500 people per year in the UK, but effective therapies may treat maturity onset diabetes and Alzheimer's disease patients as shown in figure 13 (Hirshfield and Hawkins, 2003). Accurate recognition of the fibril form, usually by genetic analysis and immunohistochemistry along with characterisation of the amount of amyloid accumulation throughout one's body are crucial in selecting suitable therapies (Lachmann et al., 2002). Presently, therapies emphasise on decreasing the supply of amyloid fibril precursor protein, while substituting and supporting the task of the involved organs (Gillmore et al., 2001).
Figure 13: Amyloidogenesis and treatment targets (Hirschfield and Hawkins, 2003)
Although amyloidosis cannot be cured, drug therapy can be used to stabilise the native structure of the precursor protein to prevent its transition to further amyloid configurations and improve survival. In secondary amyloidosis, the treatment consists of treating the underlying inflammatory disease with repeated infections by suppressing chronic inflammation and thus lessens production of reactive serum amyloid A protein (SAA) (Tan et al., 2004). Chemotherapy is often used as treatment for primary amyloidosis and at times combined with autologous stem cell transplantation in carefully selected patients. Commonly used chemotherapy drugs in killing plasma cells are melphalan, chlorambucil and cyclophosphamide (Aase et al., 2002). These drugs cause the suppression of SAA (precursor of secondary amyloid fibrils), and thereby leads to reduction of proteinuria (Dominguez & De Strooper, 2002). Liver transplantation has been the most effective therapy in hereditary amyloidosis caused by transthyretin gene alterations. Eventually, the variant transthyretin will disappear from the plasma resulting in no production of abnormal amyloid proteins in the liver and consequently the disease improves (Dominguez & De Strooper, 2002).
Masson's Trichrome stain is suitable in detecting liver cirrhosis and Congo Red stain is a sensitive more specific method for visualising hepatic amyloid. With the developing knowledge of liver cirrhosis and amyloidosis, more reproducible, accurate and non-invasive techniques been developed in histopathology laboratory to determine liver cirrhosis and amyloidosis. Appropriate therapy may substantially improve prognosis in many patients.